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https://openalex.org/W4372331227	https://www.nature.com/articles/s41598-023-34018-w.pdf	English	null	Inaccuracies of deterministic finite-element models of human middle ear revealed by stochastic modelling	Scientific reports	2,023	cc-by	11,588	OPEN Arash Ebrahimian 1,2, Hossein Mohammadi 1,2, John J. Rosowski 3,4, Jeffrey Tao Cheng 3,4 & Nima Maftoon 1,2* Arash Ebrahimian 1,2, Hossein Mohammadi 1,2, John J. Rosowski 3,4, Jeffrey Tao Cheng 3,4 Nima Maftoon 1,2* For over 40 years, finite-element models of the mechanics of the middle ear have been mostly deterministic in nature. Deterministic models do not take into account the effects of inter-individual variabilities on middle-ear parameters. We present a stochastic finite-element model of the human middle ear that uses variability in the model parameters to investigate the uncertainty in the model outputs (umbo, stapes, and tympanic-membrane displacements). We demonstrate: (1) uncertainties in the model parameters can be magnified by more than three times in the umbo and stapes footplate responses at frequencies above 2 kHz; (2) middle-ear models are biased and they distort the output distributions; and (3) with increased frequency, the highly-uncertain regions spatially spread out on the tympanic membrane surface. Our results assert that we should be mindful when using deterministic finite-element middle-ear models for critical tasks such as novel device developments and diagnosis. The middle ear plays a vital role in our hearing process by converting acoustic energy from the environment to mechanical vibrations and conducting them to the inner ear. Many studies have used different methods to model middle-ear mechanics in order to improve our fundamental understanding of the hearing process1–7. Middle-ear models can be helpful for other purposes such as predicting the hearing loss from middle ear pathologies and injuries, simulating surgeries, and developing and advancing diagnostic and treatment methods.fhi j g g p g g g Different approaches for modelling middle-ear mechanics were reviewed elsewhere8,9. The finite-element (FE) method is a powerful continuum-mechanics-based method that has been extensively used to model middle-ear mechanics starting with the pioneering work of Funnell and Laszlo10. The FE method can deal with complex geometries and different material properties and boundary conditions.f gf p p y In order to obtain reliable results from an FE model, realistic mechanical properties of different structures of the middle ear should be known. Several studies attempted to identify the mechanical properties of some of the structures in the middle ear11–17. For instance, laser Doppler vibrometry, stroboscopic holography, and FE modelling were used to estimate the viscoelastic properties of the human tympanic membrane (TM)11. www.nature.com/scientificreports www.nature.com/scientificreports OPEN Also, indentation measurements and inverse FE method were used to estimate the quasi-static Young’s modulus of the human TM12. Recently, a Bayesian inverse method was proposed that can be used to find the material prop- erties of thin structures including the TM13 using vibration measurements with holographic methods18–21. A review of the material characterization of the TM can be found elsewhere22. Additionally, several studies have employed sensitivity analysis methods to identify the most influential parameters in the middle ear. In most of these studies, local sensitivity analysis was performed by varying the value of one model parameter while keeping all other model parameters fixed at their baseline values. These one-at-a-time sensitivity analyses can study the local effects of perturbation of parameters around one point in the N-dimensional parameter space (N being the number of model parameters) but they cannot investigate the entire parameter space. Maftoon et al. performed a local sensitivity analysis of the FE model of gerbil middle ear6. Motallebzadeh et al. used both local and Morris sensitivity analysis methods to study the effects of variations of parameters of a human newborn FE model23. O’Conner et al. studied the effects of varying material properties of the human TM on the sound conduction 1Department of Systems Design Engineering, University of Waterloo, Waterloo, ON, Canada. 2Centre for Bioengineering and Biotechnology, University of Waterloo, Waterloo, ON, Canada. 3Eaton‑Peabody Laboratories, Massachusetts Eye and Ear, Boston, MA 02114, USA. 4Department of Otolaryngology‑Head and Neck Surgery, Harvard Medical School, Boston, MA 02114, USA. *email: nmaftoon@uwaterloo.ca \| https://doi.org/10.1038/s41598-023-34018-w Scientific Reports \| (2023) 13:7329 www.nature.com/scientificreports/ in the middle ear24. In general, the focus of these sensitivity analyses was to find the importance of each of the model parameters rather than studying the impacts of natural stochastic variabilities of the model parameters on the variabilities of the motions in the middle ear.h The mechanical/geometrical properties of middle-ear structures have intrinsic inter-individual variability that can be taken into account with stochastic uncertainties25. Indeed, differences observed in physiological data from different individuals (e.g., the normative study by Whittemore et al.26) can be attributed to the probabilistic distribution of the morphological features and mechanical properties of the middle-ear structures among indi- viduals. Since the first FE model of the middle ear in 197810, most FE middle-ear models in the literature were deterministic and few systematically considered the effects of these uncertainties in the model parameters on its predictions. Materials and methods Geometry, model components, and mesh. The 3D geometry was created based on the segmentation of µCT image datasets of a 73-year-old male cadaver temporal bone that included 1024 × 1012 × 1014 cubic voxels with a voxel size of 18.0828 µm. The scan was done with Xradia MicroXCT-200 at 90 kV and 8W. The segmentation process and meshing were done in 3D Slicer (www.slicer.org) software (version 4.11.20200930)27. After finalizing segmenting all parts, we used the Segment Mesher toolbox of Slicer for creating the mesh. We used Cleaver2 (www.sci.utah.edu/cibc-software/cleaver.html) meshing library for creating a conformal volumet- ric mesh for all parts of the model. A sizing field was created manually and was modified several times in order to create the desired mesh that consists of coarse elements in the regions where no considerable deformations are expected (i.e., bones) and fine elements in other regions. For the TM only, the generated volumetric mesh was converted to a surface mesh and only the medial section of the mesh (which is connected to manubrium) was used in the FE model. Thus, our model had volumetric mesh for all components in the middle ear except the TM for which we had surface mesh. The details about the number of elements will be provided later in this section.h h p The model included the TM, ossicles, anterior mallear ligament (AML), lateral mallear ligament (LML), incudostapedial and incudomallear joints (IMJ and ISJ), superior malleolar ligament (SML), stapedial annular ligament (SAL), posterior incudal ligament (PIL), and manubrial fold. The tensor tympani tendon and stapedial tendon were not included in the model as these tendons tend to have functional consequences in live ears, while the experimental data that were used for validations in this study are from cadaveric temporal bones. Also, our model does not include the middle ear cavity. The geometry and mesh of the middle-ear model are shown in Fig. 1. In the following, we discuss the motions of the anatomical landmarks highlighted in this figure. Deterministic finite‑element modelling. The material properties of the baseline model are presented in Table 1. We assumed all materials to be isotropic and elastic6. Besides, we considered nearly incompressible material properties (Poisson’s ratio of 0.49) for all soft tissues6. For the ossicles, the value of Poisson’s ratio was set to be 0.35. OPEN In deterministic FE models, the value of all model parameters are predetermined (either from experimental measurements or model sensitivity analyses) and fixed, and stochastic variations of parameters are not integrated in these models. In the present work, we developed a stochastic FE model of the human mid- dle ear to investigate the effects of different levels of natural variability in the model parameters on its outputs. Materials and methods The thickness of the TM was assumed to be uniform and it was considered to be 74 μm which is the same as the value used by Gan et al.5. The average TM thickness values used in most models in the literature are also close to this value (74 μm)29. The references for the values of the Young’s modulus and density of the model components are listed in Table 1. In addition to these a priori mate- rial properties, we determined the Rayleigh damping coefficients of all the middle-ear structures by manually adjusting them to closely replicate the experimental measurements of Voss et al.1. j g y p p To excite the model, we applied uniform pressure (with the amplitude of 1 Pa) to the entire TM area laterally. The TM annulus was considered to be fully clamped31. Also, the ligaments (AML, LML, SML, PIL, and SAL) were considered to be fixed at their distal ends where they normally connect to the wall of the middle-ear cav- ity. For all simulations except the full-field vibration patterns, we performed a transient dynamic analysis with a uniform pressure step function as the input6 and used the implicit Newmark-β scheme32. In order to have an unconditionally stable solution, we chose values of β and γ in the Newmark-β scheme to be 0.25 and 0.5, respectively6. To obtain the displacement frequency response function, we found the impulse response by dif- ferentiating the step response (with respect to time) and then used the fast Fourier transform to construct the frequency response function. The full-field vibration patterns were obtained using harmonic vibration analysis. The Code_Aster (www.code-aster.org) open-source FE code (version 14.4.0) was used for the computations. We modelled the TM using seven-node second-order TRIA7 COQUE_3D shell elements6, while all other structures (ossicles, joints, and ligaments) were modelled using ten-node second-order TETRA10 3D solid tetrahedral elements in Code_Aster. Mesh dependency. The original mesh of our model consisted of 205,322 tetrahedral elements (TETRA10 3D) for all parts of the model except the TM that was composed of 7778 shell elements (TRIA7 COQUE_3D). In order to check the mesh convergence, we used the Homard33 utility of Code_Aster to refine the original mesh. We divided each triangle into four coplanar triangles and as a result, the numbers of tetrahedral elements and tri- angular elements were increased by factors of eight and four, respectively. Materials and methods Moreover, the values of the Young’s modulus of the TM and SAL reported in Table 1 were chosen to be in the range of the values reported in the literature14,22. We used Rayleigh damping considering stiffness- proportional damping for all structures except for the TM, and manubrial fold11,28. In Table 1, α1 and α2 are the Rayleigh-damping coefficients of mass and stiffness matrices, respectively. Figure 1. 3D model of the middle ear. Constructed geometry from two different viewing angles (left and center) and mesh of the human middle-ear model (right). This model was used in our stochastic FE analysis and the motions of the highlighted structures are discussed in this study. Figure 1. 3D model of the middle ear. Constructed geometry from two different viewing angles (left and center) and mesh of the human middle-ear model (right). This model was used in our stochastic FE analysis and the motions of the highlighted structures are discussed in this study. https://doi.org/10.1038/s41598-023-34018-w Scientific Reports \| (2023) 13:7329 \| www.nature.com/scientificreports/ Table 1. Material properties of the baseline model. Structure E (MPa) Density (kg/m3) Poisson’s ratio α1 (1/s) α2 (s) TM 12 120011 0.49 700 4 × 10−6 Malleus 1400024 239024 0.3 0 4 × 10−7 Incus 1400024 215024 0.3 0 4 × 10−7 Stapes 1400024 220024 0.3 0 4 × 10−7 IMJ 3024 110024 0.49 0 13 × 10−5 ISJ 3024 110024 0.49 0 13 × 10−5 SAL 1.4 120024 0.49 0 13 × 10−5 PIL 224 120024 0.49 0 13 × 10−5 LML 224 120024 0.49 0 13 × 10−5 Manubrial fold 1.211 120011 0.49 700 4 × 10−6 SML 4.95 120030 0.49 0 13 × 10−5 AML 224 120024 0.49 0 13 × 10−5 Table 1. Material properties of the baseline model. For modelling the cochlear load, we considered a cochlear impedance of 20 GΩ which is the same value used by Gan et al.5. In our model, the surface area of the stapes footplate was measured to be 3.6 mm2 which is also in the range reported by Motallebzadeh et al.23. From these values, we calculated a viscous damping coefficient of 0.26 Ns/m that we uniformly distributed to four dashpots attached to the stapes footplate and in the direc- tion parallel to the piston-like motion6. Using dashpots to model the cochlear load is a common approach in the literature of FE modelling of the middle ear5,6,28. www.nature.com/scientificreports/ of the umbo and stapes footplate, respectively. Therefore, we used the original time step (10 μs) and time span (25 ms) for all our calculations. of the umbo and stapes footplate, respectively. Therefore, we used the original time step (10 μs) and time span (25 ms) for all our calculations. Uncertainty propagation. We made a baseline conventional deterministic FE model with a priori mate- rial parameters from the literature and validated this model against existing experimental data. We then con- sidered the probability distribution of the model parameters, sampled the 32-dimensional parameter space and propagated the uncertainties to the model outputs. We considered having uncertainties in the material proper- ties (Young’s modulus, Poisson’s ratio, and stiffness-proportional damping coefficient) for all of the components of the model as well as in the thickness of the TM and in the cochlear load. All these uncertainties existed at the same time in our simulations.ifi We quantified uncertainties using two indices: the coefficient of variation (CV): (1) CV = standard deviation mean × 100, if mean = 0 (1) nd uncertainty amplification (UA) which quantifies the amplification of the uncertainties of the outputs with espect to the uncertainties in the model parameters34: nd uncertainty amplification (UA) which quantifies the amplification of the uncertainties of the outputs with espect to the uncertainties in the model parameters34: (2) UA = CVOutput 1 n n i=1 CVInputs (2) where n is the number of uncertain model parameters. In our study, we had 32 uncertain model parameters. A UA value of greater than one indicates amplification.h where n is the number of uncertain model parameters. In our study, we had 32 uncertain model parameters. A UA value of greater than one indicates amplification.h The stochastic sets of model parameters were created by the Latin Hypercube Sampling method for each scenario (each combination of random parameters sampled from the 32-dimensional parameter space) with the mean values equal to values for the baseline model (Table 1 for material properties, TM thickness of 74 μm, and cochlear load viscous damping coefficient of 0.26 Ns/m) and CV of 10% and 20%. The ranges of variation of each model parameter for both CV values are reported in Supplementary Table S1. We used UQLab35 (ver- sion 1.3.0) for creating stochastic sets of model parameters. www.nature.com/scientificreports/ In the absence of reported probability distributions for most parameters of the model, we assumed normal distribution, which is advocated to be a suitable choice for many biological variables36. However, because the value of the Poisson’s ratio was set to 0.49 for soft tissues in the baseline model, we considered a half-normal distribution for soft tissues with the mean of 0.49 that only resulted in values less than or equal to the mean. Also, in order to reduce the number of parameters, we consid- ered all three ossicles to have the same Young’s modulus, Poisson’s ratio, and damping in our stochastic model- ling. Visualization of the sampled 32-dimensional parameter space is not directly possible but Fig. 2a shows the distribution of parameters on the planes of some pairs of parameters of the space with CV of 20%. The planes in this figure show normal and half-normal distributions as described above. Th h d l d h b f l f i g The uncertainty in the model parameter space was propagated to the output by performing FE analysis for each set of the stochastic model parameters described above. We evaluated the FE model with 1992 parameter scenarios to cover the 32-dimensional parameter space. All of the FE calculations were performed on the Niagara cluster of Digital Research Alliance of Canada (www.alliancecan.ca) with Intel Skylake (2.4 GHz, AVX512) pro- cessors running under the CentOs 7 distribution of Linux. Each single simulation of the model with the original mesh and original time step and time span (“Results”) was performed on one core using 18 GB memory. Each node of the cluster has 40 cores and 188 GiB of memory, allowing 10 parallel simulations on one node and a total number of 100 model evaluations on one node took about 14 h to complete.t p After propagating the uncertainties to the outputs, statistical analyses were done on the outputs. For the output phases, we implemented circular statistics to find values of the mean, standard deviation, skewness, and kurtosis using the Circular Statistics Toolbox of Matlab37. Using circular statistics, we consider the circular nature of all of these parameters, which is important for phase values. For instance, the circular standard deviation is analogous to linear standard deviation but it considers the cyclic nature of phase values when evaluating their variabilities. www.nature.com/scientificreports/ More details about these parameters can be found in the documentation of the Circular Statistics Toolbox37. To calculate the skewness and kurtosis of the output phase, we employed the methods described by Pewsey38 and Fisher39, respectively.l y p y Figure 2b provides a summary of the workflow of the stochastic FE analysis that we used in the current work. We submitted 2000 simulation scenarios on the cluster from which eight faced software issues and we could evaluate the FE model with 1992 parameter scenarios to cover the 32-dimensional parameter space. Materials and methods It was observed that the refined mesh does not result in substantial changes in the trend and the values of the vibration responses of the umbo and stapes footplate in the frequency range of 100 Hz to 10 kHz. We compared the results at 405 equally spaced fre- quencies in this frequency range for the displacement amplitude of the umbo and stapes footplate. For the umbo amplitude, the differences between the results obtained from the original and refined meshes were less than 1 dB at 354 frequencies, with a maximum difference of 1.40 dB at 1.24 kHz. Additionally, for the displacement ampli- tude of the stapes footplate, the difference between the results obtained from the original mesh and refined mesh was less than 1 dB at 321 frequencies, with a maximum difference of 2.11 dB at 1.17 kHz. As a trade-off between the accuracy and computational cost, we chose to perform all calculations with the original mesh. Time‑step and time‑span dependency. We chose the time span of 25 ms (which provides a frequency resolution of 24 Hz). For checking whether the response is affected by increasing this time span, we increased it to 50 ms (which provides a frequency resolution of 12 Hz) and observed less than 0.001 dB difference for both amplitudes of displacement of the umbo and stapes footplate and for all frequencies in the range of 100 Hz to 10 kHz. We chose the time step of 10 μs (the maximum frequency of 50 kHz) and decreased it to 5 μs (the maximum frequency of 100 kHz). We observed that in the frequency range of 100 Hz to 10 kHz, decreasing the time step caused maximum changes of 0.36 dB (at 7.59 kHz) and 0.44 dB (at 7.59 kHz) in the displacement amplitudes https://doi.org/10.1038/s41598-023-34018-w Scientific Reports \| (2023) 13:7329 \| www.nature.com/scientificreports/ Results Distribution of some of the uncertain model parameters and workflow of the stochastic FE analysis. Panel (a): Some of the 2D views of the 32-dimensional parameter space sampled using the Latin Hypercube method with the coefficient of variation (standard deviation/mean) of 20%. Depending on the nature of the parameter, the sampled space shows normal and half- normal distributions. We used 1992 sets of stochastic model parameters in our stochastic FE analysis. Panel (b): Workflow of the stochastic FE analysis process used to study the effects of uncertainty in the middle-ear model. In the first step, we used the values of the baseline model (reported in Table 1) as the mean values of our stochastic sets of model parameters. We then specified the amount of coefficient of variation (standard deviation/mean) of the uncertain parameters. We considered the coefficient of variation to be 10% and 20% in the current work and used the Latin Hypercube Sampling method to create stochastic sets of model parameters with normal and half-normal distributions. The next step was to solve the FE model for all of these stochastic sets of model parameters, and from here, we found the stochastic outputs of the system. We used these stochastic outputs to study the amplification of the uncertainties in the system outputs and determine the output distributions. 29 \| https://doi.org/10.1038/s41598-023-34018-w Figure 2. Distribution of some of the uncertain model parameters and workflow of the stochastic FE analysis. Panel (a): Some of the 2D views of the 32-dimensional par using the Latin Hypercube method with the coefficient of variation (standard deviation/mean) of 20%. Depending on the nature of the parameter, the sampled space show normal distributions. We used 1992 sets of stochastic model parameters in our stochastic FE analysis. Panel (b): Workflow of the stochastic FE analysis process used to stu uncertainty in the middle-ear model. In the first step, we used the values of the baseline model (reported in Table 1) as the mean values of our stochastic sets of model par specified the amount of coefficient of variation (standard deviation/mean) of the uncertain parameters. We considered the coefficient of variation to be 10% and 20% in th used the Latin Hypercube Sampling method to create stochastic sets of model parameters with normal and half-normal distributions. Results Middle‑ear model can magnify parameter uncertainties up to more than three times in the output. Figure 3a and c, respectively, present the stochastic normalized amplitude and phase of the umbo displacement for model parameters with the CV of 10%. The middle-ear resonance frequency, identified by the peak in the umbo amplitude response, was about 1.5 kHz in the deterministic baseline response and had a UA of about 0.77 in the stochastic model. At low frequencies (below the middle-ear resonance frequency), the uncer- tainty of the umbo displacement amplitude was not amplified (UA of about 0.6). Also, the value of the circular standard deviation of the phase remained close to zero at low frequencies. Above the middle-ear resonance frequency, the uncertainties were amplified in the umbo displacement output with the highest UA of 4.38 near 2.5 kHz for the amplitude and maximum values of the circular standard deviation in the phase (0.06 cycles) hap- pened near frequencies of 1.9 kHz and 3.5 kHz. https://doi.org/10.1038/s41598-023-34018-w Scientific Reports \| (2023) 13:7329 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Distribution of some of the uncertain model parameters and workflow of the stochastic FE analysis. Panel (a): Some of the 2D views of the 32-dimensional parameter space sampled using the Latin Hypercube method with the coefficient of variation (standard deviation/mean) of 20%. Depending on the nature of the parameter, the sampled space shows normal and half- normal distributions. We used 1992 sets of stochastic model parameters in our stochastic FE analysis. Panel (b): Workflow of the stochastic FE analysis process used to study the effects of uncertainty in the middle-ear model. In the first step, we used the values of the baseline model (reported in Table 1) as the mean values of our stochastic sets of model parameters. We then specified the amount of coefficient of variation (standard deviation/mean) of the uncertain parameters. We considered the coefficient of variation to be 10% and 20% in the current work and used the Latin Hypercube Sampling method to create stochastic sets of model parameters with normal and half-normal distributions. The next step was to solve the FE model for all of these stochastic sets of model parameters, and from here, we found the stochastic outputs of the system. We used these stochastic outputs to study the amplification of the uncertainties in the system outputs and determine the output distributions. Figure 2. Results The maximum UA of about 4.38 happens near 2.5 kHz for the umbo displacement amplitude. For the phase, the values of circular standard deviation are close to zero at low frequencies and the maximum circular standard deviation happens near frequencies of 1.9 kHz and 3.5 kHz. Panels (b) and (d): The CV of all uncertain model parameters was set to be 20%. The UA (Equation (2)) is about 0.6 for the umbo displacement amplitude at frequencies below the middle-ear resonance frequency (at ~ 1.5 kHz). For the amplitude, the maximum UA (3.19) happens near about 2.5 kHz and for the phase, the maximum circular standard deviation (0.09 cycles) happens at about 1.8 kHz. Increasing the uncertainty in the model parameters to the CV of 20% (Fig. 3b,d) did not lead to uncertainty amplification at low frequencies: the UA of the amplitude remained about 0.6 and the circular standard deviation values of the phase remained close to zero. Also, the UA of the middle-ear resonance frequency experienced a negligible increase (0.83 vs. 0.77). At frequencies higher than the middle-ear resonance, the increased uncer- tainty in the model parameters caused more dispersed umbo displacement responses with an average CV of about 32% for amplitude (was about 17% when CV of inputs were 10%) and an average standard deviation of 0.05 cycles in phase (was 0.02 cycles for CV of 10%). Near 1.8 kHz, the circular standard deviation of the phase shows its maximum (0.09 cycles). Also, the greatest UA peak (3.19) of the umbo displacement amplitude was in the vicinity of 2.5 kHz. In this frequency neighborhood, for some sets of model parameters, an anti-resonance occurred as can be seen in Fig. 3b and d. These anti-resonances did not exist in Fig. 3a and c where the CV of uncertain model parameters was 10%.h p The stapes displacement describes the output of the complete middle ear. Figure 4a and b show the frequency response of the stapes footplate with the CV of 10% for all uncertain model parameters. Similar to the umbo response, at low frequencies (below the middle-ear resonance at 1.5 kHz), uncertainty amplification did not occur: the UA was about 1 for the amplitude and the values of the circular standard deviation were close to zero. Results The next step was to solve the FE m stochastic sets of model parameters, and from here, we found the stochastic outputs of the system. We used these stochastic outputs to study the amplification of the unce outputs and determine the output distributions. Scientific Reports \| (2023) 13:7329 \| https://doi.org/10.1038/s41598-023-34018-w www.nature.com/scientificreports/ Figure 3. Stochastic frequency response function of the umbo displacement. Amplitude and phase of the umbo (normalized with respect to excitation pressure) are presented in this figure. The motion of the umbo is reported in the direction normal to the manubrium at the umbo. The results of individual simulations (n = 1992) are plotted with gray thin lines and because they are all close to each other, the whole response region looks like a gray shaded area. Panels (a) and (c): The CV of all uncertain model parameters was set to be 10%. The UA (Equation (2)) is about 0.6 for the umbo displacement amplitude below the middle-ear resonance frequency (at ~ 1.5 kHz). The maximum UA of about 4.38 happens near 2.5 kHz for the umbo displacement amplitude. For the phase, the values of circular standard deviation are close to zero at low frequencies and the maximum circular standard deviation happens near frequencies of 1.9 kHz and 3.5 kHz. Panels (b) and (d): The CV of all uncertain model parameters was set to be 20%. The UA (Equation (2)) is about 0.6 for the umbo displacement amplitude at frequencies below the middle-ear resonance frequency (at ~ 1.5 kHz). For the amplitude, the maximum UA (3.19) happens near about 2.5 kHz and for the phase, the maximum circular standard deviation (0.09 cycles) happens at about 1.8 kHz. Figure 3. Stochastic frequency response function of the umbo displacement. Amplitude and phase of the umbo (normalized with respect to excitation pressure) are presented in this figure. The motion of the umbo is reported in the direction normal to the manubrium at the umbo. The results of individual simulations (n = 1992) are plotted with gray thin lines and because they are all close to each other, the whole response region looks like a gray shaded area. Panels (a) and (c): The CV of all uncertain model parameters was set to be 10%. The UA (Equation (2)) is about 0.6 for the umbo displacement amplitude below the middle-ear resonance frequency (at ~ 1.5 kHz). Results For the amplitude of the umbo displacement, the absolute value of skewness is greater than 0.90 at all three frequencies with a maximum absolute value of 1.59 at Flow. Also, the values of kurtosis for the amplitude of the umbo displacement are greater than 4.12 at all three frequencies with the maximum value of 7.88 at Flow. For the phase of the umbo displacement, the value of skewness is very close to zero at all three frequencies. Additionally, the values of kurtosis of the phase of the umbo displacement are about 1.94, 12.70, and 19.72 at frequencies Flow, Fmid, and Fhigh, respectively. These findings are consistent with the results of the normality test above. y For the displacement amplitude of the stapes footplate, the value of skewness is greater than 1.17 at all three frequencies with a maximum value of 1.27 at Fmid. Furthermore, the values of kurtosis for the amplitude of stapes displacement are greater than 4.22 at all three frequencies with a maximum of 5.80 at Flow. Table 2 also shows that, like the umbo response, the values of skewness for the phase of the displacement of the stapes footplate are very close to zero at all three frequencies. Besides, the values of kurtosis for the phase of the displacement of the stapes footplate are greater than 0.88 at all three frequencies with the maximum value of 17.48 at Fmid. Uncertainties in the vibration patterns of the TM are spread in space with increased fre‑ quency. The previous results showed the propagated uncertainties only for the umbo and stapes footplate. To provide a more complete view about the stochastic motions in the model, Fig. 6 presents the vibration amplitude of the TM at the same three frequencies. This figure considers the displacement of the baseline and stochastic FE model with CV of 20% for the model parameters. For the 15,728 nodes of the TM model, we calculated the displacement amplitude and phase in the baseline model as well as their mean and standard deviation in the stochastic model. The results of the amplitude are presented in the three left columns of Fig. 6 and the results for the phase are provided in the Fig. 7. The rightmost column of Fig. 6 presents the values of CV of the displace- ment amplitude. Results For frequencies above the middle-ear resonance frequency, the model amplified the uncertainty in the stapes response (average amplitude UA: 1.72 and average phase circular standard deviation: 0.02 cycles). The maximum UA of 3.12 happened at about 2.2 kHz for the stapes displacement amplitude. Also, for the phase, the maximum circular standard deviation of 0.05 cycles happened near frequencies of 1.9 and 3.6 kHz. y pp q Also, the open-cavity stapes footplate velocity data of Voss et al. are presented in Fig. 4. We compared the sta- pes displacement amplitude of the baseline model with these experimental results and found that the maximum difference was less than 1.6 dB for all frequencies below the middle-ear resonance frequency (1.5 kHz). Also, as Fig. 4 displays, the middle-ear resonance frequency and the maximum stapes footplate displacement amplitude obtained from the model match well with the experimental data. Scientific Reports \| (2023) 13:7329 \| https://doi.org/10.1038/s41598-023-34018-w www.nature.com/scientificreports/ Figure 4c and d present the frequency response of the stapes footplate for the CV of 20% for all uncertain model parameters. At low frequencies, the UA was about 1.10 for the amplitude and the circular standard deviation values of the phase were close to zero. At 2.3 kHz, the amplitude response was most uncertain and the uncertainty in the model parameters was magnified by a factor of 2.41 while the circular standard deviation of the phase response drastically decreased at this frequency to 0.02 cycles (from 0.07 cycles at 1.8 kHz) and went up to 0.07 again near the frequency of 10 kHz. Here the anti-resonances, observed in the umbo response (Fig. 3b,d), are not present. Compared to panels (a) and (b) of Fig. 4, the increased uncertainty (CV of 20%) in the model parameters, caused an approximately similar average amplitude UA (1.58 vs. 1.72) and a higher average phase standard deviation (0.04 cycles vs. 0.02 cycles) and the stapes response was in a better agreement with the experimental results. A comparison of the stochastic model results of the stapes footplate (with CV of 20%) with several experimental measurement results in the literature is also provided in Supplementary Fig. S1. At low frequencies, middle‑ear models attenuate uncertainties and approximate a robust simple lever. Figure 4e presents the stochastic ossicular transfer function of the model with the CV of 20% for the uncertain model parameters. Results This ossicular transfer function is defined as the ratio of the stapes footplate displacement amplitude (in the piston-like direction) to the umbo displacement amplitude (in the direction normal to the manubrium at the umbo). At low frequencies (below the middle-ear resonance), the transfer func- tion can be thought as the lever ratio (at low frequencies, the mean value of the lever ratio is less than 0.4 and the UA of the lever ratio is about 0.7). However, UA increases up to a maximum of about 3.75 near 2.1 kHz. The uncertainties in the model parameters make the transfer function highly uncertain at most frequencies between 2 and 4 kHz. At the frequencies below 1 kHz, the model robustly attenuates uncertainties and portrays the mid- dle ear as a highly certain simple lever. The same trend of attenuating uncertainty at low frequencies is evident in all results of Figs. 3 and 4 as discussed earlier. The middle‑ear model is biased. To study the probability distributions of stochastic responses at low, mid, and high frequencies, we focused on three frequencies (Flow = 708 Hz, Fmid = 2.51 kHz, and Fhigh = 9.74 kHz). Figure 5 presents violin plots40 of the amplitude and circular histograms of the phase of both umbo and stapes footplate displacement at these frequencies (for CV of 20% for uncertain model parameters). The horizontal axes in the violin plots show kernel density estimations for each distribution. We used Kuiper’s normality test to check whether the distributions shown in Fig. 5 are normal (von Mises for circular distributions41). We found that for all outputs, the null hypothesis was rejected at 5% significance level except for the phase of the stapes footplate at Flow. This means that although most of the uncertain model parameters were considered to have normal distribu- tions, the output distributions may be distorted with non-normal distributions.hi The values of skewness (a measure of symmetry of a specific distribution) and kurtosis (a measure of the tail weight of a specific distribution with respect to that of a normal distribution) reported in Table 2 also confirm that most of the distributions presented in Fig. 5 are non-normal as their skewness and kurtosis values are far from the respective values for the normal distribution (skewness of zero and kurtosis of three for the amplitude42,43 and skewness and kurtosis of zero for the phase37). Results We should note that only the CV of the nodes of the TM that have mean displacement values of greater than 0.1 nm are plotted in this column. The patterns obtained from the baseline model have similarities to the mean stochastic results shown in the “mean” column. At all three frequencies, the manubrium (outlined in black at Flow) has the smallest motion in the baseline and has the smallest values of the mean and standard deviation. At Flow, the maximum deformation of about 0.25 µm occurred in the posterior side of the TM (left to the manubrium in Fig. 6), as expected44,45. Besides, at this low frequency, the standard deviation plot shows that posterior to the TM, the standard deviation of the motion can be as large as the mean or baseline displacement (also evident from the CV of about 110%). At Fmid and Fhigh, some features in the baseline pattern (one example is marked with the white circle) are not present in the mean pattern because they were smoothed out due to averag- https://doi.org/10.1038/s41598-023-34018-w Scientific Reports \| (2023) 13:7329 \| www.nature.com/scientificreports/ scientificreports/ Figure 4. Stochastic frequency response function of the stapes footplate displacement and the stochastic m function. The results of individual simulations are plotted with gray thin lines and because they are all close the whole response region looks like a gray shaded area. The stapes footplate motion is reported in the pist Also, the green line presents the experimental results of Voss et al.1 (Bone 25) converted to displacement an the viewing angle of 35°; the mean of their reported viewing angle range of 20°–50° degrees. Panels (a) and and phase of the stapes footplate (normalized with respect to excitation pressure) with the CV of of 10% fo model parameters. At frequencies below the middle-ear resonance frequency (at ~ 1.5 kHz), the UA (Equat 1 for the stapes displacement amplitude. The maximum UA (3.12) happens near 2.2 kHz for amplitude and circular standard deviation (0.05 cycles) happens near the frequencies of 1.9 kHz and 3.6 kHz for the phase (d): Amplitude and phase of the stapes footplate (normalized with respect to excitation pressure) with the C uncertain model parameters. The UA (Equation (2)) is about 1.1 for amplitude at frequencies below the mi frequency (at ~ 1.5 kHz). Results The maximum UA (2.41) happens near 2.3 kHz for the stapes displacement ampl greatest circular standard deviation happens (0.07 cycles) near frequencies of 1.8 kHz and 10 kHz for the p Stochastic middle-ear transfer function calculated using the stochastic model with CV of 20% in model pa and Fig. 4c). The maximum UA of the lever ratio (3.75) happens at about 2.1 kHz. Figure 4. Stochastic frequency response function of the stapes footplate displacement and the stochastic middle-ear transfer function. The results of individual simulations are plotted with gray thin lines and because they are all close to each other, the whole response region looks like a gray shaded area. The stapes footplate motion is reported in the piston-like direction. Also, the green line presents the experimental results of Voss et al.1 (Bone 25) converted to displacement and corrected for the viewing angle of 35°; the mean of their reported viewing angle range of 20°–50° degrees. Panels (a) and (b): Amplitude and phase of the stapes footplate (normalized with respect to excitation pressure) with the CV of of 10% for all uncertain model parameters. At frequencies below the middle-ear resonance frequency (at ~ 1.5 kHz), the UA (Equation (2)) is about 1 for the stapes displacement amplitude. The maximum UA (3.12) happens near 2.2 kHz for amplitude and the maximum circular standard deviation (0.05 cycles) happens near the frequencies of 1.9 kHz and 3.6 kHz for the phase. Panels (c) and (d): Amplitude and phase of the stapes footplate (normalized with respect to excitation pressure) with the CV of 20% for all uncertain model parameters. The UA (Equation (2)) is about 1.1 for amplitude at frequencies below the middle-ear resonance frequency (at ~ 1.5 kHz). The maximum UA (2.41) happens near 2.3 kHz for the stapes displacement amplitude and the greatest circular standard deviation happens (0.07 cycles) near frequencies of 1.8 kHz and 10 kHz for the phase. Panel (e): Stochastic middle-ear transfer function calculated using the stochastic model with CV of 20% in model parameters (Fig. 3b and Fig. 4c). The maximum UA of the lever ratio (3.75) happens at about 2.1 kHz. Scientific Reports \| (2023) 13:7329 \| https://doi.org/10.1038/s41598-023-34018-w www.nature.com/scientificreports/ Figure 5. Violin plots and circular histograms for the amplitude and phase of the umbo and stapes footplate. The results are presented at frequencies Flow, Fmid, and Fhigh. Results The CV of uncertain model parameters was set to be 20%. The horizontal axes in the violin plots show kernel density estimations for each distribution. The values of skewness and kurtosis of the distributions shown in this figure are presented in Table 2. We used Kuiper’s normality test to check whether the distributions shown in this figure are normal (von Mises for circular distributions41). Based on Kuiper test results presented in “Results”, the null hypothesis was rejected at 5% significance level except for the phase of the stapes footplate at Flow. Figure 5. Violin plots and circular histograms for the amplitude and phase of the umbo and stapes footplate. The results are presented at frequencies Flow, Fmid, and Fhigh. The CV of uncertain model parameters was set to b 20% Th h i t l i th i li l t h k l d it ti ti f h di t ib ti Th l Figure 5. Violin plots and circular histograms for the amplitude and phase of the umbo and stapes footplate g p g p p p p The results are presented at frequencies Flow, Fmid, and Fhigh. The CV of uncertain model parameters was set to be 20%. The horizontal axes in the violin plots show kernel density estimations for each distribution. The values of skewness and kurtosis of the distributions shown in this figure are presented in Table 2. We used Kuiper’s normality test to check whether the distributions shown in this figure are normal (von Mises for circular distributions41). Based on Kuiper test results presented in “Results”, the null hypothesis was rejected at 5% significance level except for the phase of the stapes footplate at Flow. Table 2. Values of skewness and kurtosis for the output distributions shown in Fig. 5. Frequency Flow (708 Hz) Fmid (2.51 kHz) Fhigh (9.74 kHz) Statistical information Skewness Kurtosis Skewness Kurtosis Skewness Kurtosis Umbo amplitude 1.59 7.88 1.25 4.12 0.90 4.63 Umbo phase 0.00 1.94 − 0.06 12.70 0.05 19.72 Stapes footplate amplitude 1.17 5.80 1.27 5.15 0.69 4.22 Stapes footplate phase 0.00 0.88 − 0.01 17.48 0.07 14.27 Table 2. Values of skewness and kurtosis for the output distributions shown in Fig. 5. ing and due to the high variations in the results at these locations. Results At the middle frequency (Fmid), the major area of the TM shows standard deviations of more than 0.02 µm except in the superior region of the TM where stand- ard deviations of down to 0.001 µm dominate the region. Consistently, the CV plot at Fmid shows that regions with high CV (≥ 50%) are apparent in all regions of the TM. Increasing the frequency to Fhigh leads to nearly even distribution of regions with high standard deviations and high CV on the entire TM, especially in comparison to the vibration patterns at Flow. The mean values of CV of the TM nodes (Fig. 6) are about 46%, 56%, and 38% for the Flow, Fmid, and Fhigh, respectively. This shows that although by increasing the frequency the regions with high CV are spatially spread out on the TM, the mean value of the CV does not follow any specific trends.h y yi The stochastic phases of vibration patterns of the TM are presented in Fig. 7. Compared to the displacement amplitude, the displacement phase has a more evenly distributed standard deviation at Flow. Also, at Flow, the vibration phase is nearly constant and highly certain over the entire TM except in a small circular region in the TM anterior with concentrated uncertainty and a maximum standard deviation of about 102°. The displacement phase pattern becomes more uncertain as the frequency increases and at Fmid several hot spots with concentrated uncertainty are present in the standard deviation plot. As the frequency increases to Fhigh the uncertainty is dispersed in the entire TM. The average values of the circular standard deviation at Fmid and Fhigh (38° and 20°, respectively), are much greater than that at Flow (4°). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. Stochastic full-field vibration patterns of the TM. Vibration patterns of the baseline model and the spatial distribution of the mean and standard deviation of the displacement amplitude of the tympanic membrane at three frequencies Flow, Fmid, and Fhigh are presented in the three left columns. The vibration patterns are reported in the direction normal to the manubrium at the umbo. Comparing the mean and baseline patterns shows that at Fmid and Fhigh, some high-frequency features are not present in the mean pattern due to the averaging and high variations in the stochastic results. The white circle shows one example of such high-frequency features. The right column shows the distribution of the coefficient of variation, excluding points where the mean values are close to zero. The manubrium is outlined in black. The purple line shows the tympanic annulus. 10 fic Reports \| (2023) 13:7329 \| https://doi.org/10.1038/s41598-023-34018-w Figure 6. Stochastic full-field vibration patterns of the TM. Vibration patterns of the baseline model and the spatial distribution of the mean and standard deviation of the displacement amplitude of the tympanic membrane at three frequencies Flow, Fmid, and Fhigh are presented in the three left columns. The vibration patterns are reported in the direction normal to the manubrium at the umbo. Comparing the mean and baseline patterns shows that at Fmid and Fhigh, some high-frequency features are not present in the mean pattern due to the averaging and high variations in the stochastic results. The white circle shows one example of such high-frequency features. The right column shows the distribution of the coefficient of variation, excluding points where the mean values are close to zero. The manubrium is outlined in black. The purple line shows the tympanic annulus. Figure 7. Stochastic full-field vibration phase of the TM. Distribution of the displacement phase of the baseline model and the spatial distribution of circular mean and circular standard deviation of the phase of the tympanic membrane. The results are presented at three frequencies Flow, Fmid, and Fhigh. The manubrium is outlined in black. Figure 6. Stochastic full-field vibration patterns of the TM. Vibration patterns of the baseline model and the spatial distribution of the mean and standard deviation of the displacement amplitude of the tympanic membrane at three frequencies Flow, Fmid, and Fhigh are presented in the three left columns. Discussions and conclusions In this study, we presented a stochastic FE model of the human middle ear that instead of generating deterministic vibrations and model outputs, provides a probabilistic description of middle-ear vibrations to account for the natural variabilities in the human middle ear. Current FE models do not have enough accuracy to be considered as acceptable tools for diagnosis and surgical planning46,47. This study is a step toward increasing the accuracy and reliability of the predictions of FE models by considering the uncertainty in the model parameters. https://doi.org/10.1038/s41598-023-34018-w Scientific Reports \| (2023) 13:7329 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ To take the variability in individual ears into account, the stochastic model considered uncertainty in the mechanical parameters as well as the thickness of the TM. We demonstrated that the middle-ear model can magnify the uncertainties in the model parameters to more than 300% in the outputs. For both CV values of 10% and 20% of the model parameters, the highest UA happened in the frequency range of 1–5 kHz for the amplitudes of the umbo and stapes displacements. This new finding enhances our existing knowledge6,48 about the sensitivity of the middle ear to variability. Uncertainty amplification that we report here was also observed in models of other organs of the body (e.g., left ventricle49). g y gt Also, for the model with the CV of 20%, for some sets of model parameters, we saw an additional anti- resonance in umbo displacement at frequencies around 2–3 kHz that does not exist in the baseline model. Based on our results, for the sample sets that lead to these anti-resonances in the umbo response, the values of the thickness or the Young’s modulus of the TM (or both of them for most samples) are small (below mean values). However, the values of other model parameters and their interactions may also have some effects in the pres- ence of these anti-resonances. The presence of anti-resonances for some sets of model parameters is consistent with the presence of similar antiresonances in some ears in experimental observations of populations of middle ears26,50. Additionally, the noisy behaviour of the transfer function (Fig. 4e) can be due to these anti-resonances for some sets of model parameters. p All the observations described above have important implications for FE models of the middle ear because in addition to inter-individual variability that makes the model parameters intrinsically uncertain, most material parameters of the models were not and cannot be measured accurately in vivo, at least with the existing methods. If conventional deterministic FE models of the middle ear are to be used for developing new medical devices or for planning therapeutic interventions, the ignored error in the values of material and geometrical parameters (uncertainty) may be amplified as errors in the model results. We observed that the umbo and stapes footplate responses and the middle-ear transfer function are less uncertain at low frequencies. www.nature.com/scientificreports/ This suggests that if, despite uncertainties, a deterministic model is used, the model predictions at low frequencies are more reliable than those at high frequencies. Moreover, by investigating stochastic vibration patterns of the TM, we observed that as the frequency of excitation increases, the regions with high uncertainty spread out more evenly on the entire TM surface and the vibration pattern becomes less certain. This suggests similar implications to what we discussed for the umbo, stapes footplate and the middle-ear transfer function. Although the current study reveals the importance of considering using stochastic models to study the mechanics of the middle ear, these models require a significantly higher computational cost in comparison to conventional deterministic FE models. One way to deal with the high computational cost is to develop and train surrogate models51 that can be used in lieu of the real FE model for stochastic simulations for some specific out- put quantities of interest. Future studies should study development and effectiveness of such surrogate models. q yf g One of the limitations of our current stochastic FE model is that we only considered normal and half-normal distributions for the model parameters. We chose these distributions based on the fact that normal distributions are suitable choices for many biological variables36. However, the exact distribution of each of the model param- eters should be determined by performing further experiments on a large number of human ears. In the absence of more accurate estimates of the value of the CV of most of the uncertain model parameters, we considered the CV values of 10% and 20%. However, additional experimental measurements should be car- ried out in order to quantify the CV of each of the model parameters. Recently, Lobato et al. tried to quantify the CV of mechanical parameters of the middle ear52 based on the ranges reported in the literature for each value. However, their study does not include all mechanical properties of the middle ear structures. For instance, CV of damping, Poisson’s ratio, and cochlear load is not reported in their work. Additionally, as Lobato et al. also mentioned in their work, even for the parameters reported in their work, the values of CV were calculated from small sample sets and therefore, those values might not be accurate enough. www.nature.com/scientificreports/ The vibration patterns are reported in the direction normal to the manubrium at the umbo. Comparing the mean and baseline patterns shows that at Fmid and Fhigh, some high-frequency features are not present in the mean pattern due to the averaging and high variations in the stochastic results. The white circle shows one example of such high-frequency features. The right column shows the distribution of the coefficient of variation, excluding points where the mean values are close to zero. The manubrium is outlined in black. The purple line shows the tympanic annulus. Figure 7. Stochastic full-field vibration phase of the TM. Distribution of the displacement phase of the baseline model and the spatial distribution of circular mean and circular standard deviation of the phase of the tympanic membrane. The results are presented at three frequencies Flow, Fmid, and Fhigh. The manubrium is outlined in black. Figure 7. Stochastic full-field vibration phase of the TM. Distribution of the displacement phase of the baseline model and the spatial distribution of circular mean and circular standard deviation of the phase of the tympanic membrane. The results are presented at three frequencies Flow, Fmid, and Fhigh. The manubrium is outlined in black. Scientific Reports \| (2023) 13:7329 \| https://doi.org/10.1038/s41598-023-34018-w www.nature.com/scientificreports/ Furthermore, to avoid excessive sophistication of performing this study, we only considered the variability in the TM thickness among all mor- phological parameters of the middle ear because that was the only morphological variability that would not need creations of new 3D models. Future studies should explore the effects of morphological variability in interaction with material variability. We have considered some simplifying assumptions to reduce the complexity of our model. For instance, our baseline model does not include the tensor tympani tendon, stapedial tendon, and the middle-ear cavity. The tendons can be activated in living ears but in ex vivo ears they are not active and the stapedial tendon is often removed in temporal-bone preparations. Voss et al. showed that removing the stapedial tendon has small effects on the mechanics of the middle ear ex vivo1. Also, the middle-ear cavity is expected to have small effects on the motions of the stapes footplate at many frequencies1,24. Moreover, although some studies have modelled the TM as an orthotropic material22, in this study we modelled the TM as an isotropic material to reduce the complexity of our model. O’Connor et al. modelled the TM using both orthotropic and isotropic materials and showed that the isotopic material model can also lead to results comparable to the ones from the orthotropic model and close to experimental measurements24. All these simplifications may have some effects on the predictions of the present stochastic finite-element analysis and they should be further investigated in follow-up studies. https://doi.org/10.1038/s41598-023-34018-w Scientific Reports \| (2023) 13:7329 \| www.nature.com/scientificreports/ Received: 11 November 2022; Accepted: 22 April 2023 Received: 11 November 2022; Accepted: 22 April 2023 References Tympanic membrane surface motions in forward and reverse middle ear transmissions. J. Acoust. Soc. Am. 145, 272–291 (2019). y g pp 21. Cheng, J. T., Maftoon, N., Guignard, J., Ravicz, M. E. & Rosowski, J. Tympanic mem middle ear transmissions. J. Acoust. Soc. Am. 145, 272–291 (2019). 22. Volandri, G., Di Puccio, F., Forte, P. & Carmignani, C. Biomechanics of the tympanic membrane. J. Biomech. 44, 1219–1236 (2011).t , , , , , g , y p J , ( ) 3. Motallebzadeh, H., Maftoon, N., Pitaro, J., Funnell, W. R. J. & Daniel, S. J. 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( ) 51. Kudela, J. & Matousek, R. Recent advances and applications of surrogate models for finite element method computations: A review. Soft Comput. 26, 13709–13733 (2022). ft p 52. Lobato, L. C., Paul, S. & Cordioli, J. A. Statistical analysis of the human middle ear mechanical properties. J. Acoust. Soc. Am. 151, 2043–2054 (2022). ft p 2. Lobato, L. C., Paul, S. & Cordioli, J. A. Statistical analysis of the human middle ear mechanical properties. J. Acoust. Soc. Am. 151 2043–2054 (2022). Acknowledgementsh g This work was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2020- 05522), the Canada Foundation for Innovation and the Ontario Research Fund-Research Innovation (38964), and the National Institute on Deafness and Other Communication Disorders (NIDCD R01DC016079), a member of the U.S. National Institutes of Health. The simulations were performed using infrastructures of Digital Research Alliance of Canada (www.alliancecan.ca). 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Eng. Phys. 31, 907–916 (2009).fii p g g y , ( ) 48. De Greef, D., Pires, F. & Dirckx, J. J. Effects of model definitions and parameter values in finite element modeling of human middle ear mechanics. Hear. Res. 344, 195–206 (2017). g g y 48. De Greef, D., Pires, F. & Dirckx, J. J. Effects of model definitions and parameter values in finite element modeling of human middle ear mechanics. Hear. Res. 344, 195–206 (2017). , ( ) 49. Osnes, H. Competing interests h p g The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-34018-w. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-34018-w. Correspondence and requests for materials should be addressed to N.M. Correspondence and requests for materials should be addressed to N.M. Author contributionst A.E.: Writing—original draft, methodology, mesh and model creation, analysis, investigation, validation.H.M.: Writing—review and editing, investigation.J.J.R.: Writing—review and editing, providing materials, investi- gation, analysis.J.T.C.: Writing—review and editing, providing materials, investigation, analysis, funding acquisition.N.M.: Conceptualization, Writing—review and editing, funding acquisition, methodology, analysis, supervision. Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-34018-w. Correspondence and requests for materials should be addressed to N.M. 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 institutional affiliations. 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https://openalex.org/W2080379275	https://europepmc.org/articles/pmc3206053?pdf=render	English	null	Status of B-Vitamins and Homocysteine in Diabetic Retinopathy: Association with Vitamin-B12 Deficiency and Hyperhomocysteinemia	PloS one	2,011	cc-by	6,873	Abstract Diabetic retinopathy (DR) is a common cause of blindness. Although many studies have indicated an association between homocysteine and DR, the results so far have been equivocal. Amongst the many determinants of homocysteine, B-vitamin status was shown to be a major confounding factor, yet very little is known about its relationship to DR. In the present study, we, therefore, investigated the status of B-vitamins and homocysteine in DR. A cross-sectional case–control study was conducted with 100 normal control (CN) subjects and 300 subjects with type-2 diabetes (T2D). Of the 300 subjects with T2D, 200 had retinopathy (DR) and 100 did not (DNR). After a complete ophthalmic examination including fundus fluorescein angiography, the clinical profile and the blood levels of all B-vitamins and homocysteine were analyzed. While mean plasma homocysteine levels were found to be higher in T2D patients compared with CN subjects, homocysteine levels were particularly high in the DR group. There were no group differences in the blood levels of vitamins B1 and B2. Although the plasma vitamin-B6 and folic acid levels were significantly lower in the DNR and DR groups compared with the CN group, there were no significant differences between the diabetes groups. Interestingly, plasma vitamin-B12 levels were found to be significantly lower in the diabetes groups compared with the CN group; further, the levels were significantly lower in the DR group compared with the DNR group. Higher homocysteine levels were significantly associated with lower vitamin-B12 and folic acid but not with other B-vitamins. Additionally, hyperhomocysteinemia and vitamin-B12 deficiency did not seem to be related to subjects’ age, body mass index, or duration of diabetes. These results thus suggest a possible association between vitamin-B12 deficiency and hyperhomocysteinemia in DR. Further, the data indicate that vitamin-B12 deficiency could be an independent risk factor for DR. Editor: German Malaga, Universidad Peruana Cayetano Heredia, Peru Editor: German Malaga, Universidad Peruana Cayetano Heredia, Peru Received June 29, 2011; Accepted October 2, 2011; Published November 1, 2011 Received June 29, 2011; Accepted October 2, 2011; Published November 1, 2011 Copyright: 2011 Satyanarayana 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. Abstract Funding: GBR received grants from the Department of Science and Technology (SR/SO/HS/0055/2008), Government of India; AS received a research fellowship from the Indian Council of Medical Research, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. grants from the Department of Science and Technology (SR/SO/HS/0055/2008), Government of India; AS received a research fellowship f Medical Research, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the Competing Interests: The authors have declared that no competing interests exist. * E-mail: geereddy@yahoo.com Status of B-Vitamins and Homocysteine in Diabetic Retinopathy: Association with Vitamin-B12 Deficiency and Hyperhomocysteinemia Alleboena Satyanarayana1, Nagalla Balakrishna1, Sujatha Pitla1, Paduru Yadagiri Reddy1, Sivaprasad Mudili1, Pratti Lopamudra1, Palla Suryanarayana1, Kalluru Viswanath2, Radha Ayyagari3, Geereddy Bhanuprakash Reddy1* e of Nutrition, Hyderabad, India, 2 Pushpagiri Vitreo Retina Institute, Hyderabad, India, 3 Ophthalmology, University of California, Sa ted States of America 1 Biochemistry, National Institute of Nutrition, Hyderabad, India, 2 Pushpagiri Vitreo Retina Institute, Hyderabad, India, 3 Ophthal Diego, San Diego, California, United States of America Citation: Satyanarayana A, Balakrishna N, Pitla S, Reddy PY, Mudili S, et al. (2011) Status of B-Vitamins and Homocysteine with Vitamin-B12 Deficiency and Hyperhomocysteinemia. PLoS ONE 6(11): e26747. doi:10.1371/journal.pone.0026747 PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26747 Competing Interests: The authors have declared that no competing interests exist. Introduction prevalence of DR in South India was reported to be 22.4% based on the Andhra Pradesh Eye Disease Study (APEDS) of self- reported diabetes [9]. The prevalence of DR was 0.5% in the general rural populations of South India and 10.5% among patients with diabetes [10]. Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes and ranks as a common cause of blindness worldwide [1,2]. Diabetic retinopathy could become a major threat to public health in the future due to the global prevalence of diabetes, which is projected to affect 438 million people by 2030 [3]. Both the duration of diabetes and its metabolic control have been identified as the risk factors most strongly associated with the development of DR [4,5]. Diabetic retinopathy occurs in 70% of all persons having diabetes for more than 15 years. While the prevalence of DR has varied (20%–60%) in different studies, a recent study indicated that the estimated prevalence was 28.5% among US adults [6]. The prevalence of DR among urban subjects with diabetes in India was reported to be about 17% [7], whereas in a clinical study it was found to be 34% among patients with type 2 diabetes (T2D) [8]. The Diabetic retinopathy is characterized by the appearance of vascular lesions of increasing severity, culminating in the growth of new vessels. Early or nonproliferative DR (NPDR) is marked by retinal vascular microaneurysms, blot hemorrhages, cotton-wool spots, loss of retinal pericytes, increased vascular retinal perme- ability, alterations in regional blood flow, and abnormal retinal microvasculature, all of which lead to retinal ischemia. Prolifer- ative DR (PDR), the more severe state, is marked by the formation of abnormal, fragile new blood vessels that are prone to hemorrhage [11,12]. Although the prevalence of DR increases with the duration of diabetes, studies have shown that intensive glycemic control can PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26747 1 November 2011 \| Volume 6 \| Issue 11 \| e26747 Diabetic Retinopathy - Vitamin-B12 Deficiency Table 1. Clinical and demographic profile of control (CN) and diabetes patients without (DNR) and with retinopathy (DR). Table 1. Clinical and demographic profile of control (CN) and diabetes patients without (DNR) and with retinopathy (DR). Introduction g p p ( ) p ( ) p y ( ) Parameter Normal (n = 100) DNR (n = 100) DR (n = 194) F –Value P –Value Age (years) 53.9969.22a 54.7669.29a 55.7568.24a 2.1 0.128 BMI 23.6363.04a 25.2164.19a 24.3264.45a 1.6 0.212 Hb (g/dL) 14.8961.73a 14.3162.16a 14.1162.32a 1.4 0.255 Duration (years) - 10.1666.92a 11.0366.92a 2.2 0.098 Glucose (mg/dL) 99.16618.86a 209.97685.10b 221.28691.36b 146.8 0.000 HbA1c (%) 5.6461.14a 8.9462.49b 10.3362.94c 86.0 0.000 Insulin (mU/mL) 29.1469.94a 38.05622.71a 34.75619.58a 1.4 0.255 Total Cholesterol (mg/dL) 169.39639.65a 167.03653.56a 178.01658.47a 0.6 0.552 Triglycerides (mg/dL) 129.78663.57a 151.99665.43b 163.00677.90b 4.3 0.015 HDL (mg/dL) 35.0268.81a 30.0969.21b 28.7067.41b 8.8 0.000 LDL (mg/dL) 112.69630.13a 110.66629.01a 117.11634.05a 0.6 0.570 Note: 1. Values are Mean 6 SD. 2. Variables of glucose, HbA1c and TC were transformed into logarithmic values due to heterogeneity of variances across groups and mean values across groups were compared by oneway ANOVA ‘F’ test with post hoc test of Tukey’s multiple comparisons. 3. Significant differences (p,0.05) of mean values between the groups are indicated by different superscript letters. doi:10.1371/journal.pone.0026747.t001 Note: 1. Values are Mean 6 SD. 2. Variables of glucose, HbA1c and TC were transformed into logarithmic values due to heterogeneity of variances across groups and mean values across groups were compared by oneway ANOVA ‘F’ test with post hoc test of Tukey’s multiple comparisons. 3. Significant differences (p,0.05) of mean values between the groups are indicated by different superscript letters. doi:10.1371/journal.pone.0026747.t001 detoxified by methionine synthetase, which is dependent on vitamin B12 and folate as coenzymes for its proper function [27,28]. Determinants of hyperhomocysteinemia such as low concentrations of folate and B-vitamin coenzymes and altered activities of enzymes involved in the breakdown of homocysteine are also associated with increased risk of cardiovascular compli- cations [26]. Nevertheless, B-vitamin status and its contribution to hyperhomocysteinemia in DR have not been examined. There- fore, we investigated the status of B-vitamins (B1, B2, B6, B12, folic acid) and homocysteine in DR. delay its development [4,5]. In principle, all patients with diabetes might be expected to develop diabetic microvascular complica- tions if hyperglycemia alone were the triggering factor for diabetic complications. It is, however, noteworthy that some patients may still develop DR even with good glycemic control. Conversely, some patients with poor glycemic control avoid this complication, notably long-surviving patients with type-1 diabetes (T1D). Therefore, the impact of strict glycemic control on prevention of diabetic complications is not that scrupulous [4,5,13]. Results This is a hospital-based case-control study consisting of T2D subjects with (DR) and without retinopathy (DNR) along with normal control (CN) subjects. The characteristics of the CN, DNR, and DR groups are shown in Table 1. The sex distribution was approximately the same in all the groups (male and female were, respectively, 58% and 42% in CN, 56% and 44% in DNR, and 55% and 45% in DR). Further, there was no significant difference between male and female subjects in all three groups with respect to demographics and measured parameters. There- fore, the data for both men and women were pooled as a whole in the respective groups. Mean age, body mass index (BMI), and hemoglobin levels were comparable between the groups. The duration of diabetes was matched for DNR and DR (p.0.05). Amongst diabetes groups, glycosylated hemoglobin (HbA1c) levels were higher in DR compared to DNR (p,0.05). While plasma total cholesterol and low-density lipoprotein (LDL) were compa- rable between the groups, triglyceride levels were higher and high- density lipoprotein (HDL) was lower in the diabetes groups compared with the CN group (Table 1). Nevertheless, the levels of triglycerides and HDL were comparable between the DNR and DR groups. Although genetic susceptibility appears to be the primary predisposing factor for DR (reviewed by Ng, 2010 [14] and Fu et al, 2010 [15]; [16]), the role of environmental factors like nutritional and dietary factors are not to be discounted. Vitamins and mineral supplementation for the management of T2D has been reported [17], but its role in the prevention and development of T2D in general and diabetic complications in particular has not been established clearly. Further, diabetes itself can alter the nutritional status [18,19], and experiments suggest that patients with diabetes are prone to deficiency of micronutrients such as magnesium, zinc, copper, manganese, and chromium. It was observed that serum ascorbic acid, B-vitamins, and possibly 1,25- dihydroxycholcalciferol concentrations are low in diabetic pa- tients. Studies indicate low plasma thiamine (vitamin B1) in both T1D and T2D patients [20], and high-dose thiamine and its derivatives such as benfotiamine can prevent the development of microvascular complications [21]. However, the levels of vitamin B1 in DR have not been investigated so far. Low concentrations of folic acid and other B vitamins are associated with increased risk of vascular damage through homocysteine. Introduction Multiple factors are likely to be involved in predisposing diabetes subjects to complications, as evidenced by many but not all patients with diabetes developing one or more microvascular complications. If the predisposing factors are known, it may be possible to delay the onset and progression of these complications. Hence, there is an obvious need to understand the risk factors that are associated with diabetic complications. PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26747 Results However, there was no significant difference in homocysteine and vitamin B12 levels between NPDR and PDR patients (14.72 vs. 14.11 mmol/L and 182 vs. 177 pg/mL, respectively). These results suggest that DR is associated with a higher prevalence of vitamin B12 deficiency. (.12 mmol/L), which was significantly different (p,0.01) between the groups: 65% in DR, 46% in DNR, and 11% in CN (Figure 2). Further, we also compared the homocysteine levels in DR patients with a shorter duration of diabetes (,5 years) with those of DNR patients with a long duration of diabetes (.5 years). The mean value of homocysteinemia in DNR of .5 years duration was 12.4 mmol/L, while it was 14.2 mmol/L in DR of ,5 years duration. In addition, when duration was controlled for in both DNR and DR groups, DR patients had significantly higher levels of homocysteine than DNR patients. Together, these results suggest an association between hyperhomocysteinemia and DR. (.12 mmol/L), which was significantly different (p,0.01) between the groups: 65% in DR, 46% in DNR, and 11% in CN (Figure 2). Further, we also compared the homocysteine levels in DR patients with a shorter duration of diabetes (,5 years) with those of DNR patients with a long duration of diabetes (.5 years). The mean value of homocysteinemia in DNR of .5 years duration was 12.4 mmol/L, while it was 14.2 mmol/L in DR of ,5 years duration. In addition, when duration was controlled for in both DNR and DR groups, DR patients had significantly higher levels of homocysteine than DNR patients. Together, these results suggest an association between hyperhomocysteinemia and DR. Since B-vitamins are linked to homocysteine metabolism and homeostasis, we then determined the B-vitamin levels. The vitamin B1 levels were marginally (but statistically significantly) Figure 2. Prevalence (%) of hyperhomocysteinemia (.12 mmol/ L) in control (CN) and diabetes patients without (DNR) and with retinopathy (DR). Data indicate percent of subjects above 12 mmol/L of the respective group. Significant differences (p,0.05) of mean values between the groups are indicated by different letters on the bars after adjusting the duration of diabetes between DNR and DR. doi:10.1371/journal.pone.0026747.g002 Homocysteine levels were inversely related (p,0.05) to vitamin B12 and folic acid but not to vitamins B1, B2, and B6, when data of all the groups were considered (Table 3). Results 3. Significant differences (p,0.05) of mean values between the groups are indicated by different superscript letters. doi:10.1371/journal.pone.0026747.t002 Figure 1. Plasma homocysteine levels. Data represent mean 6 SE in control (CN; n = 75) and diabetes patients without (DNR; n = 75) and with retinopathy (DR; n = 150). Data were transformed into log values and compared mean values across groups by oneway ANOVA ‘F’ test with post hoc test of Tukey’s multiple comparisons. Significant differences (p,0.05) of mean values between the groups are indicated by different letters on the bars after adjusting the duration of diabetes between DNR and DR. doi:10.1371/journal.pone.0026747.g001 higher in DNR compared with the CN group, but there was no significant difference between the DNR and DR groups. Furthermore, vitamin B1 levels were comparable between the DR and CN groups. While the blood levels of vitamin B2 were comparable between the groups, plasma vitamin B6 levels were significantly lower (p,0.05) in the diabetes groups (DNR and DR) compared with the CN group (Table 2). In spite of the deficiency of vitamin B6 in the diabetes groups, there was no significant difference in the mean levels of vitamin B6 between the DNR and DR groups (Table 2). Similarly, plasma folic acid levels were lower but not deficient in the diabetes groups (DNR and DR) compared with the CN group; however, the levels were comparable between the DNR and DR groups (Table 2). Plasma vitamin B12 levels were significantly lower (p,0.01) in diabetes patients (DNR and DR) compared with the CN subjects (Figure 3). Interestingly, in this study significantly lower (p,0.05) plasma vitamin B12 levels were observed in DR patients compared with DNR patients (Figure 3). The mean vitamin B12 levels in the DR group were below the normal range (200–1000 pg/mL). However, the vitamin B12 levels ranged from 20 to 1500 pg/mL with considerable overlap between the groups. Therefore, we examined the data for prevalence of vitamin B12 deficiency in the three groups. With 200 pg/mL as the cut-off level for deficiency [29–31], the prevalence of vitamin B12 deficiency was significantly different (p,0.001) between the groups; 67% in DR, 54% in DNR, and 41% in CN (Figure 4). The ratio of vitamin B12 to folic acid was also significantly different (p,0.05) between CN and DR groups, but not between the DNR and DR groups (data not shown). Results Homocysteine has been extensively studied in recent years as a biomarker as well as a risk factor for vascular diseases, including vaso-occlusive diseases of the eye [22–26]. Homocysteine is a by-product of transmethylation reactions and The mean plasma homocysteine levels were significantly higher in the DNR group compared with the CN group, and a further increase (p,0.05) was found in the DR group compared with the DNR group (Figure 1). However, there was a considerable overlap of hyperhomocysteinemia between the DNR and DR groups. Hence, we determined the prevalence of hyperhomocysteinemia PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26747 November 2011 \| Volume 6 \| Issue 11 \| e26747 2 Diabetic Retinopathy - Vitamin-B12 Deficiency Figure 1. Plasma homocysteine levels. Data represent mean 6 SE in control (CN; n = 75) and diabetes patients without (DNR; n = 75) and with retinopathy (DR; n = 150). Data were transformed into log values and compared mean values across groups by oneway ANOVA ‘F’ test with post hoc test of Tukey’s multiple comparisons. Significant differences (p,0.05) of mean values between the groups are indicated by different letters on the bars after adjusting the duration of diabetes between DNR and DR. doi:10.1371/journal.pone.0026747.g001 Table 2. Blood and plasma levels of vitamins B1, B2, B6 and folic acid of control (CN) and diabetes patients without (DNR) and with retinopathy (DR). Vitamin CN DNR DR F Value P-values B1 (ng/mL) 67.663.1a n = 45 70.163.8b n = 45 67.263.0ab n = 45 3.9 0.024 B2 (ng/mL) 23169.3a n = 45 24868.2a n = 45 23867.0a n = 45 1.99 0.143 B6 (ng/mL) 20.661.3a n = 45 13.061.1b n = 45 14.661.0b n = 60 10.1 0.000 Folic acid (ng/mL) 10.060.9a n = 75 7.860.6ab n = 75 7.260.4b n = 150 4.7 0.009 Note: 1. Values are Mean 6 SD. 2. Mean values across groups were compared by oneway ANOVA ‘F’ test with post hoc test of Tukey’s multiple comparisons. 3. Significant differences (p,0.05) of mean values between the groups are indicated by different superscript letters. doi:10.1371/journal.pone.0026747.t002 Table 2. Blood and plasma levels of vitamins B1, B2, B6 and folic acid of control (CN) and diabetes patients without (DNR) and with retinopathy (DR). Note: 1. Values are Mean 6 SD. 2. Mean values across groups were compared by oneway ANOVA ‘F’ test with post hoc test of Tukey’s multiple comparisons. Results Interestingly, irrespective of groups, homocysteine levels were significantly (p,0.05) associated with glucose and HbA1c levels but not related to subjects’ age, BMI, or duration of diabetes (Table 3). Likewise, vitamin B12 levels were significantly (p,0.05) but inversely associated with glucose, HbA1c, and homocysteine and positively related with folic acid but not with age, BMI, or duration (Table 3). Considering that plasma folic acid levels were low but Figure 2. Prevalence (%) of hyperhomocysteinemia (.12 mmol/ L) in control (CN) and diabetes patients without (DNR) and with retinopathy (DR). Data indicate percent of subjects above 12 mmol/L of the respective group. Significant differences (p,0.05) of mean values between the groups are indicated by different letters on the bars after adjusting the duration of diabetes between DNR and DR. doi:10.1371/journal.pone.0026747.g002 PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26747 3 Diabetic Retinopathy - Vitamin-B12 Deficiency Figure 3. Plasma vitamin-B12 levels. Data represent mean 6 SE in control (CN; n = 75) and diabetes patients without (DNR; n = 75) and with retinopathy (DR; n = 150). Data were transformed into log values and compared the mean values across groups by oneway ANOVA ‘F’ test with post hoc test of Tukey’s multiple comparisons. Significant differences (p,0.05) of mean values between the groups are indicated by different letters on the bars. doi:10.1371/journal.pone.0026747.g003 Table 3. Correlations of vitamin-B12 and homocysteine with demographic and other biochemical parameters. Parameter Vitamin-B12 Homocysteine r-value p-value r-value p-value Age 0.111 0.055 0.088 0.328 Duration 0.074 0.263 0.162 0.113 BMI 0.082 0.292 0.083 0.493 Glucose 20.177 0.016 0.444 0.000 HbA1c 20.192 0.034 0.247 0.047 Vitamin -B1 20.064 0.571 0.294 0.066 Vitamin -B2 20.062 0.577 0.284 0.056 Vitamin -B6 0.047 0.614 20.137 0.383 Folic acid 0.473 0.000 20.323 0.002 Vitamin -B12 - - 20.485 0.000 Homocysteine 20.485 0.000 - - Correlations (r-value) were assessed by Spearman rank correlation. While positive r-value indicates direct correlation, negative r-value indicates inverse relationship between the variables. doi:10.1371/journal.pone.0026747.t003 Table 3. Correlations of vitamin-B12 and homocysteine with demographic and other biochemical parameters. Table 3. Correlations of vitamin-B12 and homocysteine with demographic and other biochemical parameters. Figure 3. Plasma vitamin-B12 levels. Data represent mean 6 SE in control (CN; n = 75) and diabetes patients without (DNR; n = 75) and with retinopathy (DR; n = 150). Discussion Approximately 5% of the global prevalence of blindness is considered to be due to DR, with estimates of 15%–17% in developed countries [1]. Nutritional status, particularly micronu- trients, may affect the risk of DR by influencing the biochemical mechanisms underlying DR. A biochemical indicator that has generated considerable interest as a risk factor for many vascular diseases, including DR, is homocysteine. Although many studies p The results of the present study show that while lower levels of folic acid and vitamins B6 and B12 were observed in patients with diabetes irrespective of the presence of retinopathy, only vitamin B12 deficiency was associated with DR. Interestingly, a significant association was revealed between DR and hyperhomocysteinemia based on plasma homocysteine levels. Hyperhomocysteinemia has several causes, including dietary deficiencies of folic acid and vitamins B6 and B12. Furthermore, supplementation of folic acid and vitamin B12 is known to reduce homocysteine levels [23,26]. Lack of vitamin B12 is thought to be a more important determinant for increased homocysteine, particularly in older people, and it becomes the limiting nutrient for maintaining normal plasma concentrations once folate levels are optimized. It should be noted in the present study that the mean age of the subjects was 55 years and the folate levels were still in the normal range, although lower mean levels were found in patients with diabetes. In addition, plasma homocysteine in diabetes varies depending on the presence or absence of nephropathy. However, in this study the increased levels of homocysteine were independent of renal failure because we excluded DNR and DR patients with renal complications. Therefore, lower vitamin B12 status appears to be a determining factor for increased homocysteine in DR patients in this population. Further, vitamin B12 levels did not seem to be influenced by patients’ age, BMI, or Figure 4. Prevalence (%) of vitamin-B12 deficiency in control (CN) and diabetes patients without (DNR) and with retinopathy (DR). Data indicate percent of subjects below 200 pg/mL of the respective group. Proportion Z test was done to compare prevalence between groups. Significant differences (p,0.001) of mean values between the groups are indicated by different letters on the bars. doi:10.1371/journal.pone.0026747.g004 Figure 4. Prevalence (%) of vitamin-B12 deficiency in control (CN) and diabetes patients without (DNR) and with retinopathy (DR). Data indicate percent of subjects below 200 pg/mL of the respective group. Proportion Z test was done to compare prevalence between groups. Results Data were transformed into log values and compared the mean values across groups by oneway ANOVA ‘F’ test with post hoc test of Tukey’s multiple comparisons. Significant differences (p,0.05) of mean values between the groups are indicated by different letters on the bars. doi:10.1371/journal.pone.0026747.g003 Correlations (r-value) were assessed by Spearman rank correlation. While positive r-value indicates direct correlation, negative r-value indicates inverse relationship between the variables. doi:10.1371/journal.pone.0026747.t003 not deficient and glucose levels in diabetes groups were comparable irrespective of the presence of DR, the odds ratio for vitamin B12 is twice (95% CI, 1.1–3.7) in the DR group compared with the DNR group after adjusting for age and duration of diabetes. have evaluated the association between homocysteine and DR, the results are varied and inconsistent [22,25,32–34]. Amongst the many determinants of homocysteine, B-vitamin status is a major confounding factor [23,28,35,36]. However, very little is known about the extent to which B-vitamin status is associated with DR vis-a`-vis homocysteine. Considering the general prevalence of micronutrient deficiency and its contribution to many metabolic disorders, such as intrauterine growth retarda- tion, diabetes, and cardiovascular diseases in India [29–31], determining the status of micronutrients with regard to the prevalence and pathogenesis of DR is critical. Therefore, we evaluated the association of B-vitamin status and homocysteine with DR in a systematic way. To the best of our knowledge, a relationship between B-vitamin status, homocysteine, and DR has so far not been reported. Diabetic Retinopathy - Vitamin-B12 Deficiency Diabetic Retinopathy - Vitamin-B12 Deficiency NPDR and PDR [11,45,46]. The study was carried out in accordance with the guidelines of the Helsinki Declaration of 1975 and approved by the Institutional Ethics Committees of Pushpagiri Vitreo Retina Institute and National Institute of Nutrition. After obtaining written informed consent from all participants, venous blood samples were collected in EDTA tubes in the morning following an overnight fast. An aliquot of each whole blood sample was kept, while the remainder was separated into plasma and red blood cells [46]. duration of diabetes because the associations between vitamin B12 and these variables were not statistically significant. Moreover, vitamin B12 deficiency is two times as likely in the DR group compared with the DNR group after adjusting for age and duration of diabetes. Together, these results also suggest that vitamin B12 deficiency could be an independent risk factor for DR. The Age-Related Eye Disease Study (AREDS) has shown that antioxidant and trace element micronutrients can reduce the risk of developing age-related macular degeneration [37–39]. Supple- mentation with some antioxidants and micronutrients, including vitamin B1 (benfotiamine), has shown encouraging results in experimental models of DR and human studies, though the findings of clinical trials with these antioxidant micronutrients have been less conclusive [40–42]. Interestingly, a study showed that AREDS-based micronutrients proven to be beneficial in ameliorating the lesions associated with DR in experimental rats [43]. Although we have yet to analyze the association of micronutrients other than B-vitamins with DR, the results reported here imply that a deficiency or inadequacy of vitamin B12 may predispose diabetes patients to DR. Lowered levels of cobalt, an integral component of vitamin B12 (cyanocobalamine), paired with decreased dietary intake of vitamin B12 in the DR group compared with the DNR group further supports the above implication (GBR unpublished data). It should be noted that low levels of vitamin B12 have been recognized in Indians for a long time and recent studies confirm low concentrations of vitamin B12 and the implications for diabetes and cardiovascular diseases in India [29,30,44]. Our study also further substantiates the general prevalence of vitamin B12 deficiency in India; about 40% adults above 50 years are deficient and the prevalence is much higher in patients with diabetes. Statistical analysis Statistical analysis was performed using SPSS for Windows version 15.0. Mean and SD or SE values of vitamins and homocysteine, BMI, age, and duration of disease were calculated. No outliers were found in the data based on Grubb test. Comparison of mean values of these variables across groups was done by one-way ANOVA F test with post hoc Tukey test. Log transformations were also performed to stabilize the normality of the skewed variables for glucose, HbA1c, HDL, homocysteine, and vitamin B12. Proportion Z test was used for comparison of prevalence of vitamin B12 deficiency. The relationship between homocysteine and vitamin B12 with age, duration of disease, BMI, glucose, and vitamins B1, B2, B6, and folic acid was calculated by Spearman rank correlation coefficients, and risk was estimated by odds ratio with logistic regression model. Two-tailed test was considered for all statistical tests. The level of significance was p,0.05. Estimation of homocysteine and B-vitamins We employed HPLC to estimate vitamins B1, B2, and B6. The levels of vitamin B1 and B2 in whole blood and B6 in plasma were measured as total thiamine pyrophosphate, flavin adenine dinucleotide, and pyridoxal-59-phosphate, respectively, based on previously reported methods [47] using commercially available HPLC kits (Recipe Chemicals and Instruments GmbH, Ger- many). Plasma levels of vitamin B12 and folic acid were measured by a solid phase radioimmunoassay method using a commercially available kit designed for simultaneous measurement of these vitamins (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA). Radioactivity was measured by a gamma counter with a dual channel for determining 57Co and 125I simultaneously (Perkin Elmer, 3 wizard 1480, USA). Plasma total homocysteine levels were measured by HPLC using a SupelcosilTM LC-18-DB (150 mm by 4.6 mm) column according to methods reported previously [48,49]. Diabetic Retinopathy - Vitamin-B12 Deficiency This is the first study to show an association of B-vitamins with DR, and more controlled prospective studies are warranted to confirm the role of vitamin B12 deficiency in the development of DR. Study design, subjects, and sample collection y g j p This is a hospital-based case–control study conducted during April 2008 to March 2010 consisting of 300 T2D patients either with or without retinopathy (DR, n = 200 and DNR, n = 100, respectively). In addition, we recruited 100 control (CN) subjects consisting of partners, relatives, and friends of patients and employees of the National Institute of Nutrition matched for similar socioeconomic status of the T2D patients. The CN group consisted of asymptomatic subjects of age 50 years and above without any history of cardiovascular and renal complications. Subjects with T2D with and without retinopathy were recruited from patients attending the Pushpagiri Vitreo Retina Institute and were matched for duration of diabetes. Control and diabetes subjects on nutritional supplements for the last 6 months and those with a history of nephropathy (based standard renal function tests) and complications other than DR were excluded. However, many subjects with diabetes (DR and DNR) used antidiabetic medication per their physician’s advice. History or presence of diabetic complications other than DR was assessed by clinical as well as biochemical methods. All patients with diabetes underwent a complete ophthalmic examination consisting of best corrected visual acuity, slit-lamp biomicroscopy, indirect oph- thalmoscopy, and fundus fluorescein angiography. Diabetic retinopathy grading was done using the Early Treatment Diabetic Retinopathy Study adaptation of the modified Airlie House classification system, and DR was further categorized as Methods Study design, subjects, and sample collection Biochemical estimations Fasting blood glucose was estimated in plasma by the glucose oxidase–peroxidase method using a kit (BioSystems, Barcelona, Spain). Glycosylated hemoglobin (HbA1c) was estimated in whole blood by ion-exchange chromatography using a kit (BioSystems). While plasma insulin was estimated by a radioimmunoassay kit (Board of Radiation and Isotope Technology-Department of Atomic Energy, Mumbai, India), the lipid profile (total cholesterol, triglycerides, HDL) was analyzed using commercially available kits (BioSystems). Discussion Significant differences (p,0.001) of mean values between the groups are indicated by different letters on the bars. doi:10.1371/journal.pone.0026747.g004 November 2011 \| Volume 6 \| Issue 11 \| e26747 PLoS ONE \| www.plosone.org 4 References Muthayya S, Kurpad AV, Duggan CP, Bosch RJ, Dwarkanath P, et al. (2006) Low maternal vitamin B12 status is associated with intrauterine growth retardation in urban South Indians. Eur J Clin Nutr 60: 791–801. 8. Rema M, Ponnaiya M, Mohan M (1996) Prevalence of retinopathy in non- insulin dependent diabetes mellitus at a diabetes center in southern India. Diab Res Clin Pract 34: 29–36. 32. Goldstein M, Leibovitch I, Yeffimov I, Gavendo S, Sela BA, et al. (2004) Hyperhomocysteinemia in patients with diabetes mellitus with and without diabetic retinopathy. Eye 18: 460–465. 9. Dandona L, Dandona R, Naduvilath TJ, McCarty CA, Rao GN (1999) Population based assessment of diabetic retinopathy in an urban population in southern India. Br J Ophthalmol 83: 937–40. 33. Looker HC, Fagot-Campagna A, Gunter EW, Pfeiffer CM, Narayan KM, et al. (2003) Homocysteine as a risk factor for nephropathy and retinopathy in type 2 diabetes. Diabetologia 46: 766–772. p 10. Nirmalan PK, Katz J, Robin AL, Tielsch JM, Namperumalsamy P, et al. (2004) Prevalence of vitreoretinal disorders in a rural population of Southern India: the Aravind Comprehensive Eye Study. Arch Ophthalmol 122: 581–586. 34. Neugebauer S, Baba T, Kurokawa K, Watanabe T (1997) Defective homocysteine metabolism as a risk factor for diabetic retinopathy. Lancet 349: 473–474. 11. Viswanath K, Murray McGavi DD (2003) Diabetic retinopathy: Clinical findings and management. Community Eye Health 16: 21–24. 35. Selhub J, Jacques PF, Wilson PWF, Rash D, Rosenberg IH (1993) Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 270: 2693–2698. g g y y 12. Moss SE, Klein R, Klein BE (1998) The 14-year incidence of visual loss in a diabetic population. Ophthalmol 105: 998–1003. 36. Vermeulen EGJ, Stehouwer CDA, Twisk JDR, Van den Berg M, de Jong SC, et al. (2000) Effect of homocysteine lowering treatment with folic acid plus vitamin B6 on progression of subclinical atherosclerosis: a randomized, placebo- controlled trial. Lancet 355: 517–522. 13. Keenan HA, Costacou T, Sun JK, Doria A, Cavellerano J, et al. (2007) Clinical factors associated with resistance to microvascular complications in diabetic patients of extreme disease duration: the 50-year medalist study. Diabetes Care 30: 1995–1997. 14. Ng DP (2010) Human genetics of diabetic retinopathy-current perspectives. J Ophthalmol, 2010. pii: 172593. 37. References Chiu CJ, Milton RC, Gensler G, Taylor A (2007) Association between dietary glycemic index and age-related macular degeneration in nondiabetic participants in the Age-Related Eye Disease Study. Am J Clin Nutr 86: 180–188. 15. Fu YP, Hallman DM, Gonzalez VH, Klein BE, Klein R, et al. (2010) Identification of diabetic retinopathy genes through a genome-wide association study among Mexican-Americans from Starr County, Texas. J Ophthalmol, 2010. pii: 861291. 38. Giovanni JPS, Chew EY, Clemons TE (2007) Age-Related Eye Disease Study Research Group. The relationship of dietary lipid intake and age-relatedmacular degeneration in a case-control study: AREDS report No. 20. Arch Ophthalmol 125: 671–679. 16. Rema M, Saravanan G, Deepa R, Mohan V (2002) Familial clustering of diabetic retinopathy in South Indian type 2 diabetic patients. Diab Med 19: 910–916. 39. Giovanni JPS, Chew EY, Clemons TE (2007) Age-Related Eye Disease StudyResearch Group. The relationship of dietary carotenoid and vitamin A, E, and C intake with age-related macular degeneration in a case-control study: AREDS report No. 22. Arch Ophthalmol 125: 1225–1232. 17. Martini LA, Catania AS, Ferreira SR (2010) Role of vitamins and minerals in prevention and management of type 2 diabetes mellitus. Nutr Rev 68: 341–54. 18. Failla ML, Kiser RA (1981) Altered tissue content and cytosol distribution of trace metals in experimental diabetes. J Nutr 11: 1900–1909. 40. Hammes HP, Du X, Edelstein D (2003) Benfotiamine blocks three major path- ways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nat Med 9: 294–299. p 19. Mooradian AD, Morley JE (1987) Micronutrient status in diabetes mellitus. Am J Clin Nutr 45: 877–895. 41. Lee CTC, Emma I, Gayton, Chir MB, Beulens JW, et al. (2010) Micronutrients and diabetic retinopathy: A systematic review. Ophthalmol 117: 71–78. 20. Thornalley PJ, Babaei-Jadidi R, Ali AH (2007) High prevalence of low plasma thiamine concentration in diabetes linked to a marker of vascular disease. Diabetologia 50: 2164–2170. 42. Mayer-Davis EJ, Bell RA, Reboussin BA, Rushing J, Marshall JA, et al. (1998) The San Luis Valley Study: Antioxidant nutrient intake and diabetic retinopathy. Ophthalmol 105: 2264–2270. g 21. Thornalley PJ (2005) The potential role of thiamin (vitamin B1) in diabetic complications. Curr Diab Rev 1: 287–298. 22. Brazoins L, Rowley K, Itsiopoulos K, Harper CA, O’Dea K (2008) Homocysteine and diabetic retinopathy. Diabetes Care 31: 50–56. 43. References 25. Hoogeveen EK, Kostense PJ, Eysink PE, Polak BC, Beks PJ, et al. (2000) Hyperhomocysteinemia is associated with the presence of retinopathy in type 2 diabetes mellitus: the Hoorn Study. Arch Intern Med 160: 2984– 2990. 1. Resnikoff S, Pascolini D, Etya’ale D, Kocur I, Pararajasegaram R, et al. (2004) Global data on visual impairment in the year 2002. Bull World Health Organ 82: 844–851. 2. Kempen JH, O’Colmain BJ, Leske MC, Haffner SM, Klein R, et al. (2004) Eye Diseases Prevalence Research Group: The prevalence of diabetic retinopathy among adults in the United States. Arch Ophthalmol 122: 552–563. 26. Stanger O, Herrmann W, Pietrzik K, Fowler B, Geisel J, et al. (2003) DACH- LIGA homocystein (german, austrian and swiss homocysteine society): consensus paper on the rational clinical use of homocysteine, folic acid and B-vitamins in cardiovascular and thrombotic diseases: guidelines and recommendations. Clin Chem Lab Med 41: 1392–403. 3. International Diabetes Federation (2009) The Diabetes Atlas. 4th edition. Brussels. 4. The Diabetes Control and Complications Trial Research Group (1993) The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. New Engl J Med 329: 977–986. 27. Chang PK, Gordon RK, Tal J, Zeng GC, Docot BP, et al. (1996) S- Adenosylmethionine and methylation. FASEB J 10: 471–480. Adenosylmethionine and methylation. FASEB J 10: 471–480. 28. Wijekoon EP, Brosnan ME, Brosnan JT (2007) Homocysteine metabolism in diabetes. Biochem Soc Trans 35. 5. Turner R (1998) Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet 352: 837–853. 29. Misra A, Vikram NK, Pandey RM, Dwivedi M, Ahmad FU, et al. (2002) Hyperhomocysteinemia and low intakes of folic acid and vitamin B 12 in urban North India. Eur J Nutr 41: 68–77. yp ( ) 6. Zhang X, Jinan B, Saaddine, Chou CF, Cotch MF, et al. (2010) Prevalence of diabetic retinopathy in the United States, 2005–2008. JAMA 304: 649–656. 30. Yajnik CS, Deshpande SS, Lubree HG, Naik SS, Bhat DS, et al. (2006) Vitamin B12 deficiency and hyperhomocysteinemia in rural and urban Indians. J Assoc Physicians India 54: 775–782. 7. Rema M, Premkumar S, Anitha B, Deepa R, Pradeepa R, et al. (2005) Prevalence of diabetic retinopathy in urban India: the Chennai Urban Rural Epidemiology Study (CURES) eye study. Invest Ophthalmol Vis Sci 46: 2328–33. 31. Sample size for the estimation of vitamins and homocysteine Although the data for demographic and clinical parameters were collected for all 400 subjects, the actual minimum required sample size for vitamins and homocysteine was determined assuming 95% CI and 80% power and using SD of respective November 2011 \| Volume 6 \| Issue 11 \| e26747 PLoS ONE \| www.plosone.org 5 Diabetic Retinopathy - Vitamin-B12 Deficiency preparation. Part of this work was presented at the 19th Biennial Meeting of the International Society for Eye Research, July 18–23, 2010, Montreal, Canada, and a part of the work was presented at the ARVO (Association for Research in Vision and Ophthalmology) Annual Meeting, May 1–5, 2011, Fort Lauderdale, FL, USA. vitamins and homocysteine. Hence the data on vitamins and homocysteine were obtained on a subsample. Nevertheless, the sample size was increased for homocysteine and vitamin B12, for strengthening their association with DR. Author Contributions The authors are grateful to all the participants for their cooperation in this study. We thank Drs. Nilanjana Deb-Joardar, O. Muralidhar, and P. Sunitha, Pushpagiri Vitreo Retina Institute, Hyderabad, for their service in the recruitment and medical examination of study patients. The author also acknowledges the help of Ms. C. Akileshwari in the manuscript Conceived and designed the experiments: GBR. Performed the experi- ments: AS SP PYR SM PL PS KV. Analyzed the data: AS NB SP PYR SM PL PS KV RA GBR. Wrote the paper: AS NB RA GBR. 48. Pitla S, Nagalla B (2009) Gender-related differences in the relationship between plasma homocysteine, anthropometric and conventional biochemical coronary heart disease risk factors in middle-aged Indians. Ann Nutr Metab 54: 1–6. 49. Ubbink JB, Hayward Vermaak WJ, Bissbort S (1991) Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr 565: 441–446. an extension of the modified Airlie House Classification. ETDRS Report 10. Ophthalmol 98: 786–806. 46. Reddy GB, Satyanarayana A, Balakrishna N, Ayyagari R, Padma M, et al. (2008) Erythrocyte aldose reductase activity and sorbitol levels in diabetic retinopathy. Mol Vis 14: 593–601. 47. Botticher B, Botticher D (1987) Determination of B1, B2, B6 Vitamers in blood. Int J Vit Nutr Res 57: 273–278. p y 47. Botticher B, Botticher D (1987) Determination of B1, B2, B6 Vitamers in blood. Int J Vit Nutr Res 57: 273–278. 46. Reddy GB, Satyanarayana A, Balakrishna N, Ayyagari R, Padma M, et al. (2008) Erythrocyte aldose reductase activity and sorbitol levels in diabetic retinopathy. Mol Vis 14: 593–601. an extension of the modified Airlie House Classification. ETDRS Report 10. Ophthalmol 98: 786–806. PLoS ONE \| www.plosone.org Diabetic Retinopathy - Vitamin-B12 Deficiency References Kowluru RA, Kanwar M, Chan P, Zhang JP (2008) Inhibition of retinopathy and retinal metabolic abnormalities in diabetic rats with AREDS-based micronutrients. Arch Ophthalmol 126: 266–1272. 23. Wright AD, Martin N, Dodson PM (2008) Homocysteine, folates and the eye. Eye 22: 989–993. p 44. Yajnik CS, Deshpande SS, Jackson AA, Refsum H, Rao S, et al. (2008) Vitamin B12 and folate concentrations during pregnancy and insulin resistance in the offspring: the Pune Maternal Nutrition Study. Diabetologia 51: 29–38. 24. Coral K, Angayarkanni N, Gomathy N, Bharathselvi M, Pukhraj R, et al. (2009) Homocysteine levels in the vitreous of proliferative diabetic retinopathy and rhegmatogenous retinal detachment: its modulating role on lysyl oxidase. Invest Ophthalmol Vis Sci 50: 3607–12. 45. Early Treatment of Diabetic Retinopathy Study Research Group (1991) Grading diabetic retinopathy from stereoscopic colour fundus photographs - PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26747 November 2011 \| Volume 6 \| Issue 11 \| e26747 6 Diabetic Retinopathy - Vitamin-B12 Deficiency PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26747 7 7
https://openalex.org/W2507331282	https://europepmc.org/articles/pmc4981981?pdf=render	English	null	Epigenetic features of FoxP3 in children with cow’s milk allergy	Clinical epigenetics	2,016	cc-by	5,272	© 2016 The Author(s). 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. Abstract Background: DNA methylation of the Th1 and Th2 cytokine genes is altered during cow’s milk allergy (CMA). Forkhead box transcription factor 3 (FoxP3) is essential for the development and function of regulatory T cells (Tregs) and is involved in oral tolerance acquisition. We assessed whether tolerance acquisition in children with IgE-mediated CMA is associated with DNA demethylation of the Treg-specific demethylated region (TSDR) of FoxP3. Results: Forty children (aged 3–18 months) were enrolled: 10 children with active IgE-mediated CMA (group 1), 10 children who outgrew CMA after dietary treatment with an extensively hydrolyzed casein formula containing the probiotic Lactobacillus rhamnosus GG (group 2), 10 children who outgrew CMA after treatment with other formulas (group 3), and 10 healthy controls (group 4). FoxP3 TSDR demethylation and expression were measured in mononuclear cells purified from peripheral blood of the four groups of children. FoxP3 TSDR demethylation was significantly lower in children with active IgE-mediated CMA than in either children who outgrew CMA or in healthy children. Formula selection influenced the FoxP3 TSDR demethylation profile. The FoxP3 TSDR demethylation rate and expression level were correlated. Conclusions: Tolerance acquisition in children with IgE-mediated CMA involves epigenetic regulation of the FoxP3 gene. This feature could be a new target for preventive and therapeutic strategies against CMA. rds: Food allergy, Extensively hydrolyzed casein formula, Oral tolerance, Lactobacillus rhamnosus GG regulatory regions [9, 10], and, moreover, hypermethylation of the FoxP3 gene has been associated with reduced Treg function and allergy [7, 11]. * Correspondence: berni@unina.it 1Department of Translational Medical Science, University of Naples “Federico II”, Via S. Pansini, 5 80131 Naples, Italy 2CEINGE-Biotecnologie Avanzate s.c.ar.l, Via Gaetano Salvatore 486, 80131 Naples, Italy Full list of author information is available at the end of the article Epigenetic features of FoxP3 in children with cow’s milk allergy Lorella Paparo1, Rita Nocerino1, Linda Cosenza1, Rosita Aitoro1, Valeria D’Argenio2,3, Valentina Del Monaco2,3, Carmen Di Scala1, Antonio Amoroso1, Margherita Di Costanzo1, Francesco Salvatore2,3,4 and Roberto Berni Canani1,2,5* Paparo et al. Clinical Epigenetics (2016) 8:86 DOI 10.1186/s13148-016-0252-z Paparo et al. Clinical Epigenetics (2016) 8:86 DOI 10.1186/s13148-016-0252-z Open Access Open Access SHORT REPORT Background Epigenetic mechanisms have been implicated in the patho- genesis of food allergy [1]. We previously demonstrated that tolerance acquisition in children with IgE-mediated cow’s milk allergy (CMA) is driven by epigenetic modula- tion of the Th1 and Th2 cytokine genes [2]. A regulatory T cell (Treg) suppressive phenotype, characterized by stable expression of the transcription factor “Forkhead box Pro- tein 3” (FoxP3), plays a pivotal role in food tolerance [3–7]. FoxP3 messenger RNA (mRNA) expression is lower in children with atopic asthma or IgE-mediated food allergy than in healthy children [8]. FoxP3 stable expression re- quires full CpG demethylation of its transcriptional DNA methylation is a biologically and chemically stable epigenetic modification that locks in long-term gene expression patterns [12, 13]. The demethylation status of FoxP3 at a highly conserved region within the Treg-specific demethylated region (TSDR), a CpG-rich region, located on the 2nd conserved non-coding sequence of FoxP3 (CNS2), is restricted to Tregs [14, 15]. Transcriptional activity of the TSDR is essentially deter- mined by its methylation status: it is completely inactive in its methylated state, but when the TSDR is demethy- lated, transcription factors such as Ets-1 and Creb can bind to the TSDR [16] TSDR demethylated and open chromatin conformation in the Foxp3 locus leads to stable phenotype differentiated Foxp3+ Treg [17, 18]. FoxP3 TSDR demethylation in peripheral blood mononuclear * Correspondence: berni@unina.it 1Department of Translational Medical Science, University of Naples “Federico II”, Via S. Pansini, 5 80131 Naples, Italy 2CEINGE-Biotecnologie Avanzate s.c.ar.l, Via Gaetano Salvatore 486, 80131 Naples, Italy Full list of author information is available at the end of the article Paparo et al. Clinical Epigenetics (2016) 8:86 Page 2 of 6 Page 2 of 6 cells (PBMCs) has been associated with reduced atopic sensitization and asthma in children [19]. Epigenetic regu- lation of antigen-induced T cell subsets may predict a state of immune tolerance in food allergy. Indeed, DNA methy- lation of the FoxP3 gene in Tregs decreased during oral tolerance acquisition in patients with peanut allergy undergoing oral immunotherapy [19]. The aim of this study was to evaluate further the epigenetic regulation of FoxP3 gene in children with IgE-mediated CMA. discharged. In the case of a positive DBPCFC, at any test- ing dose, the patient remained under observation until symptom resolution. Study subjects We evaluated IgE-mediated CMA children (aged 3 to 18 months) consecutively referred to our tertiary Pediatric Allergy Center for oral food challenge. Oral food challenge was requested to obtain a diagnosis because of recent evidence of CMA signs and symptoms (“active CMA patients”; group 1) or to investigate the occurrence of oral tolerance acquisition after dietary treatment with an ex- tensively hydrolyzed casein formula containing the pro- biotic Lactobacillus rhamnosus GG (LGG, 1 × 106/ml CFU) (“subjects who outgrew CMA with extensively hy- drolyzed casein formula (EHCF) + LGG”; group 2) or with other formulas (“subjects who outgrew CMA with other formulas”; group 3). All patients underwent a double- blind placebo-controlled oral challenge (DBPCFC), as de- scribed previously [20]. All oral food challenges took place at our Center on two separate days with a 1-week interval. Parents of children taking antihistamine were advised to withhold these medications for at least 72 h before and during the challenge. Randomization and preparation of the challenges were performed by experienced food allergy dieticians not directly involved in the procedures. Briefly, every 20 min, successive doses (0.1, 0.3, 1, 3, 10, 30, and 100 ml) of fresh pasteurized cow’s milk containing 3.5 % fat (verum) or hypoallernic or soy formula that the child was already consuming (placebo) were administered. Full emergency equipment and medications (epinephrine, anti- histamines, and steroids) were available. The results were assessed simultaneously by three experienced pediatric al- lergists. Study subjects were scored for nine items divided into four main categories: (i) general (lowered blood pres- sure plus tachycardia); (ii) skin (rash, urticaria/angio- edema); (iii) gastrointestinal (nausea/repeated vomiting, crampy-like abdominal pain, diarrhea); and (iv) respiratory (sneezing/itching, nasal congestion/rhinorrhea, stridor de- riving from upper airway obstruction or wheezing) on a 0- to 3-point scale (0, none; 1, light; 2, moderate; and 3, se- vere). If at least two of the three physicians independently scored any item at level 3, or 2 (or more) items at level 2, the test result was considered positive. Study subjects Clinical symptoms occurring within 2 h of administering the highest dose were defined as “IgE-mediated reactions.” The infants were observed for 2 h after the final dose and then Exclusion criteria were as follows: allergic disorders or food allergies other than CMA, eosinophilic disorders of the gastrointestinal tract, food protein-induced enterocoli- tis syndrome, concomitant chronic systemic diseases, con- genital cardiac defects, active tuberculosis, autoimmune diseases, immunodeficiency, chronic inflammatory bowel diseases, celiac disease, cystic fibrosis, metabolic diseases, lactose intolerance, malignancy, chronic pulmonary dis- eases, and malformations of the gastrointestinal tract. Dur- ing the same study period, consecutive healthy children, not at risk of atopic disorders (namely, those without a first-degree relative affected by an atopic disorder), attend- ing our Center because of minimal surgical procedures served as a control group (group 4). A venous blood sample (4 ml) was collected also from these healthy subjects. They were assessed for the presence of food allergy and other allergic diseases at enrollment and 6 months after blood sampling by pediatric allergists at our Center. Background If the patient did not show any symptom within the first 24 h, parents were advised to give one single feed of 100 ml of the tested formula (verum or placebo) everyday at home for 7 days. If any symptom occurred during this period, the patient returned to the outpatient clinic on the same day. After 7 days of verum or placebo administration, the patients were examined and the parents interviewed at the Center. To rule out a false-negative challenge result, parents were asked to contact the Center if any symptoms occurred in the 7 days after the DBPCFC procedures. The challenge was considered negative if the patient tolerated the entire challenge, including the observation period. Clinical toler- ance acquisition was defined by the presence of a negative DBPCFC. Patients with a negative oral challenge (groups 2 and 3) were reassessed 4 weeks later to verify persistence of clinical tolerance. A venous blood sample (4 ml) was obtained from all patients after oral challenge. Total IgE and specific IgE against proteins and epitopes of cow’s milk Serum was obtained by centrifugation for 10 to 15 min. Serum was flash frozen and stored at −80 °C until ana- lysis. Serum total IgE and specific IgE against epitopes of cow’s milk (alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, casein, lactoferrin) were analyzed by enzymatic immunoassay (Phadia 100 ThermoFisher Scientific CAP system, Rodano Milano, Italy). Results were expressed as kilounits per liter (kU/l). DNA methylation and mRNA expression Peripheral blood mononuclear cells were isolated from whole blood samples using the Ficoll-Paque Paparo et al. Clinical Epigenetics (2016) 8:86 Page 3 of 6 (Sigma-Aldrich, St. Louis, MO, USA) method, described previously [2]. The primers used for D methylation analysis of FoxP3 TSDR are repo elsewhere [15]. High-resolution melting real-time P for methylation analysis was performed as descr previously [2]. The results of methylation ana were verified by direct sequencing (using the San method modified as follows: ddNTPs labeled w four different fluorophores) and analyzed by capi electrophoresis (the analytical specificity and sensitivit the test was >99 %). Real-time PCR was performed the LightCycler® 480 instrument (Roche Applied Scie Penzberg, Germany) using 96-well plates (Roche App Science). Briefly, RNA was extracted from the PBMC the four study groups using the Trizol protocol (Inv gen, Life Technologies Europe BV, Monza, Italy), as pr ously described [2]. The concentration and purity of R samples were measured and verified by NanoDrop 1 spectrophotometry (Thermo Scientific, Wilmington, USA). For complementary (cDNA) synthesis, 1 μg RNA was transcribed with a High Capacity cDNA Rev Transcription kit (Applied Biosystems, Foster C CA, USA) according to the manufacturer’s inst tions. The 10-μl reaction volumes contained 1 μl t plate, 10 μl SYBR Green (Applied Biosystems), 5 μM primers (FoxP3 forward primer 5′-AGCTG GTTCCGCAAGAAAC-3′; FoxP3 reverse primer TGTTCGTCCATCCTCCTTTCC-3′; GenBank Ac sion number NC_000023.11). Quantitative real-t amplifications were performed in triplicate with Table 1 The main demographic and clinical characteristics Subjects with CM at diagnosis Group 1 Number 10 Male, number (%) 7 (70) Age, months (SD) 5.5 (0.7) Body weight, kg (SD) 7.385 (964.7) Spontaneous delivery, number (%) 5 (50) Breastfeeding, ≤8 weeks, number (%) 10 (100) Symptoms at the CMA onset Gastrointestinal, n (%) 4 (40) Cutaneous, n (%) 8 (80) Respiratory, n (%) 1 (10) Total serum IgE, kU/l (SD) 260.6 (230.9) Alpha-lactalbumin, kUA/l(SD) 6 (11.2) Beta-lactoglobulin, kUA/l(SD) 4.5 (7.1) Bovine serum albumin, kUA/l(SD) 5.4 (8.7) nBCasein, kUA/l(SD) 22.7 (39.2) Lactoferrin, kUA/l(SD) 2 (6.1) (Sigma-Aldrich, St. Statistical analysis y The Kolmogorov-Smirnov test was used to determine whether variables were normally distributed. The χ2 test and Fisher’s exact test were used for categorical variables. We used the t test and one-way ANOVA to evaluate differ- ences among continuous variables. To determine which groups in the sample differ, the Bonferroni correction was performed. Pearson’s correlation coefficient “r” was used to evaluate the correlation between continuous variables. The level of significance for all statistical tests was two- sided, P < 0.05. All analyses were conducted by a statis- tician blinded to patient group assignment, using SPSS, version 19.0 for Windows (SPSS Inc., Chicago, IL, USA). Total IgE and specific IgE against proteins and epitopes of cow’s milk Louis, MO, USA) method, as described previously [2]. The primers used for DNA methylation analysis of FoxP3 TSDR are reported elsewhere [15]. High-resolution melting real-time PCR for methylation analysis was performed as described previously [2]. The results of methylation analysis were verified by direct sequencing (using the Sanger method modified as follows: ddNTPs labeled with four different fluorophores) and analyzed by capillary electrophoresis (the analytical specificity and sensitivity of the test was >99 %). Real-time PCR was performed with the LightCycler® 480 instrument (Roche Applied Science, Penzberg, Germany) using 96-well plates (Roche Applied Science). Briefly, RNA was extracted from the PBMCs of the four study groups using the Trizol protocol (Invitro- gen, Life Technologies Europe BV, Monza, Italy), as previ- ously described [2]. The concentration and purity of RNA samples were measured and verified by NanoDrop 1000 spectrophotometry (Thermo Scientific, Wilmington, DE, USA). For complementary (cDNA) synthesis, 1 μg total RNA was transcribed with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instruc- tions. The 10-μl reaction volumes contained 1 μl tem- plate, 10 μl SYBR Green (Applied Biosystems), and 5 μM primers (FoxP3 forward primer 5′-AGCTGGA GTTCCGCAAGAAAC-3′; FoxP3 reverse primer 5′- TGTTCGTCCATCCTCCTTTCC-3′; GenBank Acces- sion number NC_000023.11). Quantitative real-time amplifications were performed in triplicate with an initial incubation at 95 °C for 30 s, followed by 40 cy- cles of 95 °C for 10 s and 60 °C for 30 s, using a Light Cycler 79 HT (Applied Biosystems). The quantitative gene expression was calculated with the comparative Ct method and normalized against the Ct of glucuroni- dase (GUS) messenger as reference gene. Results The main demographic and clinical characteristics of the study population are reported in Table 1. The 10 children affected by CMA in group 1 had a positive DBPCFC at doses between 3 and 10 ml of cow’s milk. The 20 CMA children with recent evidence of oral tolerance acquisition, demonstrated by a negative DBPCFC (groups 2 and 3), were able to consume at least one full cup/day of cow’s milk without symptoms. Group 2 patients received EHCF Table 1 The main demographic and clinical characteristics of the four study groups Subjects with CMA at diagnosis Subjects outgrown CMA Healthy subjects EHCF + LGG Other formulas Group 1 Group 2 Group 3 Group 4 Number 10 10 10 10 Male, number (%) 7 (70) 7 (70) 6 (60) 4 (40) Age, months (SD) 5.5 (0.7) 16.9 (0.9) 17 (0.9) 9 (4) Body weight, kg (SD) 7.385 (964.7) 12.047 (682.4) 12.273 (776.7) 8.075 (3193.5) Spontaneous delivery, number (%) 5 (50) 2 (20) 3 (30) 4 (40) Breastfeeding, ≤8 weeks, number (%) 10 (100) 10 (100) 10 (100) 10 (100) Symptoms at the CMA onset Gastrointestinal, n (%) 4 (40) 5 (50) 3 (30) – Cutaneous, n (%) 8 (80) 8 (80) 7 (70) – Respiratory, n (%) 1 (10) 3 (30) 3 (30) – Total serum IgE, kU/l (SD) 260.6 (230.9) 255.9 (236.1) 130.4 (167.9) 0.2 (0.1) Alpha-lactalbumin, kUA/l(SD) 6 (11.2) 0.6 (0.7) 3.1 (3.9) – Beta-lactoglobulin, kUA/l(SD) 4.5 (7.1) 2.2 (3.4) 1.9 (2.6) – Bovine serum albumin, kUA/l(SD) 5.4 (8.7) 0.9 (1.8) 1.9 (3) – nBCasein, kUA/l(SD) 22.7 (39.2) 0.5 (0.4) 1.3 (2.7) – Lactoferrin, kUA/l(SD) 2 (6.1) 0 0 – Table 1 The main demographic and clinical characteristics of the four study groups Subjects with CMA Subjects outgrown CMA Paparo et al. Clinical Epigenetics (2016) 8:86 Page 4 of 6 Fig. 1 FoxP3 TSDR demethylation rate (%) (a) and FoxP3 mRNA expression versus GUS expression (b) observed in study groups (group 1, CMA children at diagnosis; group 2, children outgrown CMA after treatment with EHCF + LGG; group 3, children outgrown CMA after treatment with other formulas; group 4, healthy controls) n versus GUS expression (b) observed in study groups (group 1, CMA Fig. TSDR, Treg-specific demethylated region; AAF, amino acid-based formula; CMA, cow’s milk allergy; EHCF, extensively hydrolyzed casein formula; EHCF + LGG, extensively hydrolyzed casein formula plus Lactobacillus rhamnosus GG; FoxP3, forkhead box protein 3; IgE, immunoglobulin E; LGG, Lactobacillus rhamnosus GG; PBMCs, peripheral blood mononuclear cells; RHF, hydrolyzed rice formula; SF, soy formula; SPT, skin prick testing; Treg, regulatory T cells Acknowledgements We thank Jean Ann Gilder (Scientific Communication srl., Naples, Italy) for editing the manuscript. We thank Jean Ann Gilder (Scientific Communication srl., Naples, Italy) for editing the manuscript. Competing interests Competing interests The authors declare that they have no competing interests. We found a significant difference in the methylation status of FoxP3 TSDR gene comparing patients who ac- quired oral tolerance after treatment with EHCF + LGG vs children treated with other formulas. Similar results have been observed investigating Th1/Th2 cytokines [2]. A recent study showed that LGG in addition to vitamin D supplementation increased the number of CD4+CD25 +FoxP3+ Treg in children with allergic rhinitis [21]. Results 1 FoxP3 TSDR demethylation rate (%) (a) and FoxP3 mRNA expression versus GUS expression (b) observed in study groups (group 1, CMA children at diagnosis; group 2, children outgrown CMA after treatment with EHCF + LGG; group 3, children outgrown CMA after treatment with other formulas; group 4, healthy controls) + LGG-based treatment, and group 3 patients received the following formulas: EHCF (n = 3), soy formula (SF, n = 3), amino acid-based formula (AAF, n = 3), hydrolyzed rice formula (RHF, n = 1). The median duration of dietother- apy was 384 days (IQR 7.75) in both groups. (group 1) than in healthy controls (1.3 ± 1.3 vs 20 ± 2.1, P < 0.0001). To determine the fold expression levels, we normalized FoxP3 TSDR expression vs the transcription levels of the housekeeping gene GUS in the four study groups. As shown in Fig. 1b, FoxP3 expression was sig- nificantly lower (P < 0.0001) in active IgE-mediated CMA children than in healthy controls. Moreover, it y y g As shown in Fig. 1a, the rate of FoxP3 TSDR demeth- ylation was lower in active IgE-mediated CMA patients Fig. 2 Linear regression analysis of % FoxP3 TSDR demethylation rate (%) in PBMCs versus FoxP3 mRNA expression in study subjects regression analysis of % FoxP3 TSDR demethylation rate (%) in PBMCs versus FoxP3 mRNA expression in study subjects Fig. 2 Linear regression analysis of % FoxP3 TSDR demethylation rate (%) in PBMCs versus FoxP3 mRNA expression in study subjects Paparo et al. Clinical Epigenetics (2016) 8:86 Page 5 of 6 Page 5 of 6 Page 5 of 6 was higher in children who outgrew CMA than in children with active IgE-mediated CMA, but similar to levels in the healthy children. The FoxP3 TSDR demethylation rate was correlated with FoxP3 expression levels (Fig. 2). Notably, the FoxP3 TSDR demethylation profile stratified CMA patients according to disease state. In fact, the demethylation profile in subjects with recent evidence of tolerance acquisition was different to that of active CMA patients and differed significantly (P < 0.0001) between children treated with EHCF + LGG and children receiving other formulas (Fig. 1a). Availability of data and materials “The dataset supporting the conclusions of this article is available in the [repository name] repository [unique persistent identifier and hyperlink to dataset(s) in http:// format”. Funding This work was supported in part by the Italian Ministry of Health Grant PE-2011-02348447 and by an unrestricted grant from Mead Johnson Nutrition (Evansville, IN, USA) awarded to the University of Naples “Federico II.” Neither the Italian Ministry of Health nor Mead Johnson Nutrition had any influence on (1) the study design, (2) the collection, analysis, and interpretation of data; (3) writing the manuscript; and (4) the decision to submit the manuscript for publication. Ethics approval and consent to participate Th d d b h E h C The study was approved by the Ethics Committee of the University of Naples “Federico II” and was registered in Clinical Trials Protocol Registration System (ID number: NCT02779881). Author details 1 We demonstrated that EHCF + LGG but not EHCF alone is able to positively shape gut microbiota compos- ition increasing the abundance of butyrate-producer bacteria strains [22]. Butyrate is able to inhibit histone deacetylase 9 and 6 with subsequent demethylation of Foxp3 gene [23]. Finally, a formula-induced regulation of circulating miRNAs (microRNA-155, microRNA-148a, microRNA-29b, microRNA-21) acting on Treg differen- tiation may be also involved [24]. 1Department of Translational Medical Science, University of Naples “Federico II”, Via S. Pansini, 5 80131 Naples, Italy. 2CEINGE-Biotecnologie Avanzate s.c.ar.l, Via Gaetano Salvatore 486, 80131 Naples, Italy. 3Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, Italy. 4IRCCS-Fondazione SDN, Via E. Gianturco 113, 80143 Naples, Italy. 5European Laboratory for the Investigation of Food-Induced Diseases, University of Naples “Federico II”, Via S. Pansini 5, 80131 Naples, Italy. Received: 3 June 2016 Accepted: 2 August 2016 Received: 3 June 2016 Accepted: 2 August 2016 y Although with various caveats (the use of PBMCs, a cross-sectional design, and the relatively low number of subjects), our study sheds light on how variations in the level of FoxP3 TSDR demethylation can affect the course of CMA and demonstrates that dietary intervention influences this epigenetic mechanism. Our results also suggest that the FoxP3 TSDR demethylation rate could serve as a biomarker of oral tolerance. The novel data reported herein may serve as a basis for the develop- ment of new diagnostic and therapeutic tools for CMA. Discussion Our data show that tolerance acquisition in children with IgE-mediated CMA involves epigenetic regulation of the FoxP3 gene. We found that FoxP3 TSDR demeth- ylation is significantly increased in CMA children who acquired oral tolerance. Similarly, it has been demon- strated that farm milk exposure in children has an al- lergy preventive effect which is linked to FoxP3 TSDR demethylation in peripheral blood cells [19]. References 1. Quake C, Nadeau KC. The role of epigenetic mediation and the future of food allergy research. Semin Cell Dev Biol. 2015;43:125–30. 1. Quake C, Nadeau KC. The role of epigenetic mediation and the future of food allergy research. Semin Cell Dev Biol. 2015;43:125–30. 2. Berni Canani R, Paparo L, Nocerino R, Cosenza L, Pezzella V, Di Costanzo M, Capasso M, Del Monaco V, D’Argenio V, Greco L, Salvatore F. Differences in DNA methylation profile of Th1 and Th2 cytokine genes are associated with tolerance acquisition in children with IgE-mediated cow’s milk allergy. Clin Epigenetics. 2015;7:38. 3. Berin MC, Shreffler WG. Mechanisms underlying induction of tolerance to foods. Immunol Allergy Clin North Am. 2016;36:87–102. 4. Li Z, Li D, Tsun A, Li B. FOXP3+ regulatory T cells and their functional regulation. Cell Mol Immunol. 2015;12:558–65. 5. Weissler KA, Caton AJ. The role of T-cell receptor recognition of peptide: MHC complexes in the formation and activity of Foxp3+ regulatory T cells. Immunol Rev. 2014;259:11–22. 6. Haiqi H, Yong Z, Yi L. Transcriptional regulation of Foxp3 in regulatory T cells. Immunobiology. 2011;216:678–85. 7. Bacchetta R, Gambineri E, Roncarolo MG. Role of regulatory T cells and FOXP3 in human diseases. J Allergy Clin Immunol. 2007;120:227–35. 8. Krogulska A, Polakowska E, Wasowska-Królikowska K, Małachowska B, Młynarski W, Borowiec M. 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Authors’ contributions LP, RN, and RBC designed the study, coordinated the research team, and wrote the first draft of the report. RN, LC, VG, CDS, and MDC cared for the patients, and LP, RA, and AA performed laboratory activities. VD and FS contributed to the results analysis and revised the manuscript. RN performed statistical analysis and interpretation of data. All authors revised and approved the final version of the article. 1. Quake C, Nadeau KC. The role of epigenetic mediation and the future of food allergy research. Semin Cell Dev Biol. 2015;43:125–30. 2. Berni Canani R, Paparo L, Nocerino R, Cosenza L, Pezzella V, Di Costanzo M, Capasso M, Del Monaco V, D’Argenio V, Greco L, Salvatore F. Differences in DNA methylation profile of Th1 and Th2 cytokine genes are associated with tolerance acquisition in children with IgE-mediated cow’s milk allergy. Clin Epigenetics. 2015;7:38. 3. Berin MC, Shreffler WG. Mechanisms underlying induction of tolerance to foods. 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The role of T-cell receptor recognition of peptide: MHC complexes in the formation and activity of Foxp3+ regulatory T cells. Immunol Rev. 2014;259:11–22. Abbreviations TSDR T i 6. Haiqi H, Yong Z, Yi L. Transcriptional regulation of Foxp3 in regulatory T cells. Immunobiology. 2011;216:678–85. 7. Bacchetta R, Gambineri E, Roncarolo MG. Role of regulatory T cells and FOXP3 in human diseases. J Allergy Clin Immunol. 2007;120:227–35. 8. Krogulska A, Polakowska E, Wasowska-Królikowska K, Małachowska B, Młynarski W, Borowiec M. Decreased FOXP3 mRNA expression in children Page 6 of 6 with atopic asthma and IgE-mediated food allergy. Ann Allergy Asthma Immunol. 2015;115:415–21. with atopic asthma and IgE-mediated food allergy. Ann Allergy Asthma Immunol. 2015;115:415–21. 9. Huehn J, Polansky JK, Hamann A. Epigenetic control of FOXP3 expression: the key to a stable regulatory T-cell lineage? Nat Rev Immunol. 2009;9:83–9 9. Huehn J, Polansky JK, Hamann A. 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https://openalex.org/W4309883493	https://cancerci.biomedcentral.com/counter/pdf/10.1186/s12935-022-02775-9	English	null	Spatial metabolomics on liver cirrhosis to hepatocellular carcinoma progression	Cancer cell international	2,022	cc-by	14,999	© 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. Abstract Background: Hepatocellular carcinoma (HCC) is one of the deadliest cancers and is mainly developed from chronic liver diseases such as hepatitis-B infection-associated liver cirrhosis (LC). The progression from LC to HCC makes the detection of diagnostic biomarkers to be challenging. Hence, there have been constant efforts to improve on identify- ing the critical and predictive changes accompanying the disease progression. Methods: In this study, we looked to using the mass spectrometry mediated spatial metabolomics technique to simultaneous examine hundreds of metabolites in an untargeted fashion. Additionally, metabolic profiles were com- pared between six subregions within the HCC tissue to collect spatial information. Results: Through those metabolites, altered metabolic pathways in LC and HCC were identified. Specifically, the amino acid metabolisms and the glycerophospholipid metabolisms experienced the most changes. Many of the altered metabolites and metabolic pathways were able to be connected through the urea cycle. Conclusions: The identification of the key metabolites and pathways can expand our knowledge on HCC metabolic reprogramming and help us exam potential biomarkers for earlier detection of the malignant disease progression. Keywords: Spatial metabolomics, Liver cirrhosis, Hepatocellular carcinoma, Disease progression, Amino acid metabolism [21]. HCC usually arises from chronic liver inflamma- tion that can be caused by a variety of risk factors such as hepatitis B and/or C infections, alcohol abuse, obesity, diabetes mellitus, etc. [1, 15, 18, 29]. Particularly, the pro- gression from chronic hepatitis B infection to liver cir- rhosis then to HCC is one of the most common ways of disease occurrence. He et al. Cancer Cell International (2022) 22:366 https://doi.org/10.1186/s12935-022-02775-9 He et al. Cancer Cell International (2022) 22:366 https://doi.org/10.1186/s12935-022-02775-9 Cancer Cell International Open Access Spatial metabolomics on liver cirrhosis to hepatocellular carcinoma progression Michelle Junyi He1,2†, Wenjun Pu1,3†, Xi Wang4, Xiaoni Zhong1, Dong Zhao5, Zhipeng Zeng1, Wanxia Cai1, Jiayi Liu1, Jianrong Huang5, Donge Tang1 and Yong Dai1,6* Michelle Junyi He1,2†, Wenjun Pu1,3†, Xi Wang4, Xiaoni Zhong1, Dong Zhao5, Zhipeng Zeng1, Wanxia Cai1, Jiayi Liu1, Jianrong Huang5, Donge Tang1 and Yong Dai1,6* Background Liver cancer is currently one of the most fatal malignan- cies worldwide and poses great challenges to early diag- nosis and prognosis due to its heterogeneity. The most common form of liver cancer is hepatocellular carcinoma (HCC), which accounts for greater than 75% of the cases Hepatitis B is a life-threatening liver infection caused by the hepatitis B virus (HBV) and is transmittable through body fluid. The infection causes alterations of many cellular processes and leads to scarring of the liver, both of which greatly increase the chance of liver cirrho- sis and liver cancer. Such liver injury can lead to changes in cell signing, DNA repair, apoptosis, etc., and results in the accumulation of reactive oxygen species, and/or oncogene activation [29]. These cellular level changes often time worsen into loss of cell cycle and senescence †Michelle Junyi He and Wenjun Pu contributed equally to this work Correspondence: 512370661@qq.com; donge66@126.com; daiyong22@aliyun.com 1 Clinical Medical Research Center, The Second Clinical Medical College of Jinan University, Shenzhen People’s Hospital, No. 1017 Dongmen North Road, Shenzhen 518020, China 5 Department of Nephrology Center, Department of Liver Transplant Center, The Third People’s Hospital of Shenzhen, The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen 518100, Guangdong, China Full list of author information is available at the end of the article †Michelle Junyi He and Wenjun Pu contributed equally to this work Correspondence: 512370661@qq.com; donge66@126.com; daiyong22@aliyun.com 1 Clinical Medical Research Center, The Second Clinical Medical College of Jinan University, Shenzhen People’s Hospital, No. 1017 Dongmen North Road, Shenzhen 518020, China 5 Department of Nephrology Center, Department of Liver Transplant Center, The Third People’s Hospital of Shenzhen, The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen 518100, Guangdong, China Full list of author information is available at the end of the article AFADESI‑MSI B h i i Both positive- and negative-ion mode Air Flow-Assisted Desorption Electrospray Ionization Mass Spectrom- etry Imaging (AFADESI-MSI) were performed using the AFADESI platform (Tscience, China) coupled with the Q-Orbitrap mass spectrometer (Thermo Fisher). The AFADESI platform replaces the original ion source and provides a high-rate airflow that assists the electrospray for improved ionization and ion collection. The propelled secondary ions were then collected and transferred to the Q-Orbitrap mass spectrometer. Based on the specific properties of the compounds, different metabolites may have higher chance of being detected in different ioniza- tion modes. In the case of most DESI-MSI, positive ioni- zation mode is generally shown to have higher sensitivity and stability as the negative mode is prone to corona dis- charge [4, 26].h Now, with the development of the mass spectrometry imaging (MSI) technique, spatial metabolomics emerges as a promising direction of study. Such a study takes into account the spatial variation of metabolites in the tumor microenvironment and therefore can be useful in detect- ing the metabolic biomarkers of liver cancer. As spa- tial metabolomics is still a relatively new field of study, there has not been any systematic study that analyzes the alterations of metabolites distribution and abundance in liver diseases tissues. Hence, in this study, we used the air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) technique to examine altered metabolites and metabolic pathways in liver cirrhosis and HCC tissues compared to the healthy control. AFADESI-MSI was able to simultaneously detect hundreds of metabolites in situ with high sensitivity. Therefore, through this experiment, we were able to iden- tify key metabolites that change continuously as the dis- ease progress. The spray solutions for both the positive- and the neg- ative-ion mode were prepared by mixing acetonitrile and water (4:1, V/V). In addition, the solution for the positive- ion mode contained 0.1% formic acid. Prior to running through the mass spectrometer, the frozen samples were taken out of − 80 °C and were dried at room temperature inside the vacuum dryer for 30 min. Samples were then placed on an XY translation stage to enable continuously scanning of the sample line by line at a constant rate of Vx = 0.2 mm/s. The distance between each scanning line (Dy) was set to be 0.1 mm. The entire scanning area was 10 mm by 10 mm. © 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. He et al. Cancer Cell International (2022) 22:366 Page 2 of 16 Page 2 of 16 approved by the ethics committees of the Shenzhen Peo- ple’s Hospital (LL-KY-2021723). Informed consent about the study was collected from all participants. The flow chart of the experiment can be found in Fig. 1. Clinical data of the liver cirrhosis (LC) and HCC patients listed in Table 1 were collected one day before the operation. Sample tissues from the three different conditions of liv- ers were collected, frozen, and sectioned to prepare for Mass Spectrometry Imaging (MSI). The three samples were cancerous tissue, liver cirrhosis, and healthy liver tissue. Following the acquisition of the samples, tissues were flash-frozen in liquid nitrogen and stored at − 80 °C until sectioning. The day prior to sectioning, samples were moved from − 80 to − 20 °C to de-freeze overnight. Then, sectioning was carried out using the Leica CM1950 cryostats. The sample slices were thaw-mounted to superfrost plus positively charged slides (Thermo Fisher) and stored at − 80 °C until the MSI experiment. One of the tissue sections from each sample was stained with hematoxylin and eosin (HE) staining solution for histo- logical analysis. control, dysregulations of apoptosis and NF-κB pathway, etc. that reflect HCC progression [11, 13, 16, 29, 37]. Thus, researchers have been trying to map out the cel- lular causes of the progression from hepatitis B infection to liver cancer. AFADESI‑MSI B h i i The spray gas press was set at 0.6 MPa, the capillary temperature was at 350 °C, the spray gas flow rate was at 5 μL/min, and the extracting gas flow However, the progression of the disease usually takes various pathways and different paces in individual patients and can be affected by genetic suscep- tibilities. Therefore, the inter-and intra-personal hetero- geneity of the disease has made the identification of the exact mechanisms very challenging. Recently, with the advancement of new technologies, the identification of HCC-related biomarkers has become a promising way to decode the disease progression mech- anisms. Specifically, there has been increasing interest in metabolomics, or the study of metabolites. Global metabolomics with liquid or gas chromatography has revealed alteration in various metabolic pathways such as the TCA cycle, glycolysis, lipid synthesis, etc. [9]. Notice- ably, metabolites such as Glypican-3 [3], monounsatu- rated fatty acids, and the ratio between polyunsaturated fatty acids omega-3 and omega-6 [10] were found to be important biomarkers for the progression of liver cancer. However, global metabolomics only looks at the average of the tissues. Furthermore, most of the previous studies regarding liver cancer look at blood samples for finding key metabolites. These methods have limitations as they may omit important regional variations of the metabo- lites that mark liver disease progression or the bounda- ries of diseased tissues. Tissue collection and preparation All the sample tissues were surgery remnants acquired at Shenzhen People’s Hospital in 2021. This study was He et al. Tissue collection and preparation Cancer Cell International (2022) 22:366 Page 3 of 16 HC LC HCC Sample collection AFADESI-MSI Data analysis MSI imaging −2500 0 2500 −5000 −2500 0 2500 PC1(50.5%) PC2(21.4%) LC HCC HC PLS−DA −2000 0 2000 4000 −2500 0 2500 5000 PC1(51.3%) PC2(19.7%) HCC−Cancer HCC−Fibrosis HCC−PL HCC−PC HCC−NT HCC−FT LC−Fibrosis LC−PL HC−HL PLS−DA Multivariate analysis 99 Cancer 100 Fibrosis 88 FT 20 NT 88 PC 93 PL 93 CV 213 45 47 57 7 196 57 6 LC HCC HC p−value=0.05 0 10 20 30 −15 −10 −5 0 5 10 15 log2(FC) −log10(P−value) down−regulated not−significant up−regulated Volcano Plot GZ−11−ALL−pos−72−12 GZ−11−ALL−pos−26−74 GZ−11−ALL−pos−64−52 GZ−11−ALL−pos−17−88 GZ−11−ALL−pos−109−91 GZ−11−ALL−pos−60−42 GZ−11−ALL−pos−19−42 GZ−11−ALL−pos−95−58 GZ−11−ALL−pos−27−66 GZ−11−ALL−pos−93−49 GZ−11−ALL−pos−37−43 GZ−11−ALL−pos−65−20 GZ−11−ALL−pos−104−94 GZ−11−ALL−pos−97−90 GZ−11−ALL−pos−82−19 GZ−11−ALL−pos−45−59 GZ−11−ALL−pos−11−55 GZ−11−ALL−pos−105−91 GZ−11−ALL−pos−40−29 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GZ−14−ALL−pos−24−50 GZ−14−ALL−pos−33−16 GZ−14−ALL−pos−31−24 GZ−14−ALL−pos−18−35 GZ−14−ALL−pos−24−9 GZ−14−ALL−pos−29−23 GZ−14−ALL−pos−11−39 GZ−14−ALL−pos−19−34 GZ−14−ALL−pos−34−18 GZ−14−ALL−pos−25−43 GZ−14−ALL−pos−9−38 GZ−14−ALL−pos−28−49 GZ−14−ALL−pos−12−38 GZ−14−ALL−pos−29−47 GZ−14−ALL−pos−18−18 GZ−14−ALL−pos−26−43 GZ−14−ALL−pos−14−35 GZ−14−ALL−pos−39−27 GZ−14−ALL−pos−38−26 GZ−14−ALL−pos−34−22 GZ−14−ALL−pos−29−22 GZ−14−ALL−pos−19−10 GZ−14−ALL−pos−16−47 GZ−14−ALL−pos−25−9 GZ−14−ALL−pos−22−28 GZ−14−ALL−pos−18−33 GZ−14−ALL−pos−34−23 GZ−14−ALL−pos−16−12 GZ−14−ALL−pos−10−32 GZ−14−ALL−pos−30−28 GZ−14−ALL−pos−28−20 GZ−14−ALL−pos−15−25 GZ−14−ALL−pos−24−51 GZ−14−ALL−pos−27−45 GZ−14−ALL−pos−15−47 GZ−14−ALL−pos−31−14 GZ−14−ALL−pos−37−28 GZ−14−ALL−pos−26−45 GZ−14−ALL−pos−25−24 GZ−14−ALL−pos−31−23 GZ−14−ALL−pos−24−45 GZ−14−ALL−pos−16−28 GZ−14−ALL−pos−19−26 GZ−14−ALL−pos−32−20 GZ−14−ALL−pos−21−45 GZ−14−ALL−pos−24−29 GZ−14−ALL−pos−28−34 GZ−14−ALL−pos−17−29 GZ−14−ALL−pos−38−29 GZ−14−ALL−pos−17−41 GZ−14−ALL−pos−29−16 GZ−14−ALL−pos−27−44 GZ−14−ALL−pos−33−40 GZ−14−ALL−pos−12−48 GZ−14−ALL−pos−16−19 GZ−14−ALL−pos−31−44 GZ−14−ALL−pos−29−39 GZ−14−ALL−pos−22−27 GZ−14−ALL−pos−25−29 GZ−14−ALL−pos−31−25 GZ−14−ALL−pos−33−27 GZ−14−ALL−pos−20−50 GZ−14−ALL−pos−30−13 GZ−14−ALL−pos−28−39 GZ−14−ALL−pos−9−46 GZ−14−ALL−pos−17−23 GZ−14−ALL−pos−33−19 GZ−14−ALL−pos−22−13 GZ−14−ALL−pos−26−29 GZ−14−ALL−pos−29−33 GZ−14−ALL−pos−32−39 GZ−14−ALL−pos−23−29 GZ−14−ALL−pos−15−23 GZ−14−ALL−pos−19−22 GZ−14−ALL−pos−30−27 GZ−14−ALL−pos−25−37 GZ−14−ALL−pos−17−19 GZ−14−ALL−pos−34−34 GZ−14−ALL−pos−29−37 GZ−14−ALL−pos−14−33 GZ−14−ALL−pos−28−18 GZ−14−ALL−pos−11−30 PE(16:1(9Z)/22:5(7Z,10Z,13Z,16Z,19Z)) D−Proline 2−Furanmethanol N−Acetyl−L−glutamic acid Zeatin Anabasine N−Acetyl−L−glutamate 5−semialdehyde 4−tert−Butylphenol p−Mentha−1,3,5,8−tetraene Propionylcarnitine 5−Hydroxytryptophol 4−Trimethylammoniobutanoic acid Fosfomycin N−Methylhydantoin (±)−2−Methylbutanal L−Carnitine Norvaline Spermidine D−Tagatose 3−(3,4−dihydroxyphenyl)−2−oxopropanoic acid Beta−Guanidinopropionic acid N−Methylnicotinamide Butyrylcarnitine 4−Hydroxycyclohexylcarboxylic acid gamma−Glutamylhistidine Histidylphenylalanine Epsilon−caprolactam Lipoamide Diisobutyl phthalate Cyclohexylamine Amphetamine 1−Butylamine 2−Methoxyestradiol 2−Piperidinone N−Formyl−L−aspartate 4−Vinylphenol sulfate 3−Aminopropionaldehyde Carbamoyl phosphate 4−Pyridoxic acid 3−Hydroxysuberic acid 6−Methylmercaptopurine Isoxanthopterin 5−Methylthioribose L−Acetylcarnitine Debrisoquine Histamine Taurine Iminoaspartic acid Alanylglycine S−(3−Oxo−3−carboxy−n−propyl)cysteine 3−(3−Hydroxyphenyl)propanoic acid Tetrahydrobiopterin 2−Hydroxydecanedioic acid 5−L−Glutamyl−taurine Ferulic acid Valylmethionine Class Class GZ−11−All GZ−14−All −6 −4 −2 0 2 4 6 Metabolites detection Key metabolites Metabolites distribution HBV progression HC LC HCC Fig. Tissue collection and preparation 1 Schematics of using the AFADESI-MSI method to detect spatial distributions of metabolites in the healthy control (HC), HBV relative liver cirrhosis (LC) and liver cancer (HCC) samples Sample collection HC LC HCC Data analysis AFADESI-MSI Metabolites detection Metabolites detection MSI imaging MSI imaging Multivariate analysis Metabolites detection Key metabolites Metabolites distribution Fig. 1 Schematics of using the AFADESI-MSI method to detect spatial distributions of metabolites in the healthy control (HC), HBV relative liver cirrhosis (LC) and liver cancer (HCC) samples Fig. 1 Schematics of using the AFADESI-MSI method to detect spatial distributions of metabolites in the healthy control ( cirrhosis (LC) and liver cancer (HCC) samples rate was at 45 L/min. Additional parameters of the MSI experiment can be found in Additional file 2: Table S1. identification (ppm < 5). Differential metabolites were identified based on Student’s t test and fold change analysis. Clinical characteristics of the participants Clinical characteristics of the participants Ion intensity across each position was outputted as raw data. All raw data files were converted into “.cdf” for- mat, and data analysis was conducted using the cus- tom-developed imaging software, MassImager [12], for image reconstruction. Spatial shrunken centroid cluster- ing (based on K-Means clustering) was then performed to generate K-Means plot. These plots were compared to H&E staining images to extract the region of interest (ROI) and construct the MS profiles. Ions were com- pared to the online database HMDB (https://hmdb.ca/) and SMPD (https://www.smpdb.ca/) for metabolites Clinical data of the LC and HCC patients were collected for comparison, and significant differences were shown between many of the patients’ measurements and the reference levels. Relevant clinical data for the patients are summarized in Table 1. Particularly, bilirubin and total bile acid levels were greatly increased, reflecting the patients’ compromised ability to break down bile. In addition, prothrombin time and INR also increased in both LC and HCC patients, suggesting a reduced pro- duction of blood-clotting proteins. Generally, there was a He et al. Clinical characteristics of the participants Cancer Cell International (2022) 22:366 Page 4 of 16 Table 1 The clinical data for liver cirrhosis and HCC participates Averages are expressed as mean ± SEM TP, total protein; ALB, albumin; GLO, globulin; A/G, albumin/globulin ratio; PA, prealbumin; TB, total bilirubin; GLD, glutamate dehydrogenase; DB, direct bilirubin; ID, indirect bilirubin; TBA, total bile acid; ALT, alanine transaminase; AST, aspartate transaminase; GGT, gamma-glutamyl transferase; ALP, alkaline phosphatase; CHE, cholinesterase; LDH, lactate dehydrogenase; PT, prothrombin time; INR, international normalized ratio; APTT, activated partial thromboplastin time; FIB, fibrinogen; TT, thrombin time; AT III, antithrombin III; D-DIC, D-dimer; AMON, ammonia; HBsAg, hepatitis B virus surface antigen; HBsAb, hepatitis B virus surface antibody; HBeAg, hepatitis B virus e antigen; HBeAb, hepatitis B virus e antibody; HBcAb, hepatitis B virus core antibody; MELD, model of end stage liver disease a Reference ranges may vary with patient’s sex, age, pregnancy, etc., and may be different depending on materials and methods used Measurements LC HCC Average Referencea Change TP (g/L) 52.70 58.10 55.40 ± 2.70 63.00–79.00 ↓ ALB (g/L) 34.80 39.80 37.30 ± 2.50 35.00–50.00 – GLO (g/L) 17.90 18.30 18.10 ± 0.20 20.00–35.00 ↓ A/G 1.94 2.17 2.06 ± 0.12 1.10–2.50 – PA (mg/L) 83.00 152.00 117.50 ± 34.50 150.00–350.00 ↓ TB (μmol/L) 41.30 194.80 118.10 ± 76.75 1.71–20.50 ↑ GLD (U/L) 9.32 7.00 8.16 ± 1.16 < 7.00 ↑ DB (μmol/L) 11.00 70.00 40.50 ± 29.50 < 5.10 ↑ ID (μmol/L) 30.30 124.80 77.55 ± 47.25 3.40–12.00 ↑ TBA (μmol/L) 140.00 136.60 138.30 ± 1.70 < 10.00 ↑ ALT (U/L) 31.00 37.00 34.00 ± 3.00 7.00–55.00 – AST (U/L) 36.00 52.00 44.00 ± 8.00 8.00–48.00 – GGT (U/L) 20.00 33.00 26.50 ± 6.50 8.00–61.00 – ALP (U/L) 71.00 52.00 61.50 ± 9.50 40.00–129.00 – CHE (U/L) 3704.00 2987.00 3346.00 ± 358.50 8k–18k ↓ LDH (U/L) 216.00 933.00 574.50 ± 358.50 140.00–280.00 ↑ PT (S) 18.70 23.60 21.15 ± 2.45 11.00–13.50 ↑ PT% (%) 51.00 36.00 43.50 ± 7.50 100.00 ↓ PT INR 1.58 2.15 1.87 ± 0.29 0.80–1.10 ↑ APTT (S) 55.00 40.80 47.90 ± 7.10 21.00–35.00 ↑ FIB (g/L) 1.53 1.54 1.54 ± 0.01 2.00–4.00 ↓ TT (s) 18.10 21.70 19.90 ± 1.80 14.00–19.00 ↑ AT III (%) 39.00 41.00 40.00 ± 1.00 80.00–130.00 ↓ D-DIC (μg/mL) 0.22 > 20 10.11 ± 9.89 0.10–0.25 ↑ AMON (μmol/L) 133.00 101.00 117.00 ± 16.00 11.00–32.00 ↑ HBsAg 20.91 (+) 31.93 (+) 26.42 ± 5.51 N/A N/A HBsAb 0 (−) 0.99 (−) 0.50 ± 0.50 N/A N/A HBeAg 0.377 (+) 0.04 (−) 0.21 ± 0.17 N/A N/A HBeAb 1.54 (−) 0.01 (+) 0.78 ± 0.77 N/A N/A HBcAb 5.37 (+) 0.21 (−) 2.79 ± 2.58 N/A N/A Child–Pugh 9 13 11.00 ± 2.00 N/A N/A MELD 32 44 38.00 ± 6.00 N/A N/A Table 1 The clinical data for liver cirrhosis and HCC participates Averages are expressed as mean ± SEM TP, total protein; ALB, albumin; GLO, globulin; A/G, albumin/globulin ratio; PA, prealbumin; TB, total bilirubin; GLD, glutamate dehydrogenase; DB, direct bilirubin; ID, indirect bilirubin; TBA, total bile acid; ALT, alanine transaminase; AST, aspartate transaminase; GGT, gamma-glutamyl transferase; ALP, alkaline phosphatase; CHE, cholinesterase; LDH, lactate dehydrogenase; PT, prothrombin time; INR, international normalized ratio; APTT, activated partial thromboplastin time; FIB, fibrinogen; TT, thrombin time; AT III, antithrombin III; D-DIC, D-dimer; AMON, ammonia; HBsAg, hepatitis B virus surface antigen; HBsAb, hepatitis B virus surface antibody; HBeAg, hepatitis B virus e antigen; HBeAb, hepatitis B virus e antibody; HBcAb, hepatitis B virus core antibody; MELD, model of end stage liver disease a Reference ranges may vary with patient’s sex, age, pregnancy, etc., and may be different depending on materials and methods used scores reflected such disease progression. (See figure on next page.) Fig. 2 Total metabolites detected by AFADESI-MSI method in HC, LC and HCC tissue samples under positive ionization mode. a HE and MSI diagrams of HC, LC and HCC whole samples. b PLS-DA comparison of the AFADESI-MSI data. c, e, g Total metabolites detected by AFADESI-MSI method in HC, LC and HCC samples, respectively. d, f, h Differences between the detected metabolites under positive and negative ionization mode in HC, LC and HCC samples, respectively TP, total protein; ALB, albumin; GLO, globulin; A/G, albumin/globulin ratio; PA, prealbumin; TB, total bilirubin; GLD, glutamate dehydrogenase; DB, direct bilirubin; ID, indirect bilirubin; TBA, total bile acid; ALT, alanine transaminase; AST, aspartate transaminase; GGT, gamma-glutamyl transferase; ALP, alkaline phosphatase; CHE, cholinesterase; LDH, lactate dehydrogenase; PT, prothrombin time; INR, international normalized ratio; APTT, activated partial thromboplastin time; FIB, fibrinogen; TT, thrombin time; AT III, antithrombin III; D-DIC, D-dimer; AMON, ammonia; HBsAg, hepatitis B virus surface antigen; HBsAb, hepatitis B virus surface antibody; HBeAg, hepatitis B virus e antigen; HBeAb, hepatitis B virus e antibody; HBcAb, hepatitis B virus core antibody; MELD, model of end stage liver disease a Reference ranges may vary with patient’s sex, age, pregnancy, etc., and may be different depending on materials and methods used Clinical characteristics of the participants 2 (See legend on previous page.) HCC 0 20 40 60 80 100 0 20 40 60 80 100 120 a −2500 0 2500 −5000 −2500 0 2500 PC1(50.5%) PC2(21.4%) LC HCC HC PLS−DA HC LC HCC Alkaloids and derivatives Benzenoids 36 Hydrocarbons Lipids and lipid-like molecules 153 Nucleosides, nucleotides, and analogues 8 Organic acids and derivatives 78 Organic nitrogen compounds 11 Organic oxygen compounds 31 Organoheterocyclic compounds 55 Organosulfur compounds Phenylpropanoids and 7 Organohalogen copounds 1 polyketides 0 12 0 111 4 16 2 9 1 10 1 1 2 25 1 76 5 65 9 26 0 48 2 7 120 100 80 40 20 0 20 40 60 80 100 100 80 60 40 20 0 20 40 60 80 100 120 Alkaloids and derivatives Benzenoids Hydrocarbons Lipids and lipid-like molecules Nucleosides, nucleotides, and analogues Organic acids and derivatives Organic nitrogen compounds Organic oxygen compounds Organohalogen compounds Organoheterocyclic compounds Organosulfur compounds Phenylpropanoids and polyketides Negative mode: 167 m/z Postive mode: 266 m/z Alkaloids and derivatives 4 Benzenoids 36 Hydrocarbons 1 Lipids and lipid-like molecules 177 Nucleosides, nucleotides, and analogues 13 Organic acids and derivatives 79 Organic nitrogen compounds 12 Organic oxygen compounds 34 Organoheterocyclic compounds 58 Organosulfur compounds 1 Phenylpropanoids and polyketides 10 1 1 10 0 108 6 24 2 11 0 13 1 2 3 27 1 101 8 60 11 28 0 48 0 8 120 100 80 60 40 20 0 20 40 60 80 100 100 80 60 40 20 0 20 40 60 80 100 120 Alkaloids and derivatives Benzenoids Hydrocarbons Lipids and lipid-like molecules Nucleosides, nucleotides, and analogues Organic acids and derivatives Organic nitrogen compounds Organic oxygen compounds Organohalogen compounds Organoheterocyclic compounds Organosulfur compounds Phenylpropanoids and polyketides Negative mode: 178 m/z Postive mode: 295 m/z Alkaloids and derivatives 2 Benzenoids 41 Hydrocarbons 1 Lipids and lipid-like molecules 167 Nucleosides, nucleotides, Organic acids and derivatives 107 Organic nitrogen compounds 15 Organic oxygen compounds 41 Organoheterocyclic compounds 72 Organosulfur compounds 3 Phenylpropanoids and polyketides 10 Organohalogen copounds 3 0 10 0 110 4 24 2 11 1 12 1 2 2 33 1 89 8 90 13 34 3 63 3 9 120 100 80 60 40 20 0 20 40 60 80 100 100 80 60 40 20 0 20 40 60 80 100 120 Alkaloids and derivatives Benzenoids Hydrocarbons Lipids and lipid-like molecules Nucleosides, nucleotides, and analogues Organic acids and derivatives Organic nitrogen compounds Organic oxygen compounds Organohalogen compounds Organoheterocyclic compounds Organosulfur compounds Phenylpropanoids and polyketides Negative mode: 177 m/z Postive mode: 348 m/z b c d e h g f Intensity 2 2 10000 8000 6000 4000 10 20 30 40 50 0 10 20 30 40 50 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 120 a −2500 0 2500 −5000 −2500 0 PC1(50.5% PC2(21.4%) PLS−DA HC LC HCC Phenylpropanoids and b Intensity 10000 8000 6000 4000 10 20 30 40 50 0 10 20 30 40 50 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 120 a HC LC Intensity 10000 8000 6000 4000 10 20 30 40 50 0 10 20 30 40 50 0 20 40 60 80 100 0 20 40 60 80 100 a − HC LC HCC Phenylpropanoids and b Intensity 10000 8000 6000 4000 10 20 30 40 50 0 10 20 30 40 50 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 120 −2500 0 2500 −5000 −2500 0 2500 PC1(50.5%) PC2(21.4%) LC HCC HC PLS−DA b Alkaloids and derivatives Benzenoids 36 Hydrocarbons Lipids and lipid-like molecules 153 Nucleosides, nucleotides, and analogues 8 Organic acids and derivatives 78 Organic nitrogen compounds 11 Organic oxygen compounds 31 Organoheterocyclic compounds 55 Organosulfur compounds Phenylpropanoids and 7 Organohalogen copounds 1 polyketides 1 c 2 2 0 12 0 111 4 16 2 9 1 10 1 1 2 25 1 76 5 65 9 26 0 48 2 7 120 100 80 40 20 0 20 40 60 80 100 100 80 60 40 20 0 20 40 60 80 100 120 Alkaloids and derivatives Benzenoids Hydrocarbons Lipids and lipid-like molecules Nucleosides, nucleotides, and analogues Organic acids and derivatives Organic nitrogen compounds Organic oxygen compounds Organohalogen compounds Organoheterocyclic compounds Organosulfur compounds Phenylpropanoids and polyketides Negative mode: 167 m/z Postive mode: 266 m/z d d c 1 10 0 108 6 24 2 11 0 13 1 2 3 27 1 101 8 60 11 28 0 48 0 8 120 100 80 60 40 20 0 20 40 60 80 100 100 80 60 40 20 0 20 40 60 80 100 120 Alkaloids and derivatives Benzenoids Hydrocarbons Lipids and lipid-like molecules Nucleosides, nucleotides, and analogues Organic acids and derivatives Organic nitrogen compounds Organic oxygen compounds Organohalogen compounds Organoheterocyclic compounds Organosulfur compounds Phenylpropanoids and polyketides Negative mode: 178 m/z Postive mode: 295 m/z f Alkaloids and derivatives 4 Benzenoids 36 Hydrocarbons 1 Lipids and lipid-like molecules 177 Nucleosides, nucleotides, and analogues 13 Organic acids and derivatives 79 Organic nitrogen compounds 12 Organic oxygen compounds 34 Organoheterocyclic compounds 58 Organosulfur compounds 1 Phenylpropanoids and polyketides 10 e f e h Alkaloids and derivatives 2 Benzenoids 41 Hydrocarbons 1 Lipids and lipid-like molecules 167 Nucleosides, nucleotides, and analogues 13 Organic acids and derivatives 107 Organic nitrogen compounds 15 Organic oxygen compounds 41 Organoheterocyclic compounds 72 Organosulfur compounds 3 Phenylpropanoids and polyketides 10 Organohalogen copounds 3 g Fig. Clinical characteristics of the participants Lastly, both the LC and HCC patients were tested for HBV infection, and both patients either had past or active infection shown by their positive antibody tests. decrease in protein levels for both patients. Furthermore, almost all the observed changes were more severe in the HCC patient than the LC patient, showing a progres- sion in liver disease severity. The Child-Pugh and MELD He et al. Clinical characteristics of the participants Cancer Cell International (2022) 22:366 Page 5 of 16 a −2500 0 2500 −5000 −2500 0 2500 PC1(50.5%) PC2(21.4%) LC HCC HC PLS−DA HC LC HCC Alkaloids and derivatives Benzenoids 36 Hydrocarbons Lipids and lipid-like molecules 153 Nucleosides, nucleotides, and analogues 8 Organic acids and derivatives 78 Organic nitrogen compounds 11 Organic oxygen compounds 31 Organoheterocyclic compounds 55 Organosulfur compounds Phenylpropanoids and 7 Organohalogen copounds 1 polyketides 0 12 0 111 4 16 2 9 1 10 1 1 2 25 1 76 5 65 9 26 0 48 2 7 120 100 80 40 20 0 20 40 60 80 100 100 80 60 40 20 0 20 40 60 80 100 120 Alkaloids and derivatives Benzenoids Hydrocarbons Lipids and lipid-like molecules Nucleosides, nucleotides, and analogues Organic acids and derivatives Organic nitrogen compounds Organic oxygen compounds Organohalogen compounds Organoheterocyclic compounds Organosulfur compounds Phenylpropanoids and polyketides Negative mode: 167 m/z Postive mode: 266 m/z Alkaloids and derivatives 4 Benzenoids 36 Hydrocarbons 1 Lipids and lipid-like molecules 177 Nucleosides, nucleotides, and analogues 13 Organic acids and derivatives 79 Organic nitrogen compounds 12 Organic oxygen compounds 34 Organoheterocyclic compounds 58 Organosulfur compounds 1 Phenylpropanoids and polyketides 10 1 1 10 0 108 6 24 2 11 0 13 1 2 3 27 1 101 8 60 11 28 0 48 0 8 120 100 80 60 40 20 0 20 40 60 80 100 100 80 60 40 20 0 20 40 60 80 100 120 Alkaloids and derivatives Benzenoids Hydrocarbons Lipids and lipid-like molecules Nucleosides, nucleotides, and analogues Organic acids and derivatives Organic nitrogen compounds Organic oxygen compounds Organohalogen compounds Organoheterocyclic compounds Organosulfur compounds Phenylpropanoids and polyketides Negative mode: 178 m/z Postive mode: 295 m/z Alkaloids and derivatives 2 Benzenoids 41 Hydrocarbons 1 Lipids and lipid-like molecules 167 Nucleosides, nucleotides, and analogues 13 Organic acids and derivatives 107 Organic nitrogen compounds 15 Organic oxygen compounds 41 Organoheterocyclic compounds 72 Organosulfur compounds 3 Phenylpropanoids and polyketides 10 Organohalogen copounds 3 0 10 0 110 4 24 2 11 1 12 1 2 2 33 1 89 8 90 13 34 3 63 3 9 120 100 80 60 40 20 0 20 40 60 80 100 100 80 60 40 20 0 20 40 60 80 100 120 Alkaloids and derivatives Benzenoids Hydrocarbons Lipids and lipid-like molecules Nucleosides, nucleotides, and analogues Organic acids and derivatives Organic nitrogen compounds Organic oxygen compounds Organohalogen compounds Organoheterocyclic compounds Organosulfur compounds Phenylpropanoids and polyketides Negative mode: 177 m/z Postive mode: 348 m/z b c d e h g f Intensity 2 2 10000 8000 6000 4000 10 20 30 40 50 0 10 20 30 40 50 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 120 Fig. Identification of ROIs and differential metabolites within sub‑regions Identification of ROIs and differential metabolites within sub‑regions i AFADESI-MSI was performed on the sample tissues and the MSI profiles were constructed to analyze the global metabolic profiles. Under the positive ion mode, AFADESI- MSI was able to detect ions ranging from m/z 70–800. Both the H&E stain images and the example MSI diagrams revealed that all three liver tissues have considerable intra- and inter-sample heterogeneity. Figure 2a shows the over- lay MS images of all the detected metabolites. Based on the differential regional intensities of the ions, all three samples have considerable intra-sample variations in terms of the spatial metabolic profiles. There appeared to be meaning- ful patterns in the overall distributions of metabolites that invited us to cluster the profiles. The k-mean diagrams in Additional file 1: Fig. S1e&f then display clustering results of the metabolites and reveal that the sample tissues can be separated into different regions, possibly representing dif- ferent tissue types. From the MS images, it appeared that the three samples differed between each other, too. The partial least squares-determinant analysis (Fig. 2b) was able to separate the three tissue types (HC, LC, HCC) based on their differential metabolites. To make sense of the sepa- ration, the relative abundances of the different classes of metabolites were first examined. Overall, the compositions of the metabolites being detected in HC, LC, and HCC samples were similar, with lipids and lipid-like molecules, organic acids and derivatives, and organoheterocyclic com- pounds being the three most abundant types of metabolites (Fig. 2c, e, g). However, compared to the other two samples, lipid and lipid-like molecules had larger percentage among total metabolites in the LC sample (Fig. 2e) while organic acids and derivatives were found to have a larger abun- dance in the HCC sample (Fig. 2g). Therefore, the global metabolic profiles of the three samples suggest not only the regional diversity of liver tissues but also the metabolic dif- ferences between the three liver conditions. A total of six regions of interest (ROI) were identified by overlaying the H&E and the MSI images of the HCC sam- ple. Their region-specific metabolic profiles were analyzed to reveal any key differential metabolites. Figure 3a displays the H&E stain and MSI images for the entire HCC sam- ple and the zoomed-in images for the six ROIs: cancerous region, pseudo lobule (PL), necrotic tissue (NT), fibro- sis, pre-cancer region (PC), and fatty tissue (FT). Fig. 3 The alterations of metabolites’ spatial distributions in sub-regions of HCC sample under positive ionization mode. a HE and MSI images of different sub-regions in HCC including cancer, pseudo lobule (PL), pre-cancer (PC), fibrosis, necrotic tissue (NT), and fatty tissue (FT). b PLS-DA analysis for HCC sub-regions. c Number of metabolites detected from different HCC sub-regions. d Heatmap of significantly differentiated metabolites based on variable importance of projection > 1 (VIP > 1). e KEGG analysis of key altered metabolic pathways between the sub-regions of HCC. f Sample time series analyses of key metabolite expressions in all sub-regions of HCC (See figure on next page.) Clinical characteristics of the participants 2 (See legend on previous page.) g Fig. 2 (See legend on previous page.) Page 6 of 16 He et al. Cancer Cell International (2022) 22:366 He et al. Cancer Cell International (2022) 22:366 Identification of ROIs and differential metabolites within sub‑regions Particu- larly, the cancerous region and the necrotic tissue had the most distinct MSI profiles whereas the other regions had more comparable ion intensity patterns. The cancer region contained an overall abundance of metabolites while the necrotic region showed a lack of metabolites. The results for negative-ion mode MSI are shown in Additional file 1: Fig. S2. Under this imaging mode, the heterogeneity within the sub-regions is less visible, but there are still detect- able variabilities of the metabolite distribution patterns. Here, the pseudo lobule and the necrotic tissue appear to have the most distinct profiles. The PLS-DA result shows a similar trend, with the NT, PL, and cancer being the three most separable sub-regions within the sample (Fig. 3b). However, as seen in Fig. 3c and Additional file 1: Fig. S2c, the NT region possessed a distinct property compared to the PL and cancerous regions as having much less metabo- lites overall. Its separability seems to result from a simple absence of metabolites (Fig. 3d). The PL and cancerous regions, on the other hand, differed primarily in their metabolite distributions. The heatmap in Fig. 3d shows the overall trend of the metabolite distributions where that of the PL region and of the cancerous region seems to be complementary. Specifically, the metabolites that were more abundant in the cancer region, such as D-alanine were among the least abundant in the PL region. Metabo- lites such as L-carnitine, which were more abundant in the PL region, were much less abundant in the cancer region. The PL and cancer regions also shared some simi- larities as they both had an increase in some of the lipids and phospholipids compared to other regions. Particularly, the PL regions showed the highest abundance of phos- phocholine molecules such as PC (14:0/20:2(11Z, 14Z)) and PC (22:5(3Z, 7Z, 10Z, 13Z, 16Z)/16:1(9Z)). The fibro- sis region shared a similar profile as the PL region, both To check the consistency of the results, negative ion mode data was compared. Generally, the relative abun- dance of each class of metabolite was consistent (Fig. 2d, f, h). Yet, it was also noticeable that the negative ion mode resulted in a lower sensitivity compared to the positive ion mode for all classes of molecules other than lipid and lipid- like molecule. Hence, positive ion mode data were used in later analyses. He et al. Identification of ROIs and differential metabolites within sub‑regions Cancer Cell International (2022) 22:366 Page 7 of 16 Fig. 3 (See legend on previous page.) Fig. 3 (See legend on previous page.) Fig 3 (See legend on previous page ) Fig. 3 (See legend on previous page.) He et al. Cancer Cell International (2022) 22:366 Page 8 of 16 Page 8 of 16 metabolite distributions. Many metabolites showed a continuous increase or decrease in abundances from the HC to the LC to the cancer central veins. How- ever, some of the phospholipids and fatty acids were most abundant in the LC central vein and subsequently decreased in intensities in both the cancer and the HC central vein regions. When looking into the specific altered pathways, amino acid and fatty acid biosynthesis and metabolic pathways appeared to be the most altered pathways under positive and negative ion mode, respec- tively (Fig. 4c and Additional file 1: Fig. S3d). Time series analysis then display some of the characteristic trends of changes in distribution of key metabolites around the central vein region during disease progression. These trends match the ones shown in the heatmap. Numerous metabolites, including spermidine, taurine, histamine, ferulic acid, benzoic acid, and deoxyuridine experienced an overall continuous increase in concentration from the HC central vein to the HCC central vein. Other metabo- lites, such as isopropylmaleic acid, palmitic acid, and ana- basine, showed a reverse trend of continuous decrease in concentration from the HC to HCC central vein (Fig. 4d and e). In Fig. 4e, three typical metabolites were cho- sen to represent their clusters. The MS images and the quantification plots demonstrate that spermidine (m/z 146.165) and ferulic acid (m/z 233.0602) increased in intensities in the central vein regions as the disease pro- gress. On the other hand, anabasine (m/z 163.1127) con- tinuously decreased in the central vein region. Therefore, non-cancerous regions experience changes in metabo- lites distribution during pathological changes of the liver. demonstrated the same trends of change compared to the normal tissues. However, the PL regions showed a progres- sion of such changes. Compared to all the diseased tissues, subregions PC and FT showed highly similar overall meta- bolic profiles, with PC tissues having higher abundance of a few amino acids, phospholipids, and carboxylic acids. (See figure on next page.) Fig. 4 Metabolites’ spatial distributions in the non-cancer regions (CV) of the HC, LC and HCC samples under positive ionization mode. a HE and MSI images of the CV regions in the HC, LC and HCC samples. b PLS-DA analysis of the CV regions. c KEGG analysis of key altered metabolic pathways in the CV non-cancer regions. d Sample time series analysis of key metabolite expressions of the non-cancer regions. e Examples of key metabolites’ spatial expressions in the CV regions Identification of ROIs and differential metabolites within sub‑regions p p p y Having found out all the differential metabolites in each sub-region, a KEGG pathway enrichment plot was constructed to categorize the metabolites in terms of the pathways they are part of. Under the positive ion mode, the most significantly altered pathways as identified by the differential metabolites were the amino acid metabo- lism pathways such as the beta-alanine metabolism and the arginine and proline metabolism pathways (Fig. 3e). In addition, the choline metabolism in cancer pathway was also significantly altered. Under the negative ion mode, almost all the pathways identified were fatty acid biosynthesis and metabolism pathways (Additional file 1: Fig. S2e). Lastly, time series analysis was performed on metabolites from all the sub-regions of the HCC sample. The changes in the key metabolites along disease progression were reflected by examining the changes in their distributions in the fibrosis, PL, PC, and cancer regions. Here, differential metabolites with similar trend of changes were grouped into one cluster (Fig. 3f). Certain metabolites, such as d-proline and creatine, showed a general decrease from fibrosis to cancer regions but were particularly high in the PL region. Metabolites such as spermidine, isoleucylpro- line, deoxyuridine showed continuous decrease from fibro- sis to cancer regions. Metabolic alterations of cancer‑related regions Cancer Cell International (2022) 22:366 Page 9 of 16 b −2000 −1000 0 1000 2000 −2500 0 2500 PC1(55%) PC2(16.2%) LC-CV HCC−CV HC−CV PLS−DA d e c Cluster 1 m/z=146.165 Spermidine LC HCC LC HCC HC HCC LC HC 0 10000 20000 30000 40000 Intensity Cluster 8 m/z=137.0456 Hypoxanthine LC HCC HC Cluster 13 m/z=233.0602 Ferulic acid HCC LC HC 0 5000 10000 15000 Intensity HCC LC HC 0 10000 20000 30000 Intensity a 500μm -1.0 -0.5 0.0 0.5 1.0 HCC-CV LC-CV HC-CV Expression Abundance (Z-score) 0.2 0.4 0.6 0.8 Membership contains 29 metabolites Cluster 1 contains 18 metabolites Cluster 13 -1.0 -0.5 0.0 0.5 1.0 HCC-CV LC-CV HC-CV Expression Abundance (Z-score) 0.2 0.4 0.6 0.8 Membership Asthma Pantothenate and CoA biosynthesis Ether lipid metabolism Taurine and hypotaurine metabolism Histidine metabolism Choline metabolism in cancer beta−Alanine metabolism Arginine biosynthesis Arginine and proline metabolism 0.10 0.15 0.20 RichFactor PathwayTerm Number 1 2 3 4 5 0.01 0.02 0.03 0.04 Pvalue HCC−CV LC−CV HC−CV Isopropylmaleic acid PE(20:3/20:2(11Z,14Z)) Nitronaphthalene-oxide Palmitic acid Hexadienoic acid Dihydro-5-pentyl-furanone Coenzyme Q9 2−Maleylacetate Guaiacol Sulfogalactosylceramide Dihydronaphthalene-diol Methyl-4-nitroimidazole Anabasine Sulcatone Dimethylarginine 9−Oxo−nonanoic acid 8−Methylnonenoate Hypoxanthine Cluster 8 PC(20:5(5Z,8Z,11Z,14Z,17Z)/P-18:0) 4−Isopropylbenzoic acid Spermidine Succinyladenosine Butylparaben Dimethylurea 9-Hydroperoxyoctadeca-dienoic acid O−Desmethylangolensin 1−Methylhistamine Oxypurinol Piperidine LysoPC(16:0/0:0) Taurine PE(14:0/22:5(4Z,7Z,10Z,13Z,16Z)) 1−Phenyl−1,2−propanedione 4−Pyridoxic acid Turanose 4−Hydroxybenzyl alcohol PE(14:0/22:4(7Z,10Z,13Z,16Z)) Leukotriene E4 N−Methylnicotinamide Aminopropylcadaverine Equol Histamine Ethenyl acetate Dichloromethane 4−Trimethylammoniobutanoic acid Alanylisoleucine Iminoaspartic acid HCC−CV LC−CV HC−CV Cluster 1 HC Intensity Intensity Intensity -1.0 -0.5 0.0 0.5 1.0 HCC-CV LC-CV HC-CV 0.2 0.4 0.6 0.8 Membership contains 18 metabolites Cluster 8 Expression Abundance (Z-score) −1 −0.5 0 0.5 1 HCC−CV LC−CV HC−CV Heptaprenyl diphosphate L-Palmitoylcarnitine N-Methyltyramine Niacinamide Acetyl-methoxykynurenamine S-cysteine 1-Nitro-dihydronaphthalene Deoxyuridine Thiotepa 12,13-DHOME 9,10,13-TriHOME 12,13-EpOME p-Hydroxyfelbamate 11(R)-HETE LysoPC(18:0/0:0) Benzoic acid 3-Hydroxy-N-methylcoclaurine Ferulic acid Cluster 13 Fig. Metabolic alterations of cancer‑related regions The central vein regions of all three samples were ana- lyzed separately to see any trend of metabolic alterations within the non-cancerous regions during the disease progression. The H&E and the MSI images reveal the zoomed-in morphology around the central vein region (Fig. 4a). The CV regions of all three tissues appeared to have comparable histology in the H&E stain images. Yet, MS images revealed their different metabolic pro- files. PLS-DA analysis and heatmap of metabolites dis- tributions confirmed that the central vein regions in the three samples can be clearly separated (Fig. 4b and c) based on their differential metabolic profiles. The heat- map in Fig. 3c reveals some overall trends of change in g All the lesion regions in LC and HCC as well as a healthy control region were examined to show trends of metabolic changes within the diseased areas. First, the H&E and MSI images, heatmap of differential metabo- lites, along with the PLS-DA confirm the separability of the diseased sub-regions within the three samples based on metabolic profiles and morphologies (Fig. 5a, b). According to the PLS-DA analysis, while the most significant differences existed between the three sam- ples, the sub-regions within the samples also remain separable. Particularly, the fibrosis, PL, PC, and can- cer regions of the HCC sample showed variations He et al. Metabolic alterations of cancer‑related regions 4 (See legend on previous page.) b −2000 −1000 0 1000 2000 −2500 0 2500 PC1(55%) PC2(16.2%) LC-CV HCC−CV HC−CV PLS−DA d 500μm d d c d -1.0 -0.5 0.0 0.5 1.0 HCC-CV LC-CV HC-CV Expression Abundance (Z-score) 0.2 0.4 0.6 0.8 Membership contains 29 metabolites Cluster 1 PC(20:5(5Z,8Z,11Z,14Z,17Z)/P-18:0) 4−Isopropylbenzoic acid Spermidine Succinyladenosine Butylparaben Dimethylurea 9-Hydroperoxyoctadeca-dienoic acid O−Desmethylangolensin 1−Methylhistamine Oxypurinol Piperidine LysoPC(16:0/0:0) Taurine PE(14:0/22:5(4Z,7Z,10Z,13Z,16Z)) 1−Phenyl−1,2−propanedione 4−Pyridoxic acid Turanose 4−Hydroxybenzyl alcohol PE(14:0/22:4(7Z,10Z,13Z,16Z)) Leukotriene E4 N−Methylnicotinamide Aminopropylcadaverine Equol Histamine Ethenyl acetate Dichloromethane 4−Trimethylammoniobutanoic acid Alanylisoleucine Iminoaspartic acid HCC−CV LC−CV HC−CV Cluster 1 d c Asthma Pantothenate and CoA biosynthesis Ether lipid metabolism Taurine and hypotaurine metabolism Histidine metabolism Choline metabolism in cancer beta−Alanine metabolism Arginine biosynthesis Arginine and proline metabolism 0.10 0.15 0.20 RichFactor PathwayTerm Number 1 2 3 4 5 0.01 0.02 0.03 0.04 Pvalue e Cluster 1 m/z=146.165 Cluster 8 m/z=137.0456 Cluster 13 m/z=233.0602 contains 18 metabolites Cluster 13 -1.0 -0.5 0.0 0.5 1.0 HCC-CV LC-CV HC-CV Expression Abundance (Z-score) 0.2 0.4 0.6 0.8 Membership RichFactor HCC−CV LC−CV HC−CV Isopropylmaleic acid PE(20:3/20:2(11Z,14Z)) Nitronaphthalene-oxide Palmitic acid Hexadienoic acid Dihydro-5-pentyl-furanone Coenzyme Q9 2−Maleylacetate Guaiacol Sulfogalactosylceramide Dihydronaphthalene-diol Methyl-4-nitroimidazole Anabasine Sulcatone Dimethylarginine 9−Oxo−nonanoic acid 8−Methylnonenoate Hypoxanthine Cluster 8 −CV CV CV -1.0 -0.5 0.0 0.5 1.0 HCC-CV LC-CV HC-CV 0.2 0.4 0.6 0.8 Membership contains 18 metabolites Cluster 8 Expression Abundance (Z-score) −1 −0.5 0 0.5 1 HCC−CV LC−CV HC−CV Heptaprenyl diphosphate L-Palmitoylcarnitine N-Methyltyramine Niacinamide Acetyl-methoxykynurenamine S-cysteine 1-Nitro-dihydronaphthalene Deoxyuridine Thiotepa 12,13-DHOME 9,10,13-TriHOME 12,13-EpOME p-Hydroxyfelbamate 11(R)-HETE LysoPC(18:0/0:0) Benzoic acid 3-Hydroxy-N-methylcoclaurine Ferulic acid Cluster 13 e Cluster 1 m/z=146.165 Spermidine LC HCC LC HCC HC HCC LC HC 0 10000 20000 30000 40000 Intensity Cluster 8 m/z=137.0456 Hypoxanthine LC HCC HC Cluster 13 m/z=233.0602 Ferulic acid HCC LC HC 0 5000 10000 15000 Intensity HCC LC HC 0 10000 20000 30000 Intensity contains 18 metabolites Cluster 13 -1.0 -0.5 0.0 0.5 1.0 HCC-CV LC-CV HC-CV Expression Abundance (Z-score) 0.2 0.4 0.6 0.8 Membership RichFactor HCC−CV LC−CV HC−CV Isopropylmaleic acid PE(20:3/20:2(11Z,14Z)) Nitronaphthalene-oxide Palmitic acid Hexadienoic acid Dihydro-5-pentyl-furanone Coenzyme Q9 2−Maleylacetate Guaiacol Sulfogalactosylceramide Dihydronaphthalene-diol Methyl-4-nitroimidazole Anabasine Sulcatone Dimethylarginine 9−Oxo−nonanoic acid 8−Methylnonenoate Hypoxanthine Cluster 8 −CV CV CV HC Intensity Intensity Intensity -1.0 -0.5 0.0 0.5 1.0 HCC-CV LC-CV HC-CV 0.2 0.4 0.6 0.8 Membership contains 18 metabolites Cluster 8 Expression Abundance (Z-score) −1 −0.5 0 0.5 1 HCC−CV LC−CV HC−CV Heptaprenyl diphosphate L-Palmitoylcarnitine N-Methyltyramine Niacinamide Acetyl-methoxykynurenamine S-cysteine 1-Nitro-dihydronaphthalene Deoxyuridine Thiotepa 12,13-DHOME 9,10,13-TriHOME 12,13-EpOME p-Hydroxyfelbamate 11(R)-HETE LysoPC(18:0/0:0) Benzoic acid 3-Hydroxy-N-methylcoclaurine Ferulic acid Cluster 13 Fig. Fig. 5 Metabolites’ spatial distributions in the cancer-related regions of the HC, LC and HCC samples under positive ionization mode. a HE and MSI images of the cancer-related regions in HC, LC and HCC samples. b PLS-DA analysis of the cancer-related regions. c KEGG analysis of key altered metabolic pathways in the cancer-related regions. d Sample time series analysis of key metabolite expressions in the cancer-related regions. e Examples of key metabolites’ spatial expressions in cancer-related regions (See figure on next page.) Reconstruction of altered metabolic network Reconstruction of altered metabolic network Network maps of upregulated metabolites in both the non- cancerous and cancer-related regions were constructed to investigate the key metabolites and metabolic pathways altered in liver cirrhosis to liver cancer progression. For both the non-cancerous and cancer-related regions, amino acid metabolism pathways, especially the arginine and pro- line metabolism and the alanine, aspartate, and glutamate metabolism, locate in the center of the altered metabolic networks (Fig. 6c and d). Through these pathways, most of the upregulated metabolites were connected. In addi- tion to amino acid metabolism, the glycerophospholipid metabolism pathways connected most of the other upregu- lated metabolites, particularly in the non-cancerous region. Lastly, taking the key metabolites and metabolic pathways, a schematic of an altered networks in diseased liver tissues was reconstructed (Fig. 6e), which connects amino acid synthesis and metabolism through urea cycle. The changes in intensity are marked for the key altered metabolites along this network. For example, spermidine concentration experienced a continuous decrease from HCC PL to cancer regions. Carbamoyl-P and 3-aminopropanol, on the other hand, showed a continuous increase from the healthy tis- sues to PL to cancer. Metabolic alterations of cancer‑related regions 4 (See legend on previous page.) contains 18 metabolites Cluster 13 -1.0 -0.5 0.0 0.5 1.0 HCC-CV LC-CV HC-CV Expression Abundance (Z-score) 0.2 0.4 0.6 0.8 Membership ne V -1.0 -0.5 0.0 0.5 1.0 HCC-CV LC-CV HC-CV 0.2 0.4 0.6 0.8 Membership contains 18 metabolites Cluster 8 Expression Abundance (Z-score) −1 −0.5 0 0.5 1 HCC−C LC−CV HC−CV Heptaprenyl diphosphate L-Palmitoylcarnitine N-Methyltyramine Niacinamide Acetyl-methoxykynurenamine S-cysteine 1-Nitro-dihydronaphthalene Deoxyuridine Thiotepa 12,13-DHOME 9,10,13-TriHOME 12,13-EpOME p-Hydroxyfelbamate 11(R)-HETE LysoPC(18:0/0:0) Benzoic acid 3-Hydroxy-N-methylcoclaurine Ferulic acid Cluster 13 e Fig. 4 (See legend on previous page.) Fig. 4 (See legend on previous page.) He et al. Cancer Cell International (2022) 22:366 He et al. Cancer Cell International (2022) 22:366 Page 10 of 16 Page 10 of 16 progressively in one dimension (Fig. 5b). In Fig. 5c, the key metabolic pathways identified from the differential metabolites between the different lesion regions remain largely conserved from previous analysis of the whole tissues. Choline metabolism in cancer, beta-alanine metabolism, arginine and proline metabolism, argi- nine biosynthesis, and glycerophospholipid metabolism pathways experienced the most alterations. Time series analysis was then conducted following disease progres- sion in such order: HL of HC, PL of LC, PL of HCC, PC of HCC and the cancer region of HCC. Representa- tive metabolite clusters with continuous upward and downward trends are displayed in Fig. 5d. For exam- ple, molecules such as taurine (m/z 148.0035) has clear continuous increase in concentration from healthy to cancer regions (Fig. 5e). In contrast, metabolites like 2-furanmethanol (m/z 116.0707) were found most abundant in healthy lobule of the HC and least abun- dant in cancer region of the HCC sample. Interestingly, metabolites that showed an upward trend in abundance along disease progression such as the ones in cluster 2 and 33 seemed to experience the sharpest changes from HCC PL to PC areas. On the other hand, cluster 28 represents a downward trend in abundance where the greatest drop occurs between HC and LC tissues. progressively in one dimension (Fig. 5b). In Fig. 5c, the key metabolic pathways identified from the differential metabolites between the different lesion regions remain largely conserved from previous analysis of the whole tissues. Choline metabolism in cancer, beta-alanine metabolism, arginine and proline metabolism, argi- nine biosynthesis, and glycerophospholipid metabolism pathways experienced the most alterations. Metabolic alterations of cancer‑related regions Time series analysis was then conducted following disease progres- sion in such order: HL of HC, PL of LC, PL of HCC, PC of HCC and the cancer region of HCC. Representa- tive metabolite clusters with continuous upward and downward trends are displayed in Fig. 5d. For exam- ple, molecules such as taurine (m/z 148.0035) has clear continuous increase in concentration from healthy to cancer regions (Fig. 5e). In contrast, metabolites like 2-furanmethanol (m/z 116.0707) were found most abundant in healthy lobule of the HC and least abun- dant in cancer region of the HCC sample. Interestingly, metabolites that showed an upward trend in abundance along disease progression such as the ones in cluster 2 and 33 seemed to experience the sharpest changes from HCC PL to PC areas. On the other hand, cluster 28 represents a downward trend in abundance where the greatest drop occurs between HC and LC tissues. ROC analyses of predictive abilities of differential metabolites In this study, we conducted ADFADESI-MSI mediated spatial metabolomics to understand the metabolic repro- gramming associated with the liver cirrhosis to HCC pro- gression. The use of AFADESI-MSI enabled us to explore the regional heterogeneity of metabolite distributions. With the wide coverage and sensitivity of this MSI tech- nique [12], we were able to detect the spatial distributions of hundreds of metabolites. Then, times series analysis and KEGG pathway enrichment analysis were conducted to identify key altered metabolites and the pathways they are involved in. We discovered that many of the amino acid metabolism and biosynthesis pathways are among the most altered processes in both the cancerous and non-cancerous regions of the diseased liver. Moreover, many of the signifi- cantly altered metabolites were also discovered to be asso- ciated with the glycerophospholipid metabolism pathways specifically in the diseased tissues. Receiver Operative Characteristic (ROC) analyses were performed to determine if the metabolites described here can help differentiate between HC, LC, and HCC tissues and even between different subregions of the lesion. According to the plots, the identified differential metabolites under the positive ion mode tend to distin- guish healthy from diseased tissues with high accuracy (AUC ~ 0.9) (Fig. 6a; Additional file 1: Fig.S5c). How- ever, the predictive power of the differential metabolites decreases when differentiating between LC and HCC (AUC ~ 0.7). Such decrease in accuracy is probably due to the similar metabolic changes occurring within the LC and HCC tissues. Looking at the subregions, again, the differ- ential metabolites predict HL from cancer region and HL from PL accurately (AUC > 0.97). On the other hand, the differentiation between PL and cancer regions with any single detected metabolite appeared to be challenging as the AUCs are generally around 0.5–0.6 (Fig. 6b). i Before focusing on the diseased tissues of the samples, we examined the surrounding non-cancerous areas to under- stand the roles of the stromal cells in tumor metabolism. He et al. Cancer Cell International (2022) 22:366 Page 11 of 16 Page 11 of 16 He et al. ROC analyses of predictive abilities of differential metabolites 5 (See legend on previous page.) b c d −2000 −1000 0 1000 2000 3000 −2500 0 2500 5000 PC1(56.4%) PC2(13.7%) HCC−Cancer HCC−Fibrosis HCC−PL HCC−PC LC−Fibrosis LC−PL HC−HL PLS−DA Pantothenate and CoA biosynthesis Linoleic acid metabolism Pathogenic Escherichia coli infection Autophagy − other Ether lipid metabolism Thermogenesis Taurine and hypotaurine metabolism Insulin resistance Retrograde endocannabinoid signaling Histidine metabolism Glycerophospholipid metabolism Arginine biosynthesis Arginine and proline metabolism beta−Alanine metabolism Choline metabolism in cancer 0.10 0.15 0.20 0.25 0.30 RichFactor PathwayTerm 1 2 3 4 5 Number 0.01 0.02 0.03 0.04 Pvalue HC-HL LC-PL HCC−PL HCC−PC HCC−Cancer Dimethylurea Phenyl-1,2-propanedione Leukotriene E4 Thiotepa L−Palmitoylcarnitine Benzoic acid MG(0:0/20:3/0:0) Histamine Iminoaspartic acid Taurine Turanose 2-Furancarboxaldehyde Allyl alcohol Hydrogen carbonate 4-pyridone-3-carboxamide 4-Hydroxybenzyl alcohol Dichloromethane HC−HL LC−PL HCC−PL HCC−PC HCC−Cancer Nitronaphthalene-7,8-oxide 2,4-Hexadienoic acid 5-pentyl-2-furanone Anabasine Guaiacol N-Acetylleucine 2-Furanmethanol 3-Amino-2-piperidone −1 0 1 Methylsuccinic acid SM(d18:0/16:1(9Z)) 1-Methylhistamine PC(16:0/16:0) PE(14:0/22:4) 2-Bromoacetaldehyde 2-Methylerythritol Debrisoquine 2,6-diaminopimelate HC-HL LC-PL HCC−PL HCC−PC HCC−Cancer a −1.5 −1.0 −0.5 0.0 0.5 1.0 HC−HL LC−PL HCC−PL PC Cancer Expression Abundance (Z−score) contains 17 metabolites Cluster 2 0.2 0.4 0.6 0.8 Membership −1.5 −1.0 −0.5 0.0 0.5 1.0 HC−HL LC−PL HCC−PL PC Cancer Expression Abundance (Z−score) contains 9 metabolites Cluster 33 0.2 0.4 0.6 0.8 Membership −1.5 −1.0 −0.5 0.0 0.5 1.0 HC−HL LC−PL HCC−PL PC Cancer Expression Abundance (Z−score) contains 8 metabolites Cluster 28 0.2 0.4 0.6 0.8 Membership b −2000 −1000 0 1000 2000 3000 −2500 0 2500 5000 PC1(56.4%) PC2(13.7%) HCC−Cancer HCC−Fibrosis HCC−PL HCC−PC LC−Fibrosis LC−PL HC−HL PLS−DA b −2000 −1000 0 1000 2000 3000 −2500 0 2500 5000 PC1(56 4%) PC2(13.7%) HCC−Cancer HCC−Fibrosis HCC−PL HCC−PC LC−Fibrosis LC−PL HC−HL PLS−DA a b c d − − PC2(13 7%) a con d d PC1(56.4%) 1 2 3 4 mber HC-HL LC-PL HCC−PL HCC−PC HCC−Can Dimethylurea Phenyl-1,2-propanedione Leukotriene E4 Thiotepa L−Palmitoylcarnitine Benzoic acid MG(0:0/20:3/0:0) Histamine Iminoaspartic acid Taurine Turanose 2-Furancarboxaldehyde Allyl alcohol Hydrogen carbonate 4-pyridone-3-carboxamide 4-Hydroxybenzyl alcohol Dichloromethane −1.5 −1.0 −0.5 0.0 0.5 1.0 HC−HL LC−PL HCC−PL PC Cancer Expression Abundance (Z−score) contains 17 metabolites Cluster 2 0.2 0.4 0.6 0.8 Membership 1 0 e) contains 9 metabolites Cluster 33 Pantothenate and CoA biosynthesis Linoleic acid metabolism Pathogenic Escherichia coli infection Autophagy − other Ether lipid metabolism Thermogenesis Taurine and hypotaurine metabolism Insulin resistance Retrograde endocannabinoid signaling Histidine metabolism Glycerophospholipid metabolism Arginine biosynthesis Arginine and proline metabolism beta−Alanine metabolism Choline metabolism in cancer 0.10 0.15 0.20 0.25 0.30 RichFactor PathwayTerm Nu Pv 1 HC−HL LC−PL HCC−PL HCC−PC HCC−Cancer Nitronaphthalene-7,8-oxide 2,4-Hexadienoic acid 5-pentyl-2-furanone Anabasine Guaiacol N-Acetylleucine 2-Furanmethanol 3-Amino-2-piperidone −1 0 1 er −1.5 −1.0 −0.5 0.0 0.5 1.0 HC−HL LC−PL HCC−PL PC Cancer Expression Abundance (Z−score) contains 8 metabolites 0.2 0.4 0.6 0.8 Membership LC HCC RichFactor e r Cluster 2 m/z=148.0035 Taurine Cluster 28 m/z=116.0707 2-Furanmethanol LC HCC HC-HL LC-PL HCC-PL HCC-PC Cancer 0 5000 10000 15000 20000 Intensity HC-HL LC-PL HCC-PL HCC 0 5000 10000 15000 20000 Intensity LC HCC HC Intensity HC Intensity Fig. ROC analyses of predictive abilities of differential metabolites Cancer Cell International (2022) 22:366 b c d e −2000 −1000 0 1000 2000 3000 −2500 0 2500 5000 PC1(56.4%) PC2(13.7%) HCC−Cancer HCC−Fibrosis HCC−PL HCC−PC LC−Fibrosis LC−PL HC−HL PLS−DA Pantothenate and CoA biosynthesis Linoleic acid metabolism Pathogenic Escherichia coli infection Autophagy − other Ether lipid metabolism Thermogenesis Taurine and hypotaurine metabolism Insulin resistance Retrograde endocannabinoid signaling Histidine metabolism Glycerophospholipid metabolism Arginine biosynthesis Arginine and proline metabolism beta−Alanine metabolism Choline metabolism in cancer 0.10 0.15 0.20 0.25 0.30 RichFactor PathwayTerm 1 2 3 4 5 Number 0.01 0.02 0.03 0.04 Pvalue HC-HL LC-PL HCC−PL HCC−PC HCC−Cancer Dimethylurea Phenyl-1,2-propanedione Leukotriene E4 Thiotepa L−Palmitoylcarnitine Benzoic acid MG(0:0/20:3/0:0) Histamine Iminoaspartic acid Taurine Turanose 2-Furancarboxaldehyde Allyl alcohol Hydrogen carbonate 4-pyridone-3-carboxamide 4-Hydroxybenzyl alcohol Dichloromethane HC−HL LC−PL HCC−PL HCC−PC HCC−Cancer Nitronaphthalene-7,8-oxide 2,4-Hexadienoic acid 5-pentyl-2-furanone Anabasine Guaiacol N-Acetylleucine 2-Furanmethanol 3-Amino-2-piperidone −1 0 1 Methylsuccinic acid SM(d18:0/16:1(9Z)) 1-Methylhistamine PC(16:0/16:0) PE(14:0/22:4) 2-Bromoacetaldehyde 2-Methylerythritol Debrisoquine 2,6-diaminopimelate HC-HL LC-PL HCC−PL HCC−PC HCC−Cancer Cluster 2 m/z=148.0035 Taurine Cluster 28 m/z=116.0707 2-Furanmethanol LC HCC HC-HL LC-PL HCC-PL HCC-PC Cancer 0 5000 10000 15000 20000 Intensity HC-HL LC-PL HCC-PL HCC-PC Cancer 0 5000 10000 15000 20000 Intensity a −1.5 −1.0 −0.5 0.0 0.5 1.0 HC−HL LC−PL HCC−PL PC Cancer Expression Abundance (Z−score) contains 17 metabolites Cluster 2 0.2 0.4 0.6 0.8 Membership −1.5 −1.0 −0.5 0.0 0.5 1.0 HC−HL LC−PL HCC−PL PC Cancer Expression Abundance (Z−score) contains 9 metabolites Cluster 33 0.2 0.4 0.6 0.8 Membership −1.5 −1.0 −0.5 0.0 0.5 1.0 HC−HL LC−PL HCC−PL PC Cancer Expression Abundance (Z−score) contains 8 metabolites Cluster 28 0.2 0.4 0.6 0.8 Membership LC HCC HC Intensity HC Intensity Fig. ROC analyses of predictive abilities of differential metabolites 5 (See legend on previous page.) e Cluster 2 m/z=148.0035 Taurine Cluster 28 m/z=116.0707 LC HCC HC-HL LC-PL HCC-PL HCC-PC Cancer 0 5000 10000 15000 20000 Intensity HC Intensity Fig. 5 (See legend on previous page.) nmethanol LC HC LC 2-Furanmethanol HC HC Cluster 28 m 2-Furan Intensity HC Intensity Intensity Fig. 5 (See legend on previous page.) He et al. Cancer Cell International (2022) 22:366 Page 12 of 16 He et al. Cancer Cell International (2022) 22:366 Page 12 of 16 He et al. Cancer Cell International (2022) 22:366 First, some of the well-studied hallmarks of cancer were identified as significantly altered metabolites in our experi- ment. For example, histamine was found to have a continu- ous increase in concentrations in the central vein regions as the disease progress. Histamine has previously been iden- tified as one of the factors of tumorigenesis across many types of cancers because of its roles in immune responses, cell proliferations, angiogenesis, etc. [8, 17, 22, 24]. There- fore, the matching results reinforce the validity of our experiment. Furthermore, the transport of the amino acids have been identified as one of the major trades in metabo- lites between the cancer cells and the surrounding micro- environment [32]. Here, we found that most of the altered metabolites can be associated with amino acid metabolism or biosynthesis. The most altered amino acid metabolism pathways include the beta-alanine metabolism, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, and arginine biosynthesis pathways. Most of these metabolic pathways have been identified as altered pathways in HCC in previous LC-MS/MS studies [2, 6, 14]. However, as most of these studies used LC-MS/MS and looked at serum metabolomics, our results offer addi- tional spatial information regarding the changes of those metabolic processes. The increases in amino acid biosyn- thesis and metabolism around the cancerous region can be explained by the increased energy-consumption needs of cancer cells. Some amino acids such as glutamate are also important for DNA synthesis. Therefore, their metabolisms in the non-cancerous regions would change to provide additional supply for the cancer cells through bidirectional trades [32]. Other than through the amino acid metabo- lism pathways, the upregulated metabolites, spermidine, spermine, and 3-aminopropanal, can be linked through a polyamine catabolic enzymatic reaction. In this reaction, spermine can be oxidized into spermidine, and 3-ami- nopropanal would be converted into hydrogen peroxide (H2O2), leading to oxidative stress. Therefore, the increase in the spermine oxidase enzyme level has been associated with cancer [5, 23]. The detected changes of the associated metabolite levels may be an indication of the change in spermine oxidase enzyme level. (See figure on next page.) Fig. 6 Reconstruction of the key altered metabolic network in the HBV-related liver cirrhosis to liver cancer progression. a, b ROC analyses of key metabolites at distinguishing between diseased tissues (a) and between different subregions (b) under positive ionization mode. c The network map of key metabolic pathways in the non-cancer regions. d The network map of key metabolic pathways in the cancer-related regions. e Schematics of the related and altered metabolic pathways in the HBV infection liver disease progression 2-Furanmethanol Overall, our results show that the surrounding stroma cells of diseased livers engage in various levels of metabolic reprogramming as the lesion progresses. More importantly, the areas of cirrhosis and tumor undergo more complete metabolic changes as reflected by their altered metabolites. The pathways that con- tain the most differential metabolites are still the amino acid metabolism and biosynthesis pathways. A lot of the metabolite changes in the lesion regions are the same as in the non-cancerous regions. For example, both pro- teinogenic and non-proteinogenic amino acids experi- enced continuous changes in abundance going from liver cirrhosis to liver cancer. These upregulated amino acids serve to promote protein synthesis, DNA and RNA syn- thesis, and the conversion into other key metabolites in cancer cells [19, 35]. Therefore, the amino acid metabo- lisms act as a connection between numerous other metabolic processes, such as the purine and pyrimi- dine metabolisms and the amnioacyl-tRNA biosynthe- sis pathways, to fulfill the energy and growth needs for the tumor. In addition, we discovered that the pyrimi- dine metabolism is also altered through the increase in carbamoyl-phosphate during cancer progression. Car- bamoyl-phosphate is derived from ammonia during the first step of the urea cycle. However, the overexpression of the converting enzyme, carbamoyl-phosphate syn- thetase 1(CPS1), has been found to encourage pyrimidine biosynthesis, which is then connected to tumor prolifera- tion [30]. Since there has been study suggesting the use of CPS1 inhibitor to treat cancer [38], detecting the level of carbamoyl-phosphate, especially with spatial infor- mation, could be helpful in finding potential biomark- ers or in future drug testing. Another major aspect of metabolic reprogramming is the alteration of the glycer- ophospholipid metabolism. The changes in this pathway in the cancer-related regions are more significant than that in the non-cancerous regions. Specifically, there are significant increases in some of the phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and glycerophos- phocholine levels as the lesion develops. Glycerophos- pholipid is important in cell membrane formation and thus is commonly upregulated for cell proliferation dur- ing cancer development [20, 27]. In addition, multiple studies have associated upregulation of LPC to PC con- version enzyme with various cancers [7, 28, 31]. Further- more, it is not entirely clear why both the cancer-related He et al. 2-Furanmethanol Cancer Cell International (2022) 22:366 Page 13 of 16 R08745 R03720 R04354 R04764 R00259 R01398 R00149 R02748 R01126 R01560 R01132 R01769 R01863 R01768 R00575 R01397 R02397 R03283 R01920 R02869 R09076 R02894 R01167 R02150 R02155 R00526 R03139 R00904 R01682 R01687 R01318 R01315 R02114 R01030 R02747 R02746 R07388 R02745 R01709 R01794 R08208 R07211 R07212 R07213 R01813 R11765 R11764 R03222 R00310 4−Trimethylammoniobutanoic acid 3−Aminopropionaldehyde Carbamoyl phosphate Histamine LysoPA(P−16:0/0:0) Spermidine N−Acetyl−L−glutamic acid 4−Pyridoxic acid Tetrahydrobiopterin Inosine 5−L−Glutamyl−taurine Spermine D−Proline Taurine Protoporphyrin IX LysoPC(20:5(5Z,8Z,11Z,14Z,17Z)/0:0) N−Formyl−L−aspartate Glycerophosphocholine 2−Methoxyestradiol Hypoxanthine Alanine, aspartate and glutamate metabolism D−Amino acid metabolism Nitrogen metabolism Pyrimidine metabolism Arginine and proline metabolism Histidine metabolism Folate biosynthesis Arginine biosynthesis beta−Alanine metabolism Pantothenate and CoA biosynthesis Purine metabolism Glutathione metabolism Porphyrin metabolism Lysine degradation Taurine and hypotaurine metabolism Ether lipid metabolism Glycerophospholipid metabolism Primary bile acid biosynthesis Steroid hormone biosynthesis Vitamin B6 metabolism Degree 5 10 15 20 Type compound pathway reaction c Glutamine NH3 Glutamate Carbamoyl-P Nitrogen metabolism N-Acetylglutamate N-Acetylglutamate semialdehyde Pyrimidine metabolism Omithine Citruline Arginine Urea Cycle Omithine Proline D-proline Putrescine Spemidine Spermine 3-Aminopropanol 3-Aminopropanol Arginine and proline metabolism β-Alanine metabolism Glutathione metabolism Pantothenate and CoA biosynthesis 3.5.1.2 6.3.4.16 2.1.3.3 L-Arginosuccinate 6.3.4.5 4.3.2.1 3.5.3.1 1.14.1339 2.3.1.1 5.1.1.4 2.5.1.16 2.5.1.22 1.5.3.17 1.5.3.17 Arginine biosynthesis Arginine biosynthesis Arginine and proline metabolism β-Alanine metabolism Pantothenate and CoA biosynthesis Pathway Enzyme Expression 0.5 1.0 1.5 2.0 HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC - PL HCC PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer D−Proline 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 1 − Specificity Sensitivity AUC=0.976 Arginine and proline metabolism AUC=0.754 Spermidine 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 1 − Specificity Sensitivity HC/HCC HC/LC LC/HCC AUC=0.879 AUC=0.935 AUC=0.678 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 1 − Specificity Sensitivity HL/PL HL/Cancer PL/Cancer AUC=0.941 AUC=0.974 AUC=0.565 Spermidine 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 1 − Specificity Sensitivity AUC=0.968 AUC=0.987 AUC=0.619 D−Proline a b R07494 R07509 R08745 R03720 R00259 R01398 R00149 R02748 R01126 R01560 R01132 R01769 R01863 R01768 R00575 R01397 R02397 R03283 R01920 R02869 R09076 R02894 R01168 R01166 R01164 R01167 R04674 R02150 R02155 R03139 R00904 R01682 R01687 R02923 R02250 R02251 R01350 R08107 R12351 R12702 R07376 R07377 R01318 R01321 R01316 R01315 R01310 R02051 R02053 R02114 R01030 R02057 R04480 R02747 R02055 R01320 R02746 R02056 R07388 R02745 R01317 R07064 R07859 R07860 R01709 R03655 R08293 R08294 4−Trimethylammoniobutanoic acid Histamine LysoPC(20:5(5Z,8Z,11Z,14Z,17Z)/0:0) 3−Aminopropionaldehyde Spermidine Carbamoyl phosphate Spermine LysoPA(P−16:0/0:0) D−Proline TG(16:0/18:0/18:2(9Z,12Z)) D−Arginine N−Acetyl−L−glutamic acid Taurine PC(22:5/16:1(9Z)) 4−Pyridoxic acid 4a−Carboxy−4b−methyl−5a−cholesta−8,24−dien−3b−ol 5−L−Glutamyl−taurine Lidocaine L−Histidine 1−Methylhistamine Hypoxanthine Glycerophosphocholine Inosine Primary bile acid biosynthesis Steroid biosynthesis Alanine, aspartate and glutamate metabolism D−Amino acid metabolism Nitrogen metabolism Pyrimidine metabolism Arginine and proline metabolism Histidine metabolism Arginine biosynthesis beta−Alanine metabolism Pantothenate and CoA biosynthesis Purine metabolism Glutathione metabolism Lysine degradation Taurine and hypotaurine metabolism Glycerophospholipid metabolism Ether lipid metabolism GPI−anchor biosynthesis Glycerolipid metabolism Linoleic acid metabolism Arachidonic acid metabolism Aminoacyl−tRNA biosynthesis alpha−Linolenic acid metabolism Vitamin B6 metabolism Drug metabolism − cytochrome P450 d e Fig. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12935-022-02775-9. The online version contains supplementary material available at https://doi. org/10.1186/s12935-022-02775-9. Additional file 1: Figure S1. Total metabolite distributions in the HC, LC and HCC tissue samples. a HE and MSI diagrams of HC, LC and HCC whole samples under the negative ionization mode. b PLS-DA comparison of AFADESI-MSI data under the negative ionization mode. c, d Alterations of metabolites detected by AFADESI-MSI method in the HC, LC and HCC samples based on positive (c) and negative (d) ionization modes. e, f K-means diagrams of the HC, LC and HCC tissue samples based on positive (e) and negative (f) ionization modes. Figure S2. The alterations of metabolites’ spatial distributions in HCC sub-regions under negative ionization mode. a HE and MSI images of different sub-regions of HCC. b PLS-DA analysis of different HCC sub-regions. c Number of metabolites detected from different HCC sub-regions. d Heatmap of significantly differentiated metabolites based on VIP > 1. e KEGG analysis of key altered metabolic pathways in the sub-regions of HCC. Figure S3. Metabolites’ spatial distributions in the non-cancerous regions (CV) in the HC, LC and HCC samples under negative ionization mode. a HE and MSI images of the CV regions in the HC, LC and HCC samples. b PLS-DA analysis for the CV regions. c Heatmap of significantly differentiated metabolites under positive ionization mode in the CV regions based on VIP > 1. d KEGG analysis of key altered metabolic pathways in the CV regions. e Heatmap of significantly differentiated metabolites based on VIP > 1 under nega- tive ionization mode. f Sample time series analysis of the key metabolite expressions in the CV regions. Figure S4. Metabolites’ spatial distributions of the cancer-related regions in the HC, LC and HCC samples under nega- tive ionization mode. a HE and MSI images (negative mode) of the cancer- related regions in the HC, LC and HCC samples. b PLS-DA analysis of the cancer-related regions. c Heatmap of significantly differentiated metabo- lites in the cancer-related regions based on variable VIP > 1 under positive ionization mode. d Heatmap of significantly differentiated metabolites in the cancer-related regions based on variable VIP > 1 under negative ioniza- tion mode. e KEGG analysis of key altered metabolic pathways in cancer- related regions. Figure S5. Metabolic changes in different tissues and its predictability of disease progression. HCC: Hepatocellular carcinoma; HBV: Hepatitis B virus; MSI: Mass spectrometry imaging; AFADESI: Air flow-assisted desorption electrospray ionization; LC: Liver cirrhosis; ROI: Region of interest; MELD: Model of end stage liver disease; PL: Pseudo lobule; NT: Necrotic tissue; H&E: Hematoxylin and eosin; CPS1: Carbamoyl-phosphate synthetase; LPC: Lysophosphatidylcholine. 2-Furanmethanol Cancer Cell International (2022) 22:366 He et al. Cancer Cell International (2022) 22:366 Page 14 of 16 and non-cancerous regions saw a continuous increase in some of the antioxidant levels as the disease progress. Particularly, both taurine and ferulic acid are upregu- lated. Ferulic acid has been shown to be an effective antioxidant and anti-inflammatory compound whose anti-tumor effects have been investigated [33, 34, 36]. Taurine has also been found to have anti-tumor effect by inducing apoptosis [25, 33, 34, 39]. Therefore, it is unlikely that these metabolites are direct metabolites of cancer cells. Rather, the increase of ferulic acid and tau- rine levels may be an attempt of compensation from the surrounding regions in response to the liver damages. Lastly, our ROC analyses results validate the power of the detected metabolites in differentiating the healthy from diseased tissues. Although using any single metabolite to distinguish liver cirrhosis apart from HCC remains challenging, our results demonstrate the general progres- sive trends in metabolic changes along disease advance- ment. Hence, during future studies, it may be possible to explore the combinations of multiple key metabolites in predicting disease progression.h Author contributions Conceptualization, MJH, WJP, XW and JRH; Investigation, MJH, WJP, XW, XNZ, DZ, ZPZ, WXC and JYL; Writing—original draft, MJH and WJP; Writing—review and editing, DET and YD; Supervision, DET and YD; Funding acquisition, JRH and YD. All authors read and approved the final manuscript. Supplementary Information d Heatmap of significantly differentiated metabolites in the cancer-related regions based on variable VIP > 1 under negative ioniza- tion mode. e KEGG analysis of key altered metabolic pathways in cancer- related regions. Figure S5. Metabolic changes in different tissues and its predictability of disease progression. a Sample time series analysis of key metabolite expressions (negative mode) in the cancer-related regions. b Examples of key metabolites’ spatial expressions in cancer-related regions under negative ion mode. c ROC analyses of key metabolites at distin- guishing between diseased tissues and between different subregions (anabasine was detected under positive ionization mode, docosahexae- noic acid was detected under negative ionization mode). There are still many aspects of metabolic reprogramming to be explored. For example, future studies can work on improving the spatial resolution of the MSI technique so that finer metabolite distribution trends can be analyzed. In addition to the separation of different types of tissues, metabolic changes within each type of tissue can also be examined to help us better understand the sources of meta- bolic dysregulations. Furthermore, to better understand the causes and effects of the altered metabolic pathways, future studies can conduct multi-omics comparisons. For example, combining spatial metabolomics data with spatial transcriptome data may be able to reveal the relationships between altered gene expressions of relevant enzymes and the metabolomic phenotypes. Another potentially reward- ing study is to compare samples that have undergone differ- ent therapeutics or diets to understand the suitability and effectiveness of various treatment plans. Overall, though, our study was able to detect many key metabolites that may be able to serve as HCC biomarkers for early detection. Moreover, the time-series analysis and the spatial infor- mation provide us a better sense of the metabolic changes along disease progression and across different regions of the liver. Additional file 2: Table S1. Key parameters of the AFADESI-MSI setting. Funding The Scientific Research Youth Fund of Shenzhen Third People’s Hospital (G2021008). Guangxi Key Laboratory of Metabolic Diseases Research (20-065- 76). Shenzhen Science and Technology R&D Fund (JCYJ20190809165813331). Shenzhen Key Medical Discipline Construction Fund (SZXK059). Supplementary Information a Sample time series analysis of key metabolite expressions (negative mode) in the cancer-related regions. b Examples of key metabolites’ spatial expressions in cancer-related regions under negative ion mode. c ROC analyses of key metabolites at distin- guishing between diseased tissues and between different subregions (anabasine was detected under positive ionization mode, docosahexae- noic acid was detected under negative ionization mode). Additional file 1: Figure S1. Total metabolite distributions in the HC, LC and HCC tissue samples. a HE and MSI diagrams of HC, LC and HCC whole samples under the negative ionization mode. b PLS-DA comparison of AFADESI-MSI data under the negative ionization mode. c, d Alterations of metabolites detected by AFADESI-MSI method in the HC, LC and HCC samples based on positive (c) and negative (d) ionization modes. e, f K-means diagrams of the HC, LC and HCC tissue samples based on positive (e) and negative (f) ionization modes. Figure S2. The alterations of metabolites’ spatial distributions in HCC sub-regions under negative ionization mode. a HE and MSI images of different sub-regions of HCC. b PLS-DA analysis of different HCC sub-regions. c Number of metabolites detected from different HCC sub-regions. d Heatmap of significantly differentiated metabolites based on VIP > 1. e KEGG analysis of key altered metabolic pathways in the sub-regions of HCC. Figure S3. Metabolites’ spatial distributions in the non-cancerous regions (CV) in the HC, LC and HCC samples under negative ionization mode. a HE and MSI images of the CV regions in the HC, LC and HCC samples. b PLS-DA analysis for the CV regions. c Heatmap of significantly differentiated metabolites under positive ionization mode in the CV regions based on VIP > 1. d KEGG analysis of key altered metabolic pathways in the CV regions. e Heatmap of significantly differentiated metabolites based on VIP > 1 under nega- tive ionization mode. f Sample time series analysis of the key metabolite expressions in the CV regions. Figure S4. Metabolites’ spatial distributions of the cancer-related regions in the HC, LC and HCC samples under nega- tive ionization mode. a HE and MSI images (negative mode) of the cancer- related regions in the HC, LC and HCC samples. b PLS-DA analysis of the cancer-related regions. c Heatmap of significantly differentiated metabo- lites in the cancer-related regions based on variable VIP > 1 under positive ionization mode. 2-Furanmethanol 6 (See legend on previous page.) D−Proline 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 1 − Specificity Sensitivity AUC=0.976 AUC=0.754 Spermidine 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 1 − Specificity Sensitivity HC/HCC HC/LC LC/HCC AUC=0.879 AUC=0.935 AUC=0.678 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 1 − Specificity Sensitivity HL/PL HL/Cancer PL/Cancer AUC=0.941 AUC=0.974 AUC=0.565 Spermidine 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 1 − Specificity Sensitivity AUC=0.968 AUC=0.987 AUC=0.619 D−Proline a b Vitamin B6 metabolism Spermidine 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 1 − Specificity Sensitivity HC/HCC HC/LC LC/HCC AUC=0.879 AUC=0.935 AUC=0.678 a 1 b R08745 R03720 R04354 R04764 R00259 R01398 R00149 R02748 R01126 R01560 R01132 R01769 R01863 R01768 R00575 R01397 R02397 R03283 R01920 R02869 R09076 R02894 R01167 R02150 R02155 R00526 R03139 R00904 R01682 R01687 R01318 R01315 R02114 R01030 R02747 R02746 R07388 R02745 R01709 R01794 R08208 R07211 R07212 R07213 R01813 R11765 R11764 R03222 R00310 4−Trimethylammoniobutanoic acid 3−Aminopropionaldehyde Carbamoyl phosphate Histamine LysoPA(P−16:0/0:0) Spermidine N−Acetyl−L−glutamic acid 4−Pyridoxic acid Tetrahydrobiopterin Inosine 5−L−Glutamyl−taurine Spermine D−Proline Taurine Protoporphyrin IX LysoPC(20:5(5Z,8Z,11Z,14Z N−Formyl−L−aspartate Glycerophosphocholine 2−Methoxyestradiol Hypoxanthine Alanine, aspartate and glutamate metabolism D−Amino acid metabolism Nitrogen metabolism Pyrimidine metabolism Arginine and proline metabolism Histidine metabolism Folate biosynthesis Arginine biosynthesis beta−Alanine metabolism Pantothenate and CoA biosynthesis Purine metabolism Glutathione metabolism Porphyrin metabolism Lysine degradation Taurine and hypotaurine metabolism Ether lipid metabo Glycerophospholipid metabol Primary bile acid biosynthesis Steroid hormone biosynthesis Vitamin B6 metabolism Deg Type compound pathway reaction c Arginine biosynthesis Arginine and proline metabolism e d c e Glutamine NH3 Glutamate Carbamoyl-P Nitrogen metabolism N-Acetylglutamate N-Acetylglutamate semialdehyde Pyrimidine metabolism Omithine Citruline Arginine Urea Cycle Omithine Proline D-proline Putrescine Spemidine Spermine 3-Aminopropanol 3-Aminopropanol Arginine and proline metabolism β-Alanine metabolism Glutathione metabolism Pantothenate and CoA biosynthesis 3.5.1.2 6.3.4.16 2.1.3.3 L-Arginosuccinate 6.3.4.5 4.3.2.1 3.5.3.1 1.14.1339 2.3.1.1 5.1.1.4 2.5.1.16 2.5.1.22 1.5.3.17 1.5.3.17 Arginine biosynthesis Arginine biosynthesis Arginine and proline metabolism β-Alanine metabolism Pantothenate and CoA biosynthesis Pathway Enzyme Expression 0.5 1.0 1.5 2.0 HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC - PL HCC PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer HC-HL HCC-PL HCC-PC HCC-Cancer R01687 Taurine 5−L−Glutamyl−taurine e Fig. 6 (See legend on previous page.) 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https://openalex.org/W4211081087	https://www.atmos-chem-phys.net/16/3099/2016/acp-16-3099-2016.pdf	English	null	Atmospheric methane evolution the last 40 years	null	2,015	cc-by	20,750	Stig B. Dalsøren1, Cathrine L. Myhre2, Gunnar Myhre1, Angel J. Gomez-Pelaez3, Ole A. Søvde1, Ivar S. A. Isaksen1,4, Ray F. Weiss5, and Christina M. Harth5 Stig B. Dalsøren1, Cathrine L. Myhre2, Gunnar Myhre1, Angel J. Gomez-Pelaez3, Ole A. Søvde1, Ivar S. A. Isaksen1,4, Ray F. Weiss5, and Christina M. Harth5 1CICERO – Center for International Climate and Environmental Research Oslo, Oslo, Norway 2NILU – Norwegian Institute for Air Research, Kjeller, Norway 3Izaña Atmospheric Research Center (IARC), Meteorological State Agency of Spain (AEMET), Izaña, Spain 4University of Oslo, Department of Geosciences, Oslo, Norway 5Scripps Institution of Oceanography University of California, San Diego La Jolla, California, USA Correspondence to: Stig B. Dalsøren (stigbd@cicero.oslo.no) Received: 11 July 2015 – Published in Atmos. Chem. Phys. Discuss.: 5 November 2015 Revised: 24 February 2016 – Accepted: 26 February 2016 – Published: 9 March 2016 Received: 11 July 2015 – Published in Atmos. Chem. Phys. Discuss.: 5 November 2015 Revised: 24 February 2016 – Accepted: 26 February 2016 – Published: 9 March 2016 Abstract. Observations at surface sites show an increase in global mean surface methane (CH4) of about 180 parts per billion (ppb) (above 10 %) over the period 1984–2012. Over this period there are large ﬂuctuations in the annual growth rate. In this work, we investigate the atmospheric CH4 evolution over the period 1970–2012 with the Oslo CTM3 global chemical transport model (CTM) in a bottom- up approach. We thoroughly assess data from surface mea- surement sites in international networks and select a sub- set suited for comparisons with the output from the CTM. We compare model results and observations to understand causes for both long-term trends and short-term variations. Employing Oslo CTM3 we are able to reproduce the sea- sonal and year-to-year variations and shifts between years with consecutive growth and stagnation, both at global and regional scales. The overall CH4 trend over the period is reproduced, but for some periods the model fails to repro- duce the strength of the growth. The model overestimates the observed growth after 2006 in all regions. This seems to be explained by an overly strong increase in anthropogenic emissions in Asia, having global impact. Our ﬁndings con- ﬁrm other studies questioning the timing or strength of the emission changes in Asia in the EDGAR v4.2 emission in- ventory over recent decades. The evolution of CH4 is not only controlled by changes in sources, but also by changes in the chemical loss in the atmosphere and soil uptake. The atmospheric CH4 lifetime is an indicator of the CH4 loss. Stig B. Dalsøren1, Cathrine L. Myhre2, Gunnar Myhre1, Angel J. Gomez-Pelaez3, Ole A. Søvde1, Ivar S. A. Isaksen1,4, Ray F. Weiss5, and Christina M. Harth5 In our simulations, the atmospheric CH4 lifetime decreases by more than 8 % from 1970 to 2012, a signiﬁcant reduction of the residence time of this important greenhouse gas. Changes in CO and NOx emissions, speciﬁc humidity, and ozone col- umn drive most of this, and we provide simple prognostic equations for the relations between those and the CH4 life- time. The reduced lifetime results in substantial growth in the chemical CH4 loss (relative to its burden) and dampens the CH4 growth. Atmos. Chem. Phys., 16, 3099–3126, 2016 www.atmos-chem-phys.net/16/3099/2016/ doi:10.5194/acp-16-3099-2016 © Author(s) 2016. CC Attribution 3.0 License. Atmos. Chem. Phys., 16, 3099–3126, 2016 www.atmos-chem-phys.net/16/3099/2016/ doi:10.5194/acp-16-3099-2016 © Author(s) 2016. CC Attribution 3.0 License. 1 Introduction The atmospheric CH4 abundance has more than doubled over the industrial era. The resulting radiative forcing is second af- ter CO2 in terms of anthropogenic forcing from greenhouse gases (Myhre et al., 2013). High uncertainty remains regard- ing the contributions from speciﬁc source sectors and regions to the CH4 emissions (Neef et al., 2010; Kirschke et al., 2013; Houweling et al., 2014; Melton et al., 2013; Bruhwiler et al., 2014; Schwietzke et al., 2014; Bridgham et al., 2013; Pison et al., 2009; Ciais et al., 2013), the underlying factors con- tributing to observed trends (Dlugokencky et al., 2009, 2003; Wang et al., 2004; Kai et al., 2011; Aydin et al., 2011; Simp- son et al., 2012; Bousquet et al., 2006, 2011; Pison et al., 2013; Bergamaschi et al., 2013; Monteil et al., 2011; Ghosh et al., 2015; Nisbet et al., 2014; Fiore et al., 2006; Levin et al., 2012), and in feedbacks from the biosphere and permafrost (Bridgham et al., 2013; Melton et al., 2013; Isaksen et al., 2011; O’Connor et al., 2010). The uncertainties in our un- derstanding of current budgets, recent trends, and feedbacks limit conﬁdence in accurately projecting the future evolution of CH4. Increasing atmospheric CH4 would accelerate near- S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3100 term warming, due to its strong climate impact on a 20-year time frame (Myhre et al., 2013). Enhanced CH4 levels would also increase the ozone levels in surface air (Fiore et al., 2008, 2012; West and Fiore, 2005; Isaksen et al., 2014), and thereby worsen air pollution impacts on vegetation, crops, and human health. to 2012. However, shorter periods with declining emissions occur due to large inter-annual variability in natural emis- sions, especially from wetlands which is the largest emission sector. The inter-annual variation in wetland emissions tends to be anti-correlated with the ENSO index (Bousquet et al., 2006; Hodson et al., 2011). Low natural emissions also oc- cur due to lower global temperatures in the years after the Pinatubo eruption. In the 1990s the growth in anthropogenic emissions are small, mainly caused by the economic collapse of the former USSR. From 2000 to 2006 the total emissions are quite stable, and this is caused by decreasing wetland emissions due to dry conditions in the tropics in combination with increasing anthropogenic emissions. From 2006 there is a strong growth in total emissions due to large wetland emissions and a continuing growth of anthropogenic emis- sions. The abrupt increase in 2007 is mainly explained by high wetland emissions caused by high temperatures at high latitudes in the Northern Hemisphere, and wet conditions in the tropics (Bousquet et al., 2011). Enteric fermentation (due to ruminants) is the main anthropogenic emission sector and it grows steadily except for a period in the 1990s. Some other major anthropogenic sectors like gas, solid fuel (mostly coal) and agricultural soils (mostly rice) even decrease over shorter periods but have in common a substantial growth over the last decade. The sum of several smaller anthropogenic emission sectors (industry, residential, waste, some fossil, etc.) are also shown in Fig. 1. This sum termed “other anthropogenic sec- tors” is of the same magnitude as enteric fermentation. The growth is rather stable and moderate with some interruptions: temporary declines occur after the oil crisis in 1973 and the energy crisis in 1979. The growth is also small during the 1990s. This study seeks to increase our understanding of CH4 by providing a detailed analysis on global and regional CH4 evo- lution over the last 40 years. 2.1.1 Methane We also explore a possible impact of the recent ﬁnancial crisis using an alternative extrapolation of anthropogenic emissions for the period 2009–2012. Here, the emissions from petroleum and solid fuel production and distribution were scaled with BP Statistical Review of World Energy (http://www.bp.com/en/global/corporate/energy-economics/ statistical-review-of-world-energy.html) numbers for gas production, oil and coal consumption resulting in a drop in total emissions in 2009 (Fig. 1). However, the evolution from 2010 with this alternative extrapolation is rather similar to that for the standard extrapolation. The EDGAR v4.2 inventory was recently extended to include also the years 2009–2012. In Fig. S2 (Supplement) we compare our extrapolations with the new data and also include a comparison to ECLIPSE v5a emissions that are available for part of our study period (1990–2015, 5-year intervals). We used CH4 emissions for anthropogenic sources from EDGAR v4.2 (EC-JRC/PBL, 2011) and biomass-burning and natural sources from Bousquet et al. (2011). In addition we used soil uptake from Bousquet et al. (2011). Combina- tion of two emission inventories (EDGAR v4.2 and Bous- quet et al., 2011) makes it possible to study the impacts of many emission sectors (18 in total, see Table S1 in the Sup- plement for the sectors and speciﬁcations of the categories). The EDGAR inventory covers the period 1970–2008 while the Bousquet et al. (2011) data covers the period 1984–2009. Since we study the period 1970–2012 extrapolations were made for the years not covered by the data sets. For all years from 1970 to 1984 we used natural and biomass-burning emissions and soil uptake for 1984. For 2010–2012 we used 2009 data for these sources. For the anthropogenic emissions we extrapolated the change from the period 2007–2008 to the period 2009–2012. The rather simple extrapolations result in additional uncertainties in the model outcome for these years. Figure S1 in the Supplement shows how the emissions are included in the model for the different time periods. The to- tal emissions and emissions from major sectors are shown in Fig. 1. There is a large growth in total emissions from 1970 Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years We investigate essential natural and anthropogenic drivers controlling the atmospheric CH4 budget over the period, with a particular focus on the last 15 years. We perform a balanced analysis of both sources and sinks. The sinks depend on the atmospheric oxidation capac- ity, which is determined by complex chemical and meteoro- logical interactions. This study tries to reveal the key chem- ical components and meteorological factors affecting recent changes in the oxidation capacity. We compare model stud- ies and observations to understand causes for both long-term trends and short-term variations (year-to-year). We also ad- dress reasons for differences between observed and modelled CH4 trends. The methods used are described in Sect. 2. Sec- tion 3 presents the results from our main analysis and discuss them in a broader context related to ﬁndings from other stud- ies. Additional sensitivity studies are presented in the Sup- plement. In Sect. 4 we summarize our ﬁndings. S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3101 Figure 1. Emissions used in the model simulations. The grey shaded area is the total CH4 emissions (left y axis). The total emissions in the alternative extrapolation accounting for the ﬁnancial crisis are shown from 2006 and onwards as the grey line with markers. The other coloured lines are the CH4 emissions from the main emission sectors (right y axis). 3101 Figure 1. Emissions used in the model simulations. The grey shaded area is the total CH4 emissions (left y axis). The total emissions in the alternative extrapolation accounting for the ﬁnancial crisis are shown from 2006 and onwards as the grey line with markers. The other coloured lines are the CH4 emissions from the main emission sectors (right y axis). proxy for the different sector’s contribution to monthly mean surface CH4 concentrations. The aim was to reveal key sec- tors and regions behind recent changes in spatial distribution or temporal evolution of CH4. emissions we used GFEDv3 (van der Werf et al., 2010) for the period 1997–2012. In the period 1970–1996 we used year-2001 emissions from GFEDv3. 2001 was taken as a proxy for an average year since it has a weak ENSO index for all months (see next section for more discussion on this). Oslo CTM3 was described and evaluated by Søvde et al. (2012) and used for studying CH4 lifetime changes in Holmes et al. (2013). Oslo CTM3 is an update of Oslo CTM2 which has been used in a number of previous studies of stratospheric and tropospheric chemistry, including studies on CH4 (Dalsøren et al., 2010, 2011; Dalsøren and Isaksen, 2006; Isaksen et al., 2011). The parametrization and inter-annual variation of light- ning NOx emissions are described in Søvde et al. (2012). For other natural emissions we used emission data for 2000 for all years. The oceanic emissions of CO and NMVOCs and soil NOx emissions are from RETRO (Schultz et al., 2008). Sources for natural sulfur emissions are described in Berglen et al. (2004). The emissions from vegetation of CO and NMVOCs are from MEGANv2 (Guenther et al., 2006). Re- cently a new data set (Sindelarova et al., 2014) with MEGAN emissions covering the period 1980–2010 became available. S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years This data set was used in a sensitivity study to investigate whether inter-annual variations in CO and NMVOCs emis- sions from vegetation are important for the CH4 evolution. The Oslo CTM3 simulations were driven with 3-hourly meteorological forecast data from the European Centre for Medium-Range Weather Forecasts (ECMWF) Integrated Forecast System (IFS) model (see Søvde et al., 2012, for de- tails). These data are 36-h forecasts produced with 12 h of spin-up starting from an ERA-Interim analysis at noon on the previous day. The meteorological data used in this study cover the period 1997–October 2012. For the years ahead of 1997, year-2001 meteorology was used. 2001 was chosen since this is a year with weak ENSO index for all months. Previous studies have shown a strong inﬂuence of ENSO events on CH4 (Holmes et al., 2013; Warwick et al., 2002; Johnson et al., 2002). Initially the model was spun up in a long run with repetitive 1970 emissions until we obtained a stable atmospheric CH4 burden from one year to the next. Due to the long adjustment time of CH4 it took 27 years to get CH4 in equilibrium. After the spin up a set of simulations (Table 1) were made for the period 1970 to 2012. The “main” 2.2 Chemical transport model The emission data over the period 1970–2012 was used as input in the Oslo CTM3 model. A coupled tropospheric and stratospheric version was used. The model was run with 109 chemical active species affecting CH4 and atmospheric ox- idation capacity. In addition we added 18 passive ﬁctitious tracers for each of the CH4 emission sectors listed in Ta- ble S1. The traces were continuously emitted and then given an e-folding lifetime of 1 month, undergoing transport but not interacting chemically. The passive tracers were used as a 2.1.2 Other components Anthropogenic emissions of CO, NOx, sulfur and NMVOCs were taken from the EDGAR v4.2 inventory (EC-JRC/PBL, 2011). Similar extrapolation was done as for the CH4 emis- sions to cover the period 2009–2012. For biomass-burning Atmos. Chem. Phys., 16, 3099–3126, 2016 www.atmos-chem-phys.net/16/3099/2016/ S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 31 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3102 Figure 2. Global CH4 budget in the main Oslo CTM3 simulation over the period 1970–2012: atmospheric burden (left y axis); loss: atmo- spheric chemical destruction + soil uptake (right y axis); and total emissions (right y axis). Figure 2. Global CH4 budget in the main Oslo CTM3 simulation over the period 1970–2012: atmospheric burden (left y axis); loss: atmo- spheric chemical destruction + soil uptake (right y axis); and total emissions (right y axis). Table 1. Overview of simulations performed with the Oslo CTM3 model. Simulation name Period Characteristics Difference from main simulation Main 1970–Oct 2012 Standard emissions described in Sect. 2.1.1. Meteorology de- scribed in this section. Fixed methane 1970–Oct 2012 No prescription of methane emissions. Surface methane levels kept ﬁxed. Monthly mean 1970 levels used re- peatedly for all years Fixed meteorology 1997–Oct 2012 Year-2001 meteorology Financial∗ 2009–Oct 2012 Alternative extrapolation of anthropogenic emissions to account for the ﬁnancial crisis Bio∗ 1980–2012 Inter-annual variation in biogenic emissions of NMVOCs and CO ∗Results (and setup) from these simulations are mainly discussed in the Supplement. Table 1. Overview of simulations performed with the Oslo CTM3 model. Difference from main simulation ∗Results (and setup) from these simulations are mainly discussed in the Supplement. simulation, the period 1997–2012 was repeated using year- 2001 meteorology for all years. By comparing this run with the “main” simulation the impact of meteorological variabil- ity could be discerned. simulation includes the standard CH4 emissions described in Sect. 2.1.1. In the “ﬁnancial” simulation, the period 2009– 2012 was rerun with slightly different emissions evaluating whether the recent ﬁnancial crisis had any signiﬁcant impact on CH4 levels. With a similar purpose a “bio” simulation was performed accounting for inter-annual variation in emissions of CO and NMVOCs from vegetation. The results from the two sensitivity studies on emissions are discussed in the Sup- plement. In the “ﬁxed methane” simulation, the prescription of methane emissions was turned off and surface CH4 was kept ﬁxed at monthly mean 1970 levels (i.e., boundary con- dition of Dirichlet type instead of Neumann type) to isolate the effect of other components and meteorological factors on CH4 via changes in oxidation capacity. In the “ﬁxed met” www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3103 Figure 3. Atmospheric CH4 burden and atmospheric chemical loss for the simulation with “ﬁxed meteorology” and the “main” simulation. Figure 3. Atmospheric CH4 burden and atmospheric chemical loss for the simulation with “ﬁxed meteorology” and the “main” simulation. Figure 3. Atmospheric CH4 burden and atmospheric chemical loss for the simulation with “ﬁxed meteorolog burden and atmospheric chemical loss for the simulation with “ﬁxed meteorology” and the “main” simulation Criteria for selection were the length of measurement record (coverage over most of the time periods of interest), access to continuous time series with few gaps, time resolution (at least 2–3 measurement per month), coverage of different re- gions of the Earth, and site characteristics (e.g. elevation, to- pography, and inﬂuence of pollution episodes). The last point was evaluated in relation to the resolution of the CTM. From this analysis, 71 observational data sets from 64 stations in the WDCGG database were selected as suited for compar- isons with the CTM results. Comparisons for some of these stations are shown in Sects. 3.3 and 3.4. creased less than expected solely from the increase in CH4 emissions since a growth in the atmospheric CH4 loss oc- curred over the period. The growth in instantaneous atmo- spheric CH4 loss is almost 25 %. In the period 2001–2006 when emissions were quite stable increasing CH4 loss likely contributed to the stagnation of the CH4 growth. Interest- ingly, for 2010–2012, the loss deviates from its steady in- crease over the previous decades. A stabilization of the CH4 loss probably contributed to the continuing increase (2009– 2012) in CH4 burden after the high emission years 2007 and 2008. Due to the long response time of CH4 this change in the loss pattern might also contribute to future growth in CH4. However, there are additional uncertainties in the model bur- den and loss after 2009 due to the extrapolation of emissions after this year. 3 The methane evolution and decisive factors over the period 1970–2012 Especially after 1997 and the introduction of variation in meteorology, we see that the loss follows a different path than the burden. Comparing the main model simulation with the one with ﬁxed meteorology (Fig. 3) for the period 1997–2012 it becomes evident that inclusion of varying meteorological factors is important to take into account to understand the de- velopment of the CH4 budget. This was also shown in other studies (Johnson et al., 2002; Fiore et al., 2006; Warwick et al., 2002; Holmes et al., 2013). If there had been no varia- tion in meteorology and only changes in emissions, the CH4 loss would have been signiﬁcantly different and there would have been a stronger increase in CH4 burden after 2006. Me- teorological variability explains to a large degree much of the stabilization of CH4 loss after 2010, and might thereby explain part of the large CH4 burden increase in 2011 and 2012. Around the millennium we see a stabilization of the loss in the simulation with ﬁxed meteorology, but increased loss in the main run. This implies that meteorological vari- ations contribute to a prolonged period (2003–2006) of sta- 3.1 Global methane budget Figure 2 shows the evolution of the CH4 budget over the period 1970–2012 for the main simulation. It presents total burden and loss calculated by the forward CTM run and the emissions applied in this simulation. The total burden shown in black is balanced by the emissions (blue) and the loss (red). There is a steady growth in atmospheric CH4 burden from 1970 to the beginning of the 1990s, then a short pe- riod of decline after the Mount Pinatubo volcanic eruption in 1991. After 1994 there is a slight increase in CH4 burden towards the millennium. Then the CH4 burden is stable for 5–6 years. After 2006 there is a rapid growth in CH4 burden. The evolution of emissions and the modelled CH4 burden share many common features (Fig. 2). However, the growth in emissions is about 35 % from 1970 to 2012, while the growth in atmospheric burden is about 15 % (additional bur- den increase after 2012 due to the long response time of CH4, is not accounted for in this number). The CH4 burden in- Figure 2 shows the evolution of the CH4 budget over the period 1970–2012 for the main simulation. It presents total burden and loss calculated by the forward CTM run and the emissions applied in this simulation. The total burden shown in black is balanced by the emissions (blue) and the loss (red). There is a steady growth in atmospheric CH4 burden from 1970 to the beginning of the 1990s, then a short pe- riod of decline after the Mount Pinatubo volcanic eruption in 1991. After 1994 there is a slight increase in CH4 burden towards the millennium. Then the CH4 burden is stable for 5–6 years. After 2006 there is a rapid growth in CH4 burden. The evolution of emissions and the modelled CH4 burden share many common features (Fig. 2). However, the growth in emissions is about 35 % from 1970 to 2012, while the growth in atmospheric burden is about 15 % (additional bur- den increase after 2012 due to the long response time of CH4, is not accounted for in this number). The CH4 burden in- www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 2.3 Observations To get insights into the drivers of the changes on regional level, and reveal strengths and discrepancies in model per- formance we compared the model results to surface CH4 observations. We thoroughly assessed the surface sites pro- viding CH4 measurements to the World Data Center for Greenhouse Gases (WDCGG) (http://ds.data.jma.go.jp/gmd/ wdcgg/), and picked out a subset of sites for comparison. www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 3.2 Evolution of global mean surface methane Figure 4 compares the global mean surface CH4 in the main model simulation, to global mean surface CH4 calculated from networks of surface stations. The main picture is dis- cussed in this section while more detailed evaluations of CH4 development on continental scale, trends, and inter- annual variations are made in the following sections. The time evolution of global mean surface CH4 is very simi- lar for the three observational networks shown in Fig. 4 but there are some differences for the absolute methane level. The AGAGE (mountain and coastal sites) and NOAA ESRL (sites in the marine boundary layer) stations are distant from large pollution sources. WDCGG uses curve ﬁtting and data extension methods very similar to those developed by NOAA and many of the same stations (Tsutsumi et al., 2009), but in addition to marine boundary layer sites, WDCGG in- cludes many continental locations strongly inﬂuenced by lo- cal sources and sinks (http://www.esrl.noaa.gov/gmd/ccgg/ mbl/mbl.html). The methane emission estimates from Bous- quet et al. (2011) are optimized against atmospheric obser- vations. Since we only use their natural and biomass-burning S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years Figure 4. Global mean surface CH4 mixing ratio in the main model simulation compared to global mean surface CH4 mixing ratio calculated from the global networks AGAGE (http://agage.eas.gatech.edu/data_archive/global_mean/global_mean_md.txt), NOAA ESRL (http://www. esrl.noaa.gov/gmd/ccgg/mbl/data.php), and WDCGG (http://ds.data.jma.go.jp/gmd/wdcgg/pub/global/globalmean.html). Figure 4. Global mean surface CH4 mixing ratio in the main model simulation compared to global mean surface CH4 mixing ratio calculated from the global networks AGAGE (http://agage.eas.gatech.edu/data_archive/global_mean/global_mean_md.txt), NOAA ESRL (http://www. esrl.noaa.gov/gmd/ccgg/mbl/data.php), and WDCGG (http://ds.data.jma.go.jp/gmd/wdcgg/pub/global/globalmean.html). bilization in CH4 burden (Fig. 3). From the comparison in Fig. 3 it can also be seen that it is meteorological factors and not emissions that cause the large enhancements of CH4 loss in 1998 (El Niño event) and 2010 (warm year on global scale). Such episodes do not show up as immediate pertur- bations of the CH4 burden (Figs. 2 and 3) due to the long response time of atmospheric CH4. Meteorology and other drivers for the modelled evolution of methane loss are dis- cussed in detail in Sects. 3.5–3.6. emission inventories, we use different anthropogenic emis- sions (from EDGAR), and the OH ﬁeld in their inverse model is substantially different from our modelled OH, there is no guarantee that our model will match observations. Our model generally reproduces the different periods of growth and stagnation and the overall observed increase in concentration from 1984 to 2012 of almost 180 ppb is repli- cated. This gives us conﬁdence when evaluating the decisive drivers explaining the variable evolution over time. However, the model fails to reproduce the strength of the growth rate during some eras, for instance the growth since 2006 is over- estimated. Over the whole period the model also underes- timate the observed CH4 level. Even though there are also large uncertainties in total CH4 emission levels (Kirschke et al., 2013; Ciais et al., 2013), we ﬁnd it more likely that our model overestimates the atmospheric CH4 sink. In a re- cent model inter-comparison, the multi-model global mean CH4 lifetime was underestimated by 5–13 % (Naik et al., 2013) compared to observational estimates. Our study shows a similar underestimation of CH4 lifetime. Though the multi- model lifetime is within the uncertainty range of observa- tions, it is likely that models tend to overestimate OH abun- dances in the Northern Hemisphere (Naik et al., 2013; Strode et al., 2015; Patra et al., 2014). www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 3104 3.3 Methane evolution and emission drivers in different regions As explained below the tracers play a small role in explaining CH4 at Cape Grim and Ushuaia, where B is below 1. = B × (< total tracer > −[< total tracer >]) (1) residual, (1) + residual, where [ ] denotes longitudinal mean along a whole terres- trial parallel and <> denotes annual running mean. We are interested in the inter-annual variation of CH4, so we have carried out annual running means to remove the strong sea- sonal cycle. The subtraction of longitudinal means on each side of Eq. (1) removes the inﬂuence of differences in life- times (the mean lifetime of CH4 is around 9 years, whereas the mean lifetime of the total tracer is 1 month). B and “resid- ual” are constants (or almost constant) if the prerequisites discussed in the Supplement (Sect. S3, last paragraph) are met. We expect B to be near or equal to 1 and residual to be small. If B and residual were exactly constant, the Pear- son linear correlation coefﬁcient between < CH4 model > −[< CH4 model >] and < total tracer > −[< total tracer >] would be exactly equal to 1. The tracer approach then gives valuable information concerning the contribution to CH4 variation from recent regional–local emission or transport changes. We therefore use the correlation coefﬁcient (in- deed, its square, R2: the coefﬁcient of determination obtained where [ ] denotes longitudinal mean along a whole terres- trial parallel and <> denotes annual running mean. We are interested in the inter-annual variation of CH4, so we have carried out annual running means to remove the strong sea- sonal cycle. The subtraction of longitudinal means on each side of Eq. (1) removes the inﬂuence of differences in life- times (the mean lifetime of CH4 is around 9 years, whereas the mean lifetime of the total tracer is 1 month). B and “resid- ual” are constants (or almost constant) if the prerequisites discussed in the Supplement (Sect. S3, last paragraph) are met. We expect B to be near or equal to 1 and residual to be small. If B and residual were exactly constant, the Pear- son linear correlation coefﬁcient between < CH4 model > −[< CH4 model >] and < total tracer > −[< total tracer >] would be exactly equal to 1. 3.3 Methane evolution and emission drivers in different regions Alert, Tutuila, Mahe Island, and Key Biscayne are also remote stations that have a high B. As explained below the tracers play a small role in explaining CH4 at Cape Grim and Ushuaia, where B is below 1. achieving a good mixing (after 1–2 months) it is converted into the background component. We show how the use of a 1-month e-folding ﬁctitious tracer (total tracer) is valid as a proxy for the inhomogeneous component. The CH4 surface emissions act as the sources for the tracer. In the Supple- ment we use the continuity equation for the CH4 mole frac- tion (CH4 model) as starting point and further arguments to derive the following approximation: when performing a linear least-square ﬁt between both mag- nitudes in Eq. 1 to determine B and residual) as one criterion when selecting interesting stations for methane trend studies. Only stations where R2 is higher than 0.5 is used. This crite- rion excludes only a small number of the available stations. In addition, we use the general station selection criteria dis- cussed earlier in the manuscript (sufﬁcient coverage in the different world regions, long time series etc., see Sect. 2.3). Figure 5 shows the locations of stations used in Figs. 6–10 for detailed trend analysis and evaluation of model performance. < CH4 model > −[< CH4 model >] = B × (< total tracer > −[< total tracer >]) + residual, (1) < CH4 model > −[< CH4 model >] y p Table 2 shows R2, the constants B and residual, and RMSE from a linear ﬁt of the variables in Eq. (1). All stations except one (reason for exception at the Wendover station is discussed in the Supplement) have R2 above 0.8. Such high coefﬁcients support that the approximation in Eq. (1) is use- ful for these stations. As expected, B is usually larger than 1. The ﬁctitious tracer will underestimate somewhat the in- homogeneous recently emitted CH4, in particular at remote stations, because part of it is removed by the e-folding sink before being smoothed to the characteristic variation length of the background. Mauna Loa is probably the most remote station and located at high altitude. It has the largest B and residual. Alert, Tutuila, Mahe Island, and Key Biscayne are also remote stations that have a high B. 3.3 Methane evolution and emission drivers in different regions The tracer approach then gives valuable information concerning the contribution to CH4 variation from recent regional–local emission or transport changes. We therefore use the correlation coefﬁcient (in- deed, its square, R2: the coefﬁcient of determination obtained In the upper panels of Figs. 6–10, the model results are scaled to the observed mean CH4 level over the periods of measurements to better discern differences in trends between observations and model. The scaling procedure is explained in the Supplement. In general, the model reproduces the sea- sonal and year-to-year variations very well with high coefﬁ- Atmos. Chem. Phys., 16, 3099–3126, 2016 3.3 Methane evolution and emission drivers in different regions In the Supplement, we explain how the CH4 mole fraction can be split into two components: a quite uniform back- ground component and an inhomogeneous recently emit- ted component. The latter is advected and mixed, and when www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3105 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3105 Figure 5. Location of the 18 surface stations used in comparison between measurements and model in this section. Blue: stations in the Southern Hemisphere; orange: stations in or near North America; green: stations in or near Europe; red: stations in or near Asia. Figure 5. Location of the 18 surface stations used in comparison between measurements and model in this section. Blue: stations in the Southern Hemisphere; orange: stations in or near North America; green: stations in or near Europe; red: stations in or near Asia. when performing a linear least-square ﬁt between both mag- nitudes in Eq. 1 to determine B and residual) as one criterion when selecting interesting stations for methane trend studies. Only stations where R2 is higher than 0.5 is used. This crite- rion excludes only a small number of the available stations. In addition, we use the general station selection criteria dis- cussed earlier in the manuscript (sufﬁcient coverage in the different world regions, long time series etc., see Sect. 2.3). Figure 5 shows the locations of stations used in Figs. 6–10 for detailed trend analysis and evaluation of model performance. Table 2 shows R2, the constants B and residual, and RMSE from a linear ﬁt of the variables in Eq. (1). All stations except one (reason for exception at the Wendover station is discussed in the Supplement) have R2 above 0.8. Such high coefﬁcients support that the approximation in Eq. (1) is use- ful for these stations. As expected, B is usually larger than 1. The ﬁctitious tracer will underestimate somewhat the in- homogeneous recently emitted CH4, in particular at remote stations, because part of it is removed by the e-folding sink before being smoothed to the characteristic variation length of the background. Mauna Loa is probably the most remote station and located at high altitude. It has the largest B and residual. www.atmos-chem-phys.net/16/3099/2016/ In the 1997–1998 period, there are peaks both for the natural tracer and < total tracer > −[< total tracer >] indicating a rise in nearby natural emissions and/or trans- port from such a source. For 1987 a regional drop in natu- ral emissions has a smaller impact at Ascension compared to the whole latitude band. At Tutuila < total tracer > −[< total tracer >] decreases over time due to a relatively larger increase in the latitudinal mean anthropogenic tracers (not shown), especially enteric fermentation. This explains why the CH4 growth at the site (< CH4 model >) is slightly less than the mean latitudinal ([< CH4 model >]) growth. cients of determination, R2, for most stations (the median is 0.76, and R2 is above 0.65 for 15 of 18 stations). The model performance is lower at highly polluted sites due to large gra- dients in concentrations and non-linearity of oxidant chem- istry not fully captured by a global model with coarse reso- lution (approximately 2.8◦× 2.8◦). The model also captures the long-term evolution of CH4 seen in the observations but overestimates the increase after 2005 at most stations. The stations in the Southern Hemisphere (Fig. 6) are lo- cated far from the dominating emissions sources, and the CH4 concentration is to a large degree determined by trans- port and chemical loss. The high coefﬁcients of determina- tion ranging from 0.92 to 0.95 and reproduction of the sea- sonality and trends indicate that our model is performing ex- cellent with respect to transport and seasonal variation in the chemical loss. As seen in the mid panels, Ascension Island (Fig. 6a) and Tutuila (Fig. 6b) have negative < total tracer > −[< total tracer >]. Since these are rather remote stations, their tracer levels are below the longitudinal mean. The modelled CH4 evolution from 1990 to 2005 is well correlated with the development of the natural tracers. However, changes in natural emissions do not seem to explain the periods with large growth before 1990 and for the period 2005–2012. While the model underestimates the growth before 1990 it overestimates the growth in the recent years. The small steady increases in contributions from all anthropogenic sec- tors only has a minor contribution to the modelled CH4 in- Ushuaia (Fig. 6c) and Cape Grim (Fig. 6d) are the south- ernmost stations. www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3106 Table 2. Coefﬁcient of determination (R2) between < CH4model > −[< CH4 model >] and < total tracer > −[< total tracer >] for stations shown in Figs. 5–10. Parameters for Eq. (1) and RMSE for a linear ﬁt between < CH4 model > −[< CH4 model >] and < total tracer > −[< total tracer >]. Station Figure R2 between < CH4 model > residual B RMSE −[< CH4 model >] and < total tracer > −[< total tracer >] Ascension Island 6a 0.80 −3.01 1.21 0.74 Tutuila 6b 0.87 5.08 1.49 0.82 Cape Grim 6c 0.98 −0.15 0.97 0.05 Ushuaia 6d 0.83 −0.27 0.94 0.09 Alert 7a 0.69 −2.16 1.66 0.85 Wendover 7b 0.54 −5.74 0.78 1.07 Key Biscayne 7c 0.95 6.10 1.38 1.40 Mauna Loa 7d 0.87 18.41 1.80 1.27 Zeppelinfjellet 8a 0.91 −1.67 1.13 0.59 Pallas–Sammaltun 8b 0.95 −3.38 1.18 0.75 Mace Head 8c 0.97 −3.28 1.16 0.56 Hegyhatsal 8d 1.00 −2.46 1.15 0.96 Sede Boker 9a 0.83 5.41 1.23 0.97 Ulaan Uul 9b 0.95 1.15 1.10 0.65 Sary Taukum 9c 0.97 −8.27 1.11 0.96 Tae-ahn Peninsula 9d 0.97 0.77 1.07 1.15 Cape Rama 10a 0.92 −9.60 1.24 1.02 Mahe Island 10b 0.85 6.68 1.42 1.22 Table 2. Coefﬁcient of determination (R2) between < CH4model > −[< CH4 model >] and < total tracer > −[< total tracer >] for stations shown in Figs. 5–10. Parameters for Eq. (1) and RMSE for a linear ﬁt between < CH4 model > −[< CH4 model >] and < total tracer > −[< total tracer >]. crease for these periods. However, since these source trac- ers have an e-folding lifetime of 1 month their evolution is only representative for changes in contribution from re- gional sources. Inter-hemispheric transport occurs on longer timescales; hence, changes in large anthropogenic sources in the Northern Hemisphere most likely also had a signiﬁ- cant contribution as discussed below. At Ascension Island, extra strong inﬂuences of regional sources (< CH4 model > −[< CH4 model >] change different from zero) are mainly associated with El Niño episodes (1987, 1997–1998, and 2004–2005). Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3107 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 310 Figure 6. Evolution of CH4 and tracers at stations (a: Ascension Island, b: Tutuila, c: Cape Grim, d: Ushuaia) in the Southern Hemispher Upper panel in each ﬁgure: comparison of monthly mean surface CH4 in model and observations. The model results are scaled to th observed mean CH4 level over the periods of measurements. Mid panels: variables from Eq. (1). <> denotes annual running mean, [ denotes longitudinal mean. Left y axis: < CH4 model > and [< CH4 model >] are scaled down to be initialized to zero in the ﬁrst yea Right y axis: B × (< total tracer > −[< total tracer >]) and residual. Lower panels: Evolution of various emission tracers, see Table S1 the Supplement for detailed information. Figure 6. Evolution of CH4 and tracers at stations (a: Ascension Island, b: Tutuila, c: Cape Grim, d: Ushuaia) in the Southern Hemisphere. Upper panel in each ﬁgure: comparison of monthly mean surface CH4 in model and observations. The model results are scaled to the observed mean CH4 level over the periods of measurements. Mid panels: variables from Eq. (1). <> denotes annual running mean, [ ] denotes longitudinal mean. Left y axis: < CH4 model > and [< CH4 model >] are scaled down to be initialized to zero in the ﬁrst year. Right y axis: B × (< total tracer > −[< total tracer >]) and residual. Lower panels: Evolution of various emission tracers, see Table S1 in the Supplement for detailed information. trends are small at high southern latitudes, the distant trans- port likely originates from low latitudes in the Southern Hemisphere or the Northern Hemisphere. cisive. Distant latitudinal transport is not seen by the tracer term if it takes more than around 2 months. Such trans- port would also result in very similar < CH4 model > and [< CH4 model >] since atmospheric species with lifetime of that timescale or longer are quite homogenously distributed over latitudinal bands. Since both the emissions and their At stations in or near North America (Fig. 7) the model reproduces the observed trends with increases in the 1980s, less change in the period 1990–2005 and increase from 2006. www.atmos-chem-phys.net/16/3099/2016/ In the mid panels it can be seen that both terms on the right side in Eq. (1) are small (B × (< total tracer > −[< total tracer >] and residuals) resulting in small (< CH4 model > −[< CH4 model >]). This indicates that the contribution to CH4 from regional emissions are small and that long-range transport from other latitudes is de- www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3108 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3109 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years Figure 8. Evolution of CH4 and tracers at stations (a: Zeppelinfjellet, b: Pallas–Sammaltun, c: Mace Head, d: Hegyhatsal) in or near E See Fig. 6 caption for further description. Figure 8. Evolution of CH4 and tracers at stations (a: Zeppelinfjellet, b: Pallas–Sammaltun, c: Mace Head, d: Hegyhatsal) in or near Europe. See Fig. 6 caption for further description. coal) sector as its contribution increases from 2003 and on- wards. The same occurs for this sector at Alert (Fig. 7a). It corresponds with the start of an increase in US fugitive solid fuel emissions in the applied EDGAR v4.2 inventory. The increase in US coal emissions from 2003 to 2008 is almost 12 % in EDGAR v4.2. An increase of 28 % is found from 2005 to 2010 in the EPA inventory (EPA, 2012). At the high altitude sites Mauna Loa and Wendover (Fig. 7b and d) there are small or large increases in the contribution from all an- thropogenic sectors from the year 2000 and onwards. These stations are subject to efﬁcient transport from Asia at high altitudes. There are large emission increases after 2000 in eastern Asia in the EDGAR v4.2 inventory (Bergamaschi et al., 2013). Especially coal related emissions in China show a strong increase with a doubling from 2000 to 2008. stations are subject to efﬁcient transport from Asia at high altitudes. There are large emission increases after 2000 in eastern Asia in the EDGAR v4.2 inventory (Bergamaschi et al., 2013). Especially coal related emissions in China show a strong increase with a doubling from 2000 to 2008. At Wendover, Mauna Loa and Key Biscayne < total tracer > −[< total tracer >] decrease over the 3 decades studied (Fig. 7, mid panels). Several emission sectors contribute. The implication is a lower growth rate for < CH4 model > than for [< CH4 model >] (Fig. 7, mid At Wendover, Mauna Loa and Key Biscayne < total tracer > −[< total tracer >] decrease over the 3 decades studied (Fig. 7, mid panels). Several emission sectors contribute. The implication is a lower growth rate for < CH4 model > than for [< CH4 model >] (Fig. 7, mid www.atmos-chem-phys.net/16/3099/2016/ S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years Figure 7. Evolution of CH4 and tracers at stations (a: Alert, b: Wendover, c: Key Biscayne, d: Mauna Loa) in or near North America. See Fig. 6 caption for further description. Figure 7. Evolution of CH4 and tracers at stations (a: Alert, b: Wendover, c: Key Biscayne, d: Mauna Loa) in or near North America. See Fig. 6 caption for further description. For the latest period, the increase in the model is larger than that observed. The seasonal and year-to-year variations are well represented by the model at all stations (coefﬁ- cients of determination from 0.73 to 0.82). Key Biscayne (Fig. 7c) and Mauna Loa (Fig. 7d) have relatively large neg- ative < total tracer > −[< total tracer >] which shows that these are background stations and that important emission sources exist at their latitude. The tracer difference is quite small and negative at Alert (Fig. 7b) and since the resid- ual is quite close to zero, this may indicate small sources at the station latitude. The contribution from natural emis- sions is decisive for year-to-year variations at all four stations in Fig. 7, and the inﬂuence of emission from the gas sector increases gradually. Key Biscayne situated in the boundary layer (Fig. 7c) is mostly inﬂuenced by emissions from the American continent, and the rest of the anthropogenic sec- tors have moderately declining impact after 1990. However, this decline occurs only initially for the solid fuel (mainly www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 3110 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years The contribution from natural emissions and re- cent regional coal mining peaked in 2007. A quite strong CH4 enhancement occurs for 2009–2010 in both the model and observations. The longitudinal mean tracers for individ- ual sectors are almost stable to declining (not shown) while contribution from the < gas > and some other tracers show a small maximum (lower panel Fig. 8a and b). Pallas has a similar pattern. The runs with ﬁxed meteorology suggest en- hanced transport from Russia passing major gas ﬁelds and Pallas. The model has larger discrepancies at Hegyhatsal, a semi- polluted site in central Europe (Fig. 8d). Despite seasonal is- sues the model performance is reasonable for the long-term CH4 changes. In years with high contributions from natu- ral sources, the seasonal maxima tend to be too high in the model. It could be that the coarse model resolution results in too much transport from nearby wetlands or that the emission inventory has overly large natural emissions in surround- ing regions. < total tracer > −[< total tracer >] is very large and positive meaning that the station is very sensitive to emissions close upwind. The evolution of < CH4 model > therefore deviates strongly from the longitudinal mean [< CH4 model >]. The deviation starts in 1996 when a sharp in- crease in natural emission occurs. From 2003 to 2008 there Mace Head (Fig. 8c) is a rural background coastal site in Europe. The result of < total tracer > −[< total tracer >] is quite large and negative, suggesting important emission sources along the station’s latitude. In the beginning of the 1990s, there is a mismatch between declining model concen- trations and the increase found from the observations. Some of the decrease in the model is due to decreasing contri- butions from solid fuel (mainly coal), enteric fermentation and other regional anthropogenic sources. The station ex- periences unusual meteorological conditions in the ENSO S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3111 Figure 10. Evolution of CH4 and tracers at stations (a: Cape Rama, b: Mahe Island) in background/outﬂowing air in or near Asia. See Fig. 6 caption for further description. Figure 10. Evolution of CH4 and tracers at stations (a: Cape Rama, b: Mahe Island) in background/outﬂowing air in or near Asia. See Fig. 6 caption for further description. Figure 10. Evolution of CH4 and tracers at stations (a: Cape Rama, b: Mahe Island) in background/outﬂowin caption for further description. by natural source contribution in the model falling from a pe- riod maximum in 2004 to low values in 2005–2006. This is also the case for the sub-Arctic site Pallas (Fig. 8b) located in a region characterized by forest and wetlands. Gas, enteric fermentation and various other small regional anthropogenic sources seems to contribute to the CH4 increase at Zeppelin after 2006. The contribution from natural emissions and re- cent regional coal mining peaked in 2007. A quite strong CH4 enhancement occurs for 2009–2010 in both the model and observations. The longitudinal mean tracers for individ- ual sectors are almost stable to declining (not shown) while contribution from the < gas > and some other tracers show a small maximum (lower panel Fig. 8a and b). Pallas has a similar pattern. The runs with ﬁxed meteorology suggest en- hanced transport from Russia passing major gas ﬁelds and Pallas. year 1997, as there are abrupt shifts in concentrations of CH4 and several of the anthropogenic tracers having small year-to-year variations in emissions. Similarly, there seems to be transport of less polluted air masses to the station in 2004 compared to earlier years resulting in lower CH4 con- centration in measurements and model in 2004 and 2005. Several regional sources seem to have small contributions to the modelled and observed CH4 increases from 2006 to 2009. After 2009 we extrapolate emission trends due to lack of emission inventories and this may be the reason why the model doesn’t reproduce the observed levelling off in growth in 2010 and 2011. by natural source contribution in the model falling from a pe- riod maximum in 2004 to low values in 2005–2006. This is also the case for the sub-Arctic site Pallas (Fig. 8b) located in a region characterized by forest and wetlands. Gas, enteric fermentation and various other small regional anthropogenic sources seems to contribute to the CH4 increase at Zeppelin after 2006. S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years Evolution of CH4 and tracers at stations (a: Sede Boker, b: Ulaan Uul, c: Sary Taukum, d: Tae-ahn Peninsula) near Asian emission See Fig. 6 caption for further description. Figure 9. Evolution of CH4 and tracers at stations (a: Sede Boker, b: Ulaan Uul, c: Sary Taukum, d: Tae-ahn Peninsula) near Asian emission sources. See Fig. 6 caption for further description. variability in the period 1997–1999 (Morimoto et al., 2006) was due to ﬂuctuations in wetland and biomass-burning emissions. Our modelled variation in the natural source tracer conforms to the ﬂuctuations deduced from the isotopic mea- surements of Morimoto et al. (2006). Seasonal tracer analysis (not shown) is in agreement with the conclusion of Fisher et al. (2011), who found that wetlands and gas are the main con- tributors in summer and winter, respectively. A CH4 concen- tration drop from 2004 to 2006 seems to mainly be explained panels); i.e. other locations (for Asian stations, see discus- sion below) at the same latitudes have a larger trend in CH4. There are large ﬂuctuations of tracer transport to Mauna Loa in 1997–1998 and 2010–2011 that strongly impacts < CH4 model >. The observations also show changes in growth and seasonal pattern during these years. At the Arctic site Zeppelin (Fig. 8a), located on the coast of western Svalbard, there is a small CH4 increase both in model and observations up to 2004. A large part of the CH4 www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 B. Dalsøren et al.: Atmospheric methane evolution the last 40 years Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3112 is a period with stable to declining modelled CH4 concentra- tions. This is caused by decreasing central European emis- sions particularly from enteric fermentation and the category “other anthropogenic sectors” together with decreasing or ﬂuctuating natural sources. ern Hemispheric (NH and SH) air masses (Bhattacharya et al., 2009). Mixed with regional ﬂuxes and varying chemical loss, this results in large seasonal variation. During the sum- mer monsoon, the station is located south of the inter-tropical convergence zone. Air arriving during this period (June to September) represent tropical or SH oceanic air masses and the station is upwind of Mahe Island (Fig. 10b). During the winter monsoon the situation is opposite. There is outﬂow from the continent affecting both Cape Rama and Mahe Is- land. The ENSO event in 1997 seems to have opposite effects on modelled and observed CH4 variability at Cape Rama. Despite that, the model does a reasonable job in reproduc- ing the measurements. Most regional tracers show stable to upward levels over the period of comparison and likely con- tribute to a small fraction of the modelled CH4 trend. At Mahe Island in the SH (Fig. 10b), the CH4 concentration peaks sharply during NH winter when the station is inﬂu- enced by outﬂow from continental Asia. The station is there- fore an indicator of inﬂow to the SH. This feature is well captured by the model. Over the last decade, there is a small and continuous rise in the levels of all anthropogenic tracers at the station. This coincides with large emission increases in Asia, suggesting that the recent development in Asia has some inﬂuence on the SH. g In general, the model reproduces the features in the ob- servations over and near Asia quite well (Figs. 9 and 10) with coefﬁcient of determination in the range of 0.24–0.84. For the trends, the overestimation after 2006 is higher here than modelled in other world regions (Figs. 6–8). Gas is the major cause of increases in CH4 in Israel (Sede Boker, Fig. 9a). The increase of the < gas > tracer is much larger than for the longitudinal mean [< gas >], suggesting im- portant emission increases from nearby gas ﬁelds. Small changes in regional natural emissions and in the category “other anthropogenic sources” (lower panel) are correlated with the modelled year-to-year variations (upper panel). The station in Kazakhstan (Fig. 3.4 Methane evolution and emission drivers over distinct time periods Figure 11 compares the latitudinal distribution of surface CH4 in the model and observations. Generally, the model and the observational approach reveal the same pattern and characteristics both in time and space, although some clear differences are evident. From 1985 to the early 1990s, there is a homogeneous growth in the observations (Fig. 11b). The model (Fig. 11a) also has growth over the same period but a distinct period (1987–1988) with no growth, correspond- ing to smaller emissions from wetlands and biomass burn- ing (Fig. 1). 1987–1988 were El Niño years, and there is a tendency of low wetland emissions for those years, e.g. an anti-correlation between wetland emissions and ENSO in- dex (Hodson et al., 2011). One possibility is that our ap- plied emission inventory for natural CH4 sources (Bousquet et al., 2011) has overly large variability in wetland emissions in the 1980s and overly strong reductions in wetland emis- sions in 1987–1988. Bousquet et al. (2006) state that bias in OH inferred from methyl chloroform (CH3CCl3) obser- vations (Bousquet et al., 2005) could account for some of the variability that they attributed to wetland emissions. Later ﬁndings (Montzka et al., 2011) support this. If OH changes are set to zero instead of the large variability in the 1980s, suggested by early CH3CCl3 studies (Bousquet et al., 2005), the ﬂuctuations in wetland emissions are dampened by 50 %. On the other hand, the model simulation has no year-to-year variation in meteorology before 1997, and the meteorology used corresponds to the year 2001, which has a weak ENSO Regional solid fuel emissions (mainly coal) is the main cause of last-decade-modelled CH4 increase in eastern con- tinental Asia (Ulaan Uul and Tae-ahn Peninsula, Fig. 9b and d), but gas and other reginal anthropogenic sectors also con- tribute. There is large growth in < CH4 model > for Ulaan Uul in 2006–2007 and 2010 mainly due to peaks in the contribution from solid fuel sources, but also other anthro- pogenic sectors have a role in this. Similar pattern appears for Tae-ahn Peninsula in 2009. The ﬁrst peak at Ulaan Uul is also partly seen in the observations, but the existence of the latest episode and the event at Tae-ahn Peninsula is less clear from the measurements. Our tracer analysis for Minamitori- shima (not shown), a background station affected by outﬂow from the Asian continent indicates less continental outﬂow in 2007. S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 9c) is downwind of large sources (< total tracer > −[< total tracer >] large and positive), and the modelled CH4 increase after 2005 is much larger than for the longitudinal mean. Also at this station, the CH4 trend is heavily inﬂuenced by gas, although not to the same extent as in Israel. Other regional anthropogenic emission changes also contribute somewhat to the modelled CH4 increase over recent years. High natural emissions in 2008–2009 also had an impact. Since we use repetitive year-2009 natural emis- sions for the latter years, it could be that the contribution from this source is too large after 2009. Unfortunately, the modelled CH4 increase cannot be conﬁrmed by measure- ments since data at the station is missing after 2008. www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3113 Figure 11. CH4 year-to-year variation (ppb) in surface CH4 in model (a) compared to the levels of surface CH4 estimated from observa- tions (b) in various latitudinal bands based on the NOAA ESRL network of surface stations (Ciais et al., 2013, and data set provided by Edward J. Dlugokencky, personal communication, 2015). Figure 11. CH4 year-to-year variation (ppb) in surface CH4 in model (a) compared to the levels of surface CH4 estimated from observa- tions (b) in various latitudinal bands based on the NOAA ESRL network of surface stations (Ciais et al., 2013, and data set provided by Edward J. Dlugokencky, personal communication, 2015). index. Therefore, during the 1987–1988 El Niño, the mete- orology used is less representative than for other years with weaker ENSO. In the two periods of CH4 growth before and after 1987–1988, the CH4 increase is strong in the model (Fig. 11a) in the Northern Hemisphere and might be over- estimated. However, it might be that the model is able to bet- ter capture latitudinal gradients, as only a few measurement sites are available to make latitudinal averages for the 1980s. In 1992 and 1993 there is a pause in the CH4 growth in the measurements (Fig. 11b) at all latitudes. This pause has been explained as a consequence of the Mount Pinatubo volcanic eruption in 1991 (Dlugokencky et al., 1996; Bekki and Law, 1997; Bânda et al., 2013). The eruption results in an initial increase in the CH4 growth rate (less OH) lasting for half a year. This is due to backscattering by volcanic stratospheric aerosols, which reduces the UV radiation to the troposphere. After that, the growth rate due to Pinatubo becomes negative (more OH plus less natural methane emissions are the domi- nating effects) reaching a minimum after 2 years (1993), be- fore levelling off towards zero after 5 years. The main cause of the OH increase is reduction in stratospheric ozone allow- ing more UV radiation to the troposphere. In contrast to the measurements the model shows a stronger decrease in CH4 after the eruption, and the pause in CH4 growth is longer. This might be due the fact that the model does not fully in- clude all factors affecting CH4 related to the Mount Pinatubo eruption. S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years Reduced emissions are implicitly included in the natural CH4 emission inventories, but changes in meteorol- ogy (temperature, water vapour, etc.) and volcanic SO2 and sulphate aerosols in the stratosphere, are not accounted for in the simulations. In the period 1994–1997 the model struggles to reproduce the latitudinal distribution of growth (Fig. 11). The model seems to have overly large growth in the Tropics probably due to a small but signiﬁcant growth in wetland and biomass-burning emissions in the period (Fig. 1). In the next paragraphs, we study whether the model is able to reproduce CH4 measurements when we split the time frame into shorter epochs that measured distinct different growth rates. The splits are made within the period 1998– 2009 when our simulations have both inter-annual variation in meteorology and complete emission data (no extrapola- tions made). We have only included observation sites that have measurements available for all months within the given time period, see Sect. 2.3 for details about data selection. Figure 12 shows the modelled CH4 growth in the CTM in the period 1998–2000, compared to the observed changes at various sites. The model seems to slightly underestimate increases at several stations. The largest underestimation oc- cur in eastern Asia. In parts of eastern Asia and some other regions in the Northern Hemisphere there are declines in modelled CH4 concentrations caused by decreased contribu- tion from several anthropogenic sectors. Increased emissions Atmos. Chem. Phys., 16, 3099–3126, 2016 3.4 Methane evolution and emission drivers over distinct time periods For these polluted continental sites the correlation coefﬁcients are lower than for the other stations. The coarse resolution of the model has problems resolving large gradi- ents in concentrations and non-linearity of oxidant chemistry. At Tae-ahn Peninsula < CH4 model > starts increasing in 2005, while the increase at Ulaan Uul ﬁrst starts in 2006. At Ulaan Uul decreasing regional natural emissions over the pe- riod 2000–2005 seems to compensate for the large increase of solid fuel emissions from around 2000. For Cape Rama in India (Fig. 10a, the observations show signatures of both Northern Hemispheric and South- www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years www.atmos-chem-phys.net/16/3099/2016/ www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3114 Figure 12. (Upper panel) Mean year-to-year growth (ppb yr−1) in surface CH4 in Oslo CTM3 over the period 1998–2000. The 32 circles show the observed growth rates over the same period. The stations picked for comparison are based on the criteria described in Sect. 2.3, and only observation sites that have measurements available for all months within the given time is included. (a–f) Mean year-to-year growth ppb yr−1) of emission tracers in the same period. (a) Natural (wetlands + other natural + biomass burning), (b) enteric, (c) agricultural soils, (d) gas, (e) solid fuel, (f) the sum of all other anthropogenic tracers. Figure 12. (Upper panel) Mean year-to-year growth (ppb yr−1) in surface CH4 in Oslo CTM3 over the period 1998–2000. The 32 circles show the observed growth rates over the same period. The stations picked for comparison are based on the criteria described in Sect. 2.3, and only observation sites that have measurements available for all months within the given time is included. (a–f) Mean year-to-year growth ppb yr−1) of emission tracers in the same period. (a) Natural (wetlands + other natural + biomass burning), (b) enteric, (c) agricultural soils, (d) gas, (e) solid fuel, (f) the sum of all other anthropogenic tracers. Wetland and biomass burning sources seem to play the key role for the variations in the model from 1997 to 2000 (Fig. 12a). They were particularly large in 1998 due to the 1997–1998 El Niño (Chen and Prinn, 2006; Simpson et al., 2002; Dlugokencky et al., 2001; Bousquet et al., 2006; Pison et al., 2013; Spahni et al., 2011; Hodson et al., 2011). Simp- son et al. (2002) also conclude that the increase in observed surface CH4 between 1996 and 2000 was driven primarily by a large growth in 1998. Both model and measurements have the strongest growth (Fig. 12) in the Southern Hemi- sphere, which had large wetland emissions in 1998 (Bous- quet et al., 2006; Dlugokencky et al., 2001). In the model, slowly rising anthropogenic emissions in the Southern Hemi- sphere also seems to contribute (Fig. 12b–f). Natural emis- sions (Fig. 12a) are also important for the irregular pattern seen at mid-to-high northern latitudes. www.atmos-chem-phys.net/16/3099/2016/ 2.3, and only observation sites that have measurements available for all months within the given time is included. (a–f) Mean year-to-year growth (ppb yr−1) of emission tracers in the same period. (a) Natural (wetlands + other natural + biomass burning), (b) enteric, (c) agricultural soils, (d) gas, (e) solid fuel, (f) the sum of all other anthropogenic tracers. Figure 13. (Upper panel) Mean year-to-year growth (ppb yr−1) in surface CH4 in Oslo CTM3 over the period 2001–2006. The 25 circles show the observed growth rates over the same period. The stations picked for comparison is based on the criteria described in Sect. 2.3, and only observation sites that have measurements available for all months within the given time is included. (a–f) Mean year-to-year growth (ppb yr−1) of emission tracers in the same period. (a) Natural (wetlands + other natural + biomass burning), (b) enteric, (c) agricultural soils, (d) gas, (e) solid fuel, (f) the sum of all other anthropogenic tracers. model and measurements (Fig. 11) is very similar for the Southern Hemisphere but there are larger differences for the Northern Hemisphere. creases in anthropogenic emissions and decreased wetland emissions together with moderate increasing OH can explain the stagnation in CH4 growth from 2000. Bergamaschi et al. (2013), assuming constant OH, also ﬁnds a decrease in wetland emissions but that a large increase in anthropogenic emissions ﬁrst occurs from 2006 and beyond. Uncertainty in wetland emissions in the period is well illustrated by Pi- son et al. (2013). Using different methods to estimate global wetland emissions from 2000 to 2006, Pison et al. (2013) ﬁnds either a decrease or an increase. On the other hand, in- crease in both wetland and anthropogenic emission would not conform to the observed stable global mean CH4 levels in this period. Spahni et al. (2011) found a small decrease in wetland emissions from 1999–2004 followed by an in- crease from 2004 to 2008. Our model results from simula- tions with declining natural emissions and increasing anthro- pogenic emissions (Fig. 1) reproduce the measurements in most regions (Fig. 13). Eastern Asian stations are exceptions. Gas and solid fuels (coal) (Fig. 13d, e) are causing much of the modelled increases over southern and eastern Asia. During 2000–2006 the CH4 growth levelled off and there was a period with stagnation in global mean growth rate (Fig. 13). www.atmos-chem-phys.net/16/3099/2016/ This is expected due to the 1997–1998 ENSO-event, showing a dip in high north- ern wetland emissions in 1997 followed by unusual large emissions in 1998 (Bousquet et al., 2006; Dlugokencky et al., 2001). During the ENSO event, the zonal pattern in the from gas ﬁelds in Russia, the Middle East, and in several anthropogenic tracers over India explain why these are the regions in the Northern Hemisphere with largest modelled CH4 increase. Earlier studies ﬁnd that a low CH4 growth rate in the 1990s is mostly caused by lower fugitive fossil fuel emissions from oil and gas industries, mainly due to the collapse of the So- viet Union (Bousquet et al., 2006; Simpson et al., 2012; Dlu- gokencky et al., 2003; Aydin et al., 2011). Another important factor is decreased emissions from rice paddies. Lower emis- sions from agricultural soils last until around the year 2000 in the EDGAR v4.2 inventory (Fig. 1) and are also evident in Fig. 12c. Kai et al. (2011) exclude fossil fuel emissions as the primary cause of the slowdown of CH4 growth. According to their isotopic studies, it is more likely long-term reductions in agricultural emissions from rice crops in Asia, or alterna- tively another microbial source in the Northern Hemisphere that is the major factor. Another isotope study (Levin et al., 2012) disagrees and ﬁnds that both fossil and microbial emis- sions were quite stable. www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3115 Figure 13. (Upper panel) Mean year-to-year growth (ppb yr−1) in surface CH4 in Oslo CTM3 over the period 2001–2006. The 25 circles show the observed growth rates over the same period. The stations picked for comparison is based on the criteria described in Sect. 2.3, and only observation sites that have measurements available for all months within the given time is included. (a–f) Mean year-to-year growth (ppb yr−1) of emission tracers in the same period. (a) Natural (wetlands + other natural + biomass burning), (b) enteric, (c) agricultural soils, (d) gas, (e) solid fuel, (f) the sum of all other anthropogenic tracers. Figure 13. (Upper panel) Mean year-to-year growth (ppb yr−1) in surface CH4 in Oslo CTM3 over the period 2001–2006. The 25 circles show the observed growth rates over the same period. The stations picked for comparison is based on the criteria described in Sect. www.atmos-chem-phys.net/16/3099/2016/ The agreement between the zonal averages from the model and the measurement approach is excellent, both with regards to timing and strength of the growth (Figs. 11 and 13). The 2002–2003 anomaly in the Northern Hemi- sphere is captured by the model (Fig. 11) and explained by enhanced emissions from biomass burning in Indonesia and boreal Asia (Bergamaschi et al., 2013; Simpson et al., 2006; van der Werf et al., 2010). The EDGAR v4.2 inventory applied here and in other stud- ies (e.g. Bergamaschi et al., 2013) show that global anthro- pogenic emissions rise substantially, especially in Asia after the year 2000. This increase in the anthropogenic emissions is compensated by a drop in northern tropical wetland emis- sions associated with years of dry conditions (Bousquet et al., 2006, 2011). Monteil et al. (2011) ﬁnd that moderate in- www.atmos-chem-phys.net/16/3099/2016/ www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3116 Figure 14. (Upper panel) Mean year-to-year growth (ppb yr−1) in surface CH4 in Oslo CTM3 over the period 2007–2009. The 36 circles show the observed growth rates over the same period. The stations picked for comparison are based on the criteria described in Sect. 2.3, and only observation sites that have measurements available for all months within the given time is included. (a–f) Mean year-to-year growth (ppb yr−1) in mole fraction of emission tracers in the same period. (a) Natural (wetlands + other natural + biomass burning), (b) enteric, (c) agricultural soils, (d) gas, (e) solid fuel, (f) the sum of all other anthropogenic tracers. Figure 14. (Upper panel) Mean year-to-year growth (ppb yr−1) in surface CH4 in Oslo CTM3 over the period 2007–2009. The 36 circles show the observed growth rates over the same period. The stations picked for comparison are based on the criteria described in Sect. 2.3, and only observation sites that have measurements available for all months within the given time is included. (a–f) Mean year-to-year growth (ppb yr−1) in mole fraction of emission tracers in the same period. (a) Natural (wetlands + other natural + biomass burning), (b) enteric, (c) agricultural soils, (d) gas, (e) solid fuel, (f) the sum of all other anthropogenic tracers. Since the observation at the eastern Asian stations close to large anthropogenic sources show smaller changes it is plau- sible that the emission growth is overly strong in the applied EDGAR v4.2 inventory, for this region. However, it is difﬁ- cult to be conclusive since the few observation sites available are situated in zones with sharp gradients in modelled con- centration changes. The EDGAR v4.2 emissions from the region increase gradually between 2000 and 2008, with a larger growth rate after 2002. Findings from Bergamaschi et al. (2013) question this as they suggest a large increase mostly since 2006. can be seen at downwind stations over and near northern America and in the Southern Hemisphere (Seychelles) (see Figs. 6 and 7). For the Southern Hemisphere a small steady increase in several regional anthropogenic emissions also contributes. 3.5 Changes in methane lifetime The modelled evolution of CH4 is not only decided by changes in sources but also changes in the atmospheric CH4 loss and soil uptake. The CH4 lifetime is an indicator of the CH4 loss. The lifetime is dependent on the efﬁciency of soil uptake (Curry, 2009) as well as on concentrations of atmo- spheric chemical components reacting with CH4, including the kinetic rates of the corresponding reactions. It also de- pends on how efﬁciently the emitted CH4 is transported be- tween regions with differences in loss rate. Our prescribed ﬁelds for soil uptake (Bousquet et al., 2011) are responsi- ble for about 5 % of the loss and the difference between the year with smallest and largest soil uptake is only 2 %. The main reactant removing CH4 chemically in the atmosphere is OH, but there is also a small loss due to reactions with excited atomic oxygen (O1D) and chlorine (Lelieveld et al., 1998; Crutzen, 1991). Due to the limited inﬂuence of soil uptake, chlorine, and O1D we will hereafter focus on the role of changes in OH and the kinetic loss rate for this reac- tion. A number of components (CO, NOx, NMVOCs, CH4, SO2, aerosols, meteorological factors, solar radiation) con- trol the atmospheric OH level and the kinetic loss rate (Dal- søren and Isaksen, 2006; Lelieveld et al., 2004; Holmes et al., 2013; Levy, 1971). Due to the extremely high reactivity of OH, measurements on large scale are impossible (Heard and Pilling, 2003). Forward models have been employed to cal- culate the OH evolution over time on global scale. Another alternative is inverse models in combination with observa- tions of 14CO , CH3CCl3 or other long-lived species reacting with OH. This section discusses the modelled evolution of CH4 lifetime in this study and compares it to ﬁndings from other relevant studies on CH4 lifetime and OH change. In the section thereafter we try to identify the key drivers behind the modelled changes in CH4 lifetime. p Using the EDGAR v4.0 inventory as input to a CTM and observations of CH4 and its isotopic composition Monteil et al. (2011) led to the conclusion that a reduction of biomass burning and/or of the growth rate of fossil fuel emissions is needed to explain the observed growth after 2005. The differ- ences between the EDGAR v4.0 and EDGAR v4.2 used in this study are moderate. S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3117 from EDGAR v4.2 and natural from Bousquet et al., 2011) that both have overly large growth in the period 2006–2008. South America is mainly explained by variations in natural emissions (Fig. 14a). Other studies (Kirschke et al., 2013; Rigby et al., 2008; Bergamaschi et al., 2013; Bousquet et al., 2011; Dlugo- kencky et al., 2009; Crevoisier et al., 2013; Bruhwiler et al., 2014) attribute the resumed strong growth of observed (Dlugokencky et al., 2009; Rigby et al., 2008; Frankenberg et al., 2011; Sussmann et al., 2012; Crevoisier et al., 2013) global CH4 levels after 2006 to increases in both natural and anthropogenic emissions. However, the share of natural vs. anthropogenic contribution varies in the different stud- ies. The studies agree that abnormally high temperatures at high northern latitudes in 2007 and increased tropical rainfall in 2007 and 2008 resulted in large wetland emissions these years. There is also a likely contribution from forest ﬁres in the autumn of 2006 due to drought in Indonesia (Bergam- aschi et al., 2013; Worden et al., 2013). Top-down (Bergam- aschi et al., 2013; Bousquet et al., 2006, 2011; Kirschke et al., 2013; Bruhwiler et al., 2014) and bottom-up studies (EC- JRC/PBL, 2011; Schwietzke et al., 2014; Höglund-Isaksson, 2012; EPA, 2012) suggest steady moderate to substantial in- creases in anthropogenic emissions in the period 2007–2009. Much of this is due to intensiﬁcation of oil and shale gas ex- traction in the United States and coal exploitation in China. Extrapolating anthropogenic emissions that likely have overly strong growth probably explain why the model also overestimates the CH4 growth from 2009 to 2012. Mismatch between the spatial distributions of the model and measure- ments (Fig. 11) on regional scales from 2009 to 2012 are ex- pected due to the extrapolation of anthropogenic emissions and use of constant 2009 natural and biomass-burning emis- sions. Of these, especially wetland emissions have large spa- tial and temporal variation from year to year. 3.5 Changes in methane lifetime Other bottom-up inventories (EPA, 2012; Höglund-Isaksson, 2012; Schwietzke et al., 2014) re- port lower increases in anthropogenic emissions, see also comparison with ECLIPSE emission in the Supplement. Us- ing the mean of the EPA and EDGAR v4.2 inventory for an- thropogenic emissions, Kirschke et al. (2013) ﬁnd that ei- ther is the increase in fossil fuel emissions overestimated by inventories, or the sensitivity of wetland emissions to tem- perature and precipitation is too large in wetland emission models. Schwietzke et al. (2014) and the top-down studies by Bergamaschi et al. (2013) and Bruhwiler et al. (2014) conclude that the EDGAR v4.2 emission inventory overesti- mates the recent emission growth in Asia. This is especially the case for coal mining in China. From our results above, it is plausible that overly high growth of fossil fuel emissions, in particular in Asia, is the reason why the recent CH4 growth is higher in our model than for the observations. However, in 2007 and 2008 much of the increase in the model in the Northern Hemisphere is driven by high natural wetland emis- sions. Our natural emissions are from Bousquet et al. (2011) who attributes much of the 2007–2008 increase in total emis- sions to wetlands. According to Bergamaschi et al. (2013) a substantial fraction of the total increase is attributed to an- thropogenic emissions. There is therefore a possibility that we could combine two emission inventories (anthropogenic The overall picture from the main simulation (blue lines Fig. 15) is that there is a clear decrease in the CH4 lifetime over the last 4 decades, more than 8 % from 1970 to 2012 and a similar increase in OH concentration. Of particular impor- tance are large increases in OH over Southeast Asia, mainly due to strong growth in NOx emissions. From 2000–2010 the modelled tropospheric OH column increase by 10–20 % over China and India (not shown). In Fig. 15, the reaction rate www.atmos-chem-phys.net/16/3099/2016/ For the Arctic stations the responsible sectors for the recent increase and their geographical origin varies but high wetland emissions in 2007–2008, gas in Russia, and coal and other anthropogenic emissions in Asia seem to play a central roles (Figs. 7, 8 and 14). For North America anthro- pogenic emissions increase in the central and eastern US and decrease in the eastern parts (Fig. 14). A similar west–east gradient is seen over the continent for natural sources but this is likely temporary due to special conditions in 2007– 2008. These factors, together with the distant contributions from rising emissions in eastern Asia explain the modelled CH4 trends. In central Europe there is a decline in modelled CH4 due to a combination of declining emissions from en- teric fermentation, solid fuels (coal), and several other an- thropogenic sectors (Fig. 14b, d, f), and ﬂuctuations in nat- ural emissions (Fig. 14a). A decrease over a small region of The period 2007 to 2009 is characterized by strong growth in observed global mean growth rate and even stronger growth in the model (Figs. 11 and 14). The model overes- timation seems to occur almost everywhere. Due to the long lifetime of CH4, strong increase in regional emissions has a global impact. Increases in anthropogenic sources in Asia (e.g. Figs. 9, 14b–f), in particular, natural gas in the Middle East and solid fuel (coal) in eastern Asia have large contribu- tions. The inﬂuence from emission increases in these regions www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3118 Figure 15. Evolution of yearly global average atmospheric instantaneous CH4 lifetime in the main and ﬁxed methane simulations (left y axis). Evolution of yearly global average atmospheric OH concentration in the main simulation (right y axis) using the reaction rate with CH4 as averaging kernel. Figure 15. Evolution of yearly global average atmospheric instantaneous CH4 lifetime in the main and ﬁxed methane simulations (left y axis). Evolution of yearly global average atmospheric OH concentration in the main simulation (right y axis) using the reaction rate with Figure 15. Evolution of yearly global average atmospheric instantaneous CH4 lifetime in the main and ﬁxed methane simulations (left y axis). Evolution of yearly global average atmospheric OH concentration in the main simulation (right y axis) using the reaction rate with CH4 as averaging kernel. the 1980s and a large negative trend for the 1990s were in- ferred from CH3CCl3 observations (Prinn et al., 2005, 2001; Krol and Lelieveld, 2003; Bousquet et al., 2005; Montzka et al., 2000). These studies also found large inter-annual vari- ability of OH. However, the studies were debated (Krol and Lelieveld, 2003; Lelieveld et al., 2006; Bousquet et al., 2005; Wang et al., 2008) and it was shown that largely reduced vari- ations and trends are possible within the uncertainties bonds of the CH3CCl3 emission inventory. In a more recent anal- ysis of CH3CCl3, measurements for the period 1998–2007 Montzka et al. (2011) ﬁnd small inter-annual OH variability and trends and attribute previously estimated large year-to- year OH variations before 1998 to uncertainties in CH3CCl3 emissions and representation issues due to the sparse obser- vation network. Kai et al. (2011) ﬁnd that relatively stable dD-CH4 suggested small changes in the OH sink between 1998 and 2005. Rigby et al. (2008) ﬁnds declining OH from 2004 to 2007. Bousquet et al. (2011) also ﬁnds a decline in 2007 and 2008, compared to 2006. However the decline is much less than that found by Rigby et al. (2008). Holmes et al. (2013) concludes that better understanding of system- atic differences between different CH3CCl3 observation net- works is required before using them as constraints on inter- annual variability of CH4 lifetime and OH. Using 14CO Man- ning et al. (2005) ﬁnds no signiﬁcant long-term trend in OH in the Southern Hemisphere but short-term large variations persisting for a few months. www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3119 Figure 16. Development in atmospheric CH4 lifetime and key parameters known to inﬂuence CH4 lifetime. All variables values are relative to 1970. (To make it apparent in the ﬁgure, temperature variations are relative to the Celsius scale). Figure 16. Development in atmospheric CH4 lifetime and key parameters known to inﬂuence CH4 lifetime. All variables values are relative to 1970. (To make it apparent in the ﬁgure, temperature variations are relative to the Celsius scale). and the increase of stratospheric chlorine (larger loss through reaction with Cl). seen in years with high incidences of ﬁres resulting in large CO emissions. This is typical for ENSO episodes (1997– 1998) and warm years (2010). Agreement with observed CO trends (see comparison in Supplement Sect. S5) indicates that the modelled changes of CO and OH, and applied CO emissions are internally consistent. It is evident from the above discussion that there are uncer- tainties related to all methods (models, CH3CCl3, and 14CO) and missing consensus on OH trends. To increase under- standing and facilitate discussion, it is important not to stop by a derived number for change in OH or methane lifetime, but investigate the major drivers for the changes. The next section address drivers in this model study. y Holmes et al. (2013) found formulas for predicting CH4 lifetime due to changes in meteorology using some of the factors shown in Fig. 16. It is only from 1997 that our simu- lations include inter-annual variation in meteorology. We ﬁnd that variations in global averaged speciﬁc humidity and tem- perature are highly correlated with each other and a 6-month delayed ENSO index. This is reasonable as this is a typical response time for physical and chemical signals to propa- gate from one hemisphere to the other. High temperature and speciﬁc humidity, meaning high water vapour content, is for instance found in the ENSO year 1998 and warm year 2010 (Fig. 16). Variations in these parameters are important for the CH4 lifetime since the reaction rate (k) between OH and CH4 is highly temperature dependent and water vapour is a pre- cursor of OH (Levy, 1971). The production of OH is also de- pendent on UV radiation and thereby the atmospheric ozone column absorbing such radiation (Rohrer and Berresheim, 2006). S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years The highest UV radiation is found at low latitudes and the ozone burden between 40◦S and 40◦N is regarded as a useful indicator (Holmes et al., 2013). The emissions of NOx from lightning are dependent on a number of meteorological factors and thereby quite variable from year to year (Fig. 16). 3.6 Major drivers for changes in the methane lifetime Figure 16 shows the evolution of main factors known to de- termine atmospheric CH4 lifetime. The factors chosen are based on the study by Dalsøren and Isaksen (2006) and Holmes et al. (2013). Using the NOx / CO emission ratio and linear regression analysis (Dalsøren and Isaksen, 2006) found a simple equa- tion describing the evolution of OH resulting from emission changes in the period 1990–2001. In general, CO emission increases lead to an overall reduction in current global av- eraged OH levels. An increase in NOx emissions increases global OH as long as it takes place outside highly polluted regions. In this study the general picture is that the NOx / CO emission ratio increases over the 1970–2012 period (Fig. 16). Despite the general increase, periods of declining ratio can be seen both after the oil crisis in 1973 and the energy crisis in 1979. This occurs since NOx emissions are more affected than CO emissions. After 1997 when we include year-to-year variation in emissions from vegetation ﬁres the NOx / CO emission ratio is more variable. Large drops in ratio can be In this section we investigate whether simpliﬁed expres- sions for the evolution of CH4 lifetime can be found based on Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years Like CH3CCl3 there are uncer- tainties related to inferring OH from 14CO (Krol et al., 2008). Ghosh et al. (2015) does not consider trends in OH but any- way they ﬁnd a decrease in CH4 lifetime over the last century and attribute it to temperature increase (larger reaction rate) with methane is used as an averaging kernel to examine the OH change relevant for changes in methane lifetime. There is a very strong anti-correlation between the evolution of OH and methane lifetime suggesting causality. This is especially the case for the period 1970–1997 run without inter-annual variation in meteorology resulting in a static CH4 + OH re- action rate (k) for these years. The lifetimes in the ﬁxed CH4 run (red line) and the main CH4 run (blue line) are highly cor- related. This is another way of illustrating that OH (k × OH), and not the CH4 burden itself, is driving the long-term evo- lution and year-to-year variations of CH4 lifetime. However, some inﬂuence from CH4 ﬂuctuations is evident in a few of the years studied (mainly in the 1980s), with large variations in CH4 emissions (Fig. 1). CH4 itself is important for its own lifetime length (blue line well above red line), due to the de- crease in the OH concentration produced by the reaction with the CH4. Other forward models also suggest a similar decrease in CH4 lifetime due to an increase in global OH concentrations the recent decades (Karlsdóttir and Isaksen, 2000; Dentener et al., 2003; Wang et al., 2004; Dalsøren and Isaksen, 2006; Fiore et al., 2006; John et al., 2012; Holmes et al., 2013; Naik et al., 2013). However, some of these studies focus on the effect of certain factors (emissions or meteorology) and do not cover changes in all central physical and chem- ical parameters affecting CH4 lifetime. Using observations of CH4 and its isotopic composition, Monteil et al. (2011) ﬁnd that moderate (< 5 % per decade) increases in global OH over the period 1980–2006 are needed to explain the ob- served slowdown in the growth rate of atmospheric CH4 at the end of that period. In contrast, large increases in OH in www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years www.atmos-chem-phys.net/16/3099/2016/ CH4 lifetime (yr) = 11.9 −21.4 × (NOx/CO)emissions. It should be noted that speciﬁc humidity and temperature have almost identical year-to-year variation, and it is there- fore not given which of these parameters should be used. This conﬁrms the analysis from previous sections suggesting that CH4 itself has small inﬂuence on the variation in CH4 lifetime during this period. The same seems to be the case for variations in ozone column. A similar simple equation was found by Dalsøren and Isaksen (2006). This suggests that near-future variation of CH4 lifetime due to changes in emis- sions can be predicted solely by looking at the ratio of NOx to CO emissions. However, it should be noted that the region of emission change is important (Berntsen et al., 2006). This is especially the case for NOx emissions due to the short at- mospheric NOx lifetime. For instance, changes in NOx emis- www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 3120 S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 year Figure 17. CH4 lifetime evolution 1970–1996. Comparison of the main model simulation (blue line) with CH4 lifetime from the simple model (red line) obtained from multiple linear regression. Figure 17. CH4 lifetime evolution 1970–1996. Comparison of the main model simulation (blue line) with CH4 lifetime from the simple model (red line) obtained from multiple linear regression. the parameters in Fig. 16. Such equations could be very use- ful for fast prediction of future development of CH4 lifetime and CH4 burden. Since we study different time periods than Dalsøren and Isaksen (2006) and Holmes et al. (2013), and both emissions and meteorology are perturbed in our simu- lations, it is not obvious that simpliﬁed equations would be statistically valid. sions at low latitudes with moderate pollution levels (OH re- sponse is non-linear) would have profound impacts on CH4 lifetime due to the temperature dependency of the reaction between CH4 and OH. The blue line in Fig. 18 shows the lifetime over the pe- riod 1997–2012 as predicted by the main model run. The red line shows the best ﬁt from a simple parametric model. Be- cause the main CTM run for this period include year-to-year variation in meteorology, the simple regression model need more parameters to reproduce the evolution. Still, a simpli- ﬁed equation (R2 = 0.99) is statistically valid, predicting the CH4 lifetime by a linear combination of the parameters spe- ciﬁc humidity (q), NOx / CO emission ratio (NOx / CO)e, lightning NOx emissions (LNOx)e, and O3 column: Figure 17 shows the results of multiple linear regression analysis performed to describe the CH4 lifetime over the pe- riod 1970 to 1996. For this period, ﬁxed year-to-year mete- orology was used in the main model simulation. This means that parameters like lightning NOx, temperature, and speciﬁc humidity (Fig. 16) can be kept out of the regression analy- sis. The equation best reproducing (R2 = 0.99) the lifetime evolution from the main run (Fig. 17) and having statistical signiﬁcant linear relations between its parameters and CH4 lifetime is the following: CH4 lifetime (yr) = 0.07 × O3column −4.80 × (NOx/CO)e −0.04 × q −1.21 × (LNOx)e. CH4 lifetime (yr) = 11.9 −21.4 × (NOx/CO)emissions. S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years 3121 Figure 18. CH4 lifetime evolution 1997–2012. Comparison of main model simulation (blue line) with CH4 lifetime from simple model (red line) obtained from multiple linear regression. Figure 18. CH4 lifetime evolution 1997–2012. Comparison of main model simulation (blue line) with CH4 lifetime from simple model (red line) obtained from multiple linear regression. 2013; Nisbet et al., 2014). As the quality and detail level of models, input data, and measurements progress, the chances of understanding more pieces in the big puzzle increase. This study is an effort in such a perspective. 2006 in both hemispheres. From 2006, the model overesti- mates the growth in all regions, in particular in Asia. Large emission growth in Asia inﬂuences the CH4 trends in most world regions. Our ﬁndings support other studies, suggesting that the recent growth in Asian anthropogenic emissions is too high in the EDGAR v4.2 inventory. Based on our model results and the comparison between ECLIPSE and EDGAR v4.2 emissions in the Supplement (Sect. S2) we also ques- tion the Asian emission trends in the 1990s and beginning of the 2000s in the EDGAR v4.2 inventory, although the lim- ited number of measurement sites in Asia makes it difﬁcult to validate this. In our bottom-up approach, a global chemical transport model (CTM) was used to study the evolution of atmospheric CH4 over the period 1970–2012. The study includes a thor- ough comparison with CH4 measurements from surface sta- tions covering all regions of the globe. The seasonal varia- tions are reproduced at most stations. The model also repro- duces much the observed evolution of CH4 on both inter- annual and decadal time scales. Variations in wetland emis- sions are the major drivers for year-to-year variation of CH4. Regarding trends, the causes are much debated, as discussed in the previous sections. Consensus is neither reached on the relative contribution from individual emission sectors, nor on the share of natural vs. anthropogenic sources. The fact that our simulations capture much of the observed regional changes indicates that our transport and chemistry schemes perform well and that applied emission inventories are rea- sonable with regard to temporal, spatial, sectoral, and nat- ural vs. anthropogenic distribution of emissions. However, there are some larger discrepancies in model performance questioning the accuracy of the CH4 emission data in cer- tain regions and periods. 4 Summary and conclusions Uncertainties in physical and chemical processes in models, input data on emissions and meteorology, and limited spatial and temporal coverage of measurement data, have made it hard for both bottom-up and top-down studies to settle the global CH4 budget, untangle the causes for recent trends, and predict future evolution (Ciais et al., 2013; Kirschke et al., Atmos. Chem. Phys., 16, 3099–3126, 2016 www.atmos-chem-phys.net/16/3099/2016/ S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years S. B. Dalsøren et al.: Atmospheric methane evolution the last 40 years Potential ﬂaws in emission data are pinpointed for recent years when our model simulations are more complete with regard to input data (e.g. emissions, variable meteorology, etc.) and there are more measurements available for comparison. After a period of stable CH4 levels from 2000 to 2006, observations show increasing levels from The modelled evolution of CH4 is also dependent on changes in the atmospheric CH4 loss. The CH4 lifetime is an indicator of the CH4 loss. In our simulations, the CH4 life- time decreases by more than 8 % from 1970 to 2012. The rea- son for the large change is increased atmospheric oxidation capacity. Such changes are in theory driven by complex inter- actions between a number of chemical components and me- teorological factors. However, our analysis reveals that key factors for the development are changes in speciﬁc humidity, NOx / CO emission ratio, lightning NOx emissions, and to- tal ozone column. It is statistically valid to predict the CH4 lifetime by a combination of these parameters in a simple equation. The calculated change in CH4 lifetime is within the range reported by most other bottom-up model studies. How- ever, ﬁndings from these studies do not fully agree with top- down approaches using observations of CH3CCl3 or 14CO. Without the calculated increase in oxidation capacity, the CH4 growth over the last decades would have been much Atmos. Chem. Phys., 16, 3099–3126, 2016 The Supplement related to this article is available online at doi:10.5194/acp-16-3099-2016-supplement. Berntsen, T., Fuglestvedt, J., Myhre, G., Stordal, F., and Berglen, T.: Abatement of Greenhouse Gases: Does Location Matter?, Climatic Change, 74, 377–411, doi:10.1007/s10584-006-0433- 4, 2006. Bhattacharya, S. K., Borole, D. V., Francey, R. J., Allison, C. E., Steele, L. P., Krummel, P., Langenfelds, R., Masarie, K. A., Ti- wari, Y. K., and Patra, P. K.: Trace gases and CO2 isotope records from Cabo de Rama, India, Curr. Sci., 97, 1336–1344, 2009. Acknowledgements. This work was funded by the Norwegian Research Council project GAME (Causes and effects of Global and Arctic changes in the Methane budget), grant no. 207587, under the program NORKLIMA, and the EU project ACCESS (Arctic Climate Change Economy and Society). ACCESS received funding from the European Union under grant agreement no. 265863 within the Ocean of Tomorrow call of the European Commission Seventh Framework Programme. We are grateful to Phillipe Bousquet for providing and sharing data sets on methane emissions. The work and conclusions of the paper could not been achieved without globally distributed observational data and we acknowledge all data providers, and the great efforts of AGAGE, NOAA ESRL, and The World Data Centre for Greenhouse Gases (WDCGG) under the GAW programme for making data public and available. Speciﬁc thanks go to Nina Paramonova, Hsiang J. Wang, Simon O’Doherty, Yasunori Tohjima, Edward J. Dlugokencky, who are the PIs of the observation data shown in Figs. 6–10 and 12–14. We also thank Edward J. Dlugokencky for sharing the observational based data set for Fig. 11, and WDCGG and Paul Novelli for sharing CO data sets used in Fig. S5 of the supplement. Bousquet, P., Hauglustaine, D. A., Peylin, P., Carouge, C., and Ciais, P.: Two decades of OH variability as inferred by an in- version of atmospheric transport and chemistry of methyl chlo- roform, Atmos. Chem. Phys., 5, 2635–2656, doi:10.5194/acp-5- 2635-2005, 2005. Bousquet, P., Ciais, P., Miller, J. B., Dlugokencky, E. 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Increasing CH4 loss also likely contributed to the stagnation of CH4 growth in the period 2001–2006. Inter- estingly, over the last few years, the loss deviates from its steady increase over the previous decades. Much of this de- viation seems to be caused by variation in meteorology. Our simulations reveal that accounting for variation in meteorol- ogy has a strong effect on the atmospheric CH4 loss. This in turn affects both inter-annual and long-term changes in CH4 burden. A stabilization of the CH4 loss, mainly due to me- teorological variability, likely contributed to a continuing in- crease (2009–2012) in CH4 burden after high emission years in 2007 and 2008. Due to the long response time of CH4 this could also contribute to future CH4 growth. However, there are extra uncertainties in the model results after 2009 due to lack of comprehensive emission inventories. A new inven- tory or update of existing ones with sector–vice separation of emission for recent years (2009–2015) would be a very valuable piece for model studies trying to close the gaps in the CH4 puzzle. It will also provide important fundament for more accurate predictions of future CH4 levels and various mitigation strategies. higher. Increasing CH4 loss also likely contributed to the stagnation of CH4 growth in the period 2001–2006. Inter- estingly, over the last few years, the loss deviates from its steady increase over the previous decades. Much of this de- viation seems to be caused by variation in meteorology. Our simulations reveal that accounting for variation in meteorol- ogy has a strong effect on the atmospheric CH4 loss. This in turn affects both inter-annual and long-term changes in CH4 burden. A stabilization of the CH4 loss, mainly due to me- teorological variability, likely contributed to a continuing in- crease (2009–2012) in CH4 burden after high emission years in 2007 and 2008. Due to the long response time of CH4 this could also contribute to future CH4 growth. However, there are extra uncertainties in the model results after 2009 due to lack of comprehensive emission inventories. A new inven- tory or update of existing ones with sector–vice separation of emission for recent years (2009–2015) would be a very valuable piece for model studies trying to close the gaps in the CH4 puzzle. It will also provide important fundament for more accurate predictions of future CH4 levels and various mitigation strategies. www.atmos-chem-phys.net/16/3099/2016/ Atmos. Chem. Phys., 16, 3099–3126, 2016 S. B. 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https://openalex.org/W2775350907	https://portalseer.ufba.br/index.php/nit/article/download/23120/23120	Portuguese	null	EFICIÊNCIA DO INSTITUTO FEDERAL BAIANO: ANÁLISE DOS GRUPOS DE PESQUISA E PROPRIEDADE INDUSTRIAL	Cadernos de Prospecção	2,017	cc-by	4,634	Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 D.O.I.: http://dx.doi.org/10.9771/cp.v10i3.23120 RESUMO Este estudo trata de uma pesquisa documental com o objetivo de identificar os grupos de pesquisa e estudo ligados ao IF Baiano registrados no diretório dos grupos de pesquisa do CNPq, bem como, as patentes e programa de computador depositado na base de dados do INPI. Foram identificados 17 grupos, 12 depósitos de patentes e 01 programa de computador associado à instituição. Estas bases permitem inferir a área de contribuição da produção científico-tecnológica do IF Baiano conforme será apresentado e discutido nos resultados. Com os resultados possibilitou maior visibilidade a pesquisa e produção do conhecimento oriunda do instituto em questão. Palavra-chave: Instituto Federal. Propriedade Intelectual. Prospecção. Ayalla Oliveira Chaves1; Gustavo Pereira da Cruz2 Ayalla Oliveira Chaves1; Gustavo Pereira da Cruz2 1, 2 Universidade Estadual de Santa Cruz – UESC, Ilhéus, BA, Brasil 1, 2 Universidade Estadual de Santa Cruz – UESC, Ilhéus, BA, Brasil Rec.: 16/07/2017 Ace.: 05/09/2017 * Autor para correspondência: ayalla.chaves@gmail.com INTRODUÇÃO A rede federal de educação existe desde 1909 e devido às mudanças de políticas governamentais, em 2008, os centros federais de educação tecnológica - CEFETs, juntamente com as unidades descentralizadas de ensino – UNEDs, as escolas agrotécnicas, as escolas técnicas federais e as escolas vinculadas a universidades formaram os Institutos Federais de Educação, Ciência e Tecnologia (BRASIL, 2016a). Os institutos cobrem todo o território nacional e desde 2002 ocorre à expansão com a construção de novas unidades e a federalização de outras, totalizando 644 unidades em funcionamento até 2016 e contabilizando 38 institutos federias em todo o país conforme dados do Ministério da Educação (BRASIL, 2016b). Dentre os institutos, no estado da Bahia, o Instituto Federal de Educação, Ciência e Tecnologia Baiano - IF Baiano, criado em 2008, instituído através da Lei 11.892, é constituído por 14 campi em atividade nas cidades de Alagoinhas, Bom Jesus da Lapa, Catu, Governador Mangabeira, Guanambi, Itaberaba, Itapetinga, Santa Inês, Senhor do Bonfim, Serrinha, Teixeira de Freitas, Uruçuca e Xique-Xique, sendo sua sede em Salvador. De acordo com PERUCCHI; GARCIA (2012 p 52): “[...] exige-se que os professores/pesquisadores tanto das Universidades quanto dos Institutos Federais, Ciências e Tecnologia (IFs) desenvolvam pesquisas que devem ser disseminadas para a sociedade, mostrando resultados, a pertinência e relevância de suas ações, através da produção científica, de produtos, processos e serviços tecnológicos. Por meio desses conhecimentos produzidos, difundidos e democratizados constrói o desenvolvimento sustentável integrado” Com quase uma década de atuação do IF Baiano no setor educação, questiona-se: Qual a contribuição do IF Baiano para o processo de inovação tecnológica? Para tal, torna-se indispensável caracterizar os grupos de pesquisa certificados, as áreas de estudos, os produtos desenvolvidos, quantidade de discentes e docentes envolvidos no processo de produção de pesquisa, conhecimento e desenvolvimento de tecnologias. Instalado no parque tecnológico da Bahia em Salvador, e hierarquicamente ligado a pró-reitoria de pesquisa e inovação, o Núcleo de Inovação Tecnológica – NIT do IF Baiano (2017) é um dos membros entre as organizações participantes da Rede de Núcleo de Inovação Tecnológica do Nordeste – Rede NIT NE desde 2011, sendo seu objetivo apoiar as ações que tenham por fundamento a inovação tecnológica em todos os segmentos da ciência e da tecnologia, bem como, as matérias tratadas pelas leis que regulam propriedade intelectual no Brasil. ABSTRACT This paper is a documentary research with the aim of identifying the research and study groups connected to IF Baiano that are registered in the directory of the CNPq research groups, as well as, the patents and computer program applied in INPI's database. 17 groups, 12 patents applied and a computer program associated with the institution were identified. These bases allow us to infer an area of contribution of the scientific and technological production of IF Baiano as presented and discussed in the results. With the results, it was possible to have a greater visibility of the research and production of knowledge derived from the office studied. Keywords: Federal Institute. Intellectual property. Prospective study. Área tecnológica: Propriedade intelectual. Transferência de tecnologia. * Autor para correspondência: ayalla.chaves@gmail.com 438 Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 D.O.I.: http://dx.doi.org/10.9771/cp.v10i3.23120 CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade INTRODUÇÃO Este estudo tem como objetivo identificar os grupos de pesquisa – GP certificados no diretório de grupos de Pesquisa do Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq e os inventos submetidos à proteção junto ao Instituto Nacional de Propriedade Industrial – INPI, considerando a produção científica, patentes e programas de computador como indicador de potencial inovador da instituição. 439 D.O.I.: http://dx.doi.org/10.9771/cp.v10i3.23120 Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 OS GRUPOS DE PESQUISA E A PRODUÇÃO DO CONHECIMENTO A pesquisa científica no Brasil teve início a partir do século XX. Para inventariar, organizar e tornar pública a produção documental, em 1951 surgiu o Conselho Nacional de Pesquisa, atualmente denominado Conselho Nacional de Desenvolvimento Científico e Tecnológico, agência do Ministério da Ciência, Tecnologia, Inovações e Comunicações. Sob sua égide está a base de dados corrente do diretório de grupos de pesquisa, instrumento que permite de forma acessível e segura traçar o perfil dos grupos ativos que desenvolvem pesquisa no país. De acordo com o CNPq (2017) “O grupo de pesquisa foi definido como um conjunto de indivíduos organizados hierarquicamente [...] deve, portanto, organizar-se em torno de uma liderança (eventualmente duas), e estar "abrigado" em uma instituição previamente autorizada pelo CNPq.” Na contemporaneidade, vivemos a era da informação e do conhecimento, para acompanhar esta vertente nas Instituições de ensino superior e tecnológico os pesquisadores se dividem entre as atividades de ensino, promoção de atividades de extensão e desenvolvimento de pesquisa. “No Brasil, as atividade de produção de conhecimentos em pesquisa vêm sendo desenvolvidas por equipes de pesquisadores titulados ou em formação, organizados sob a designação de grupos de pesquisa.” (BARBOSA, 2009, p 444) Segundo os dados do censo, realizado pelo CNPq (2016), o número de grupos de pesquisa e estudo cresceu 6% se comparado ao ano de 2014. Por vez, quando se compara o crescimento da quantidade de grupos de pesquisa por região, é percebido que o Nordeste cresceu em 7% se comparado o censo 2016 ao de 2014 e os valores absolutos continuam em crescente crescimento em todas as unidades federativas CNPq (2016). CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial. CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial A PROPRIEDADE INTELECTUAL E O INCENTIVO A INOVAÇÃO Conforme a Organização Mundial da Propriedade Intelectual (OMPI), a propriedade intelectual corresponde à soma dos direitos relativos às obras literárias, artísticas e científicas, às interpretações dos artistas intérpretes e às execuções dos artistas instrumentistas, aos fonogramas e às emissões de radiodifusão, às invenções em todos os domínios da atividade humana, às descobertas científicas, aos desenhos e modelos industriais, às marcas industriais, comerciais e de serviço, bem como às firmas comerciais e denominações comerciais, à proteção contra a concorrência desleal e todos os outros direitos inerentes à atividade intelectual nos domínios industrial, científico, literário e artístico. Importante ressaltar, para fins deste estudo, duas áreas: propriedade industrial e direito autoral. A primeira abrange modelo de utilidade e patentes de invenção, e segundo abrange os programas de computador, ambos evidenciados nesta pesquisa. No Brasil, atua desde 1970, a autarquia federal denominada Instituto Nacional da Propriedade Industrial, vinculada ao Ministério da Indústria, Comércio Exterior e Serviços. Entre os serviços prestados pelo INPI (2017), estão os registros de marcas, desenhos industriais, indicações geográficas, programas de computador e topografias de circuitos, as concessões de patentes e as averbações de contratos de franquia e das distintas modalidades de transferência de tecnologia. Para garantir o direito de uso do criador em relação a inovação tecnológica desenvolvida, no Brasil, foram instituídas a Lei nº 9.279/1996 que regula direitos e obrigações relativos à propriedade 440 Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 D.O.I.: http://dx.doi.org/10.9771/cp.v10i3.23120 industrial e a Lei nº 10.973/2004 que dispõe sobre incentivos à inovação e à pesquisa científica e tecnológica no ambiente produtivo e dá outras providências. industrial e a Lei nº 10.973/2004 que dispõe sobre incentivos à inovação e à pesquisa científica e tecnológica no ambiente produtivo e dá outras providências. No contexto globalizado, a produção intelectual brasileira ainda precisa de largo desenvolvimento no âmbito da concentração de esforços em prol da inovação, políticas públicas de fomento e subsídio às instituições de ensino superior e demais instituições de pesquisa, bem como, meios para viabilizar a transferência de tecnologias escola-empresa. CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial. METODOLOGIA Este estudo é uma pesquisa documental, exploratória e descritiva de acordo com Godoy (1995, p.21): “[...] a pesquisa documental representa uma forma que pode se revestir de um caráter inovador, trazendo contribuições importantes no estudo de alguns temas. Além disso, os documentos normalmente são considerados importantes fontes de dados para outros tipos de estudos qualitativos, merecendo, portanto atenção especial.” “[...] a pesquisa documental representa uma forma que pode se revestir de um caráter inovador, trazendo contribuições importantes no estudo de alguns temas. Além disso, os documentos normalmente são considerados importantes fontes de dados para outros tipos de estudos qualitativos, merecendo, portanto atenção especial.” Sendo significativo ressaltar que: CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial RESULTADOS E DISCUSSÕES O IF Baiano com sua estrutura pulverizada pelo estado da Bahia está presente em diferentes territórios de identidade ofertando educação profissional e tecnológica gratuito do nível médio- técnico até pós-graduação strictu senso. Está em funcionamento 18 cursos de nível técnico distribuídos entre seus 14 campi, nas modalidades de ensino médio integrado, programa nacional de integração da educação profissional com a educação básica na modalidade de jovens e adultos - PROEJA e subsequente, enquadrados nos seguintes eixos tecnológicos: gestão e negócio; controle e processos industriais; desenvolvimento educacional e social; produção alimentícia; ambiente, saúde e segurança; recursos naturais e informação e comunicação. A instituição atua, também, na oferta de ensino superior com 12 cursos de graduação, são eles: tecnológico em agroecologia, tecnológico em agroindústria, bacharelado em agronomia, tecnológico em análise e desenvolvimento de sistemas, licenciatura em biologia, licenciatura em ciências agrárias, licenciatura em ciências da computação, licenciatura geografia, tecnológico gestão de turismo, licenciatura em química e bacharelado em zootecnia, distribuídos entre suas unidades. Além de curso de curta duração, oferecidos esporadicamente para comunidade externa e interna, a instituição oferta aperfeiçoamento profissional por intermédio dos cursos de pós-graduação lato senso, a saber: especialização de desenvolvimento sustentável no semiárido, educação científica e popularização da ciência, educação de jovens e adultos com necessidades especiais, educação no campo, inovação social, língua brasileira de sinais, e inovação social com ênfase em economia solidária e agroecologia. Quanto à pós-graduação strictu senso está em atividade o mestrado profissional em produção vegetal do semiárido. O fomento a pesquisa e inovação são evidenciadas através dos editais anuais de apoio aos projetos desenvolvidos na instituição contemplando os custos de execução e bolsas a discentes evolvidos. Porém, pelo panorama dos grupos de pesquisa e estudo em atividade, os docentes são a maioria entre os participantes destes, atingindo um percentual de 68,10% contra 31,09% de discentes envolvidos com a produção de conhecimento científico. A quantidade de pesquisadores docentes se sobrepõe significativamente a quantidade de alunos engajados nos grupos de pesquisa promovidos pela instituição, sendo totalizados 158 e 74, respectivamente. Sendo significativo ressaltar que: “[...] esquecemos que os documentos constituem uma rica fonte de dados. O exame de materiais de natureza diversa, que ainda não receberam um tratamento analítico, ou que podem ser reexaminados, buscando-se novas e/ ou interpretações complementares, constitui o que estamos denominando pesquisa documental.” (Ibidem, 1995). “[...] esquecemos que os documentos constituem uma rica fonte de dados. O exame de materiais de natureza diversa, que ainda não receberam um tratamento analítico, ou que podem ser reexaminados, buscando-se novas e/ ou interpretações complementares, constitui o que estamos denominando pesquisa documental.” (Ibidem, 1995). A coleta foi realizada na base de dados do Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico, foram investigados núcleos de pesquisa vinculados ao Instituto Federal de Educação, Ciências e Tecnologia Baiano, utilizando as palavras “grupo”, “estudo”, e “baiano”, no campo termo de busca. A coleta foi realizada em 07/06/2017. Foram incluídos os grupos atualizados no período dede 2010 a 2017. Neste estudo foram sistematizadas as seguintes informações: localização, grupo, líder, área predominante, ano de formação, linhas, quantidade de estudantes e pesquisadores cadastrados, equipamentos e software, distribuídos em colunas usadas na planilha. A segunda etapa de coleta foi realizada na base de dados de Instituto Nacional de Propriedade Industrial, foram investigados patentes e programa de computador vinculado ao Instituto Federal de Educação, Ciências e Tecnologia Baiano, utilizando a expressão “Instituto Federal Baiano”, no campo nome do depositante. A busca foi realizada em 05/07/2017. Neste estudo foram sistematizadas as seguintes informações: número do pedido, data de depósito, título, classificação de infraestrutura da chave pública – ICP, data de concessão, nome do inventor, distribuídos em colunas usadas na planilha. As informações foram tabuladas no Excel versão 2007 e apresentado informações absoluta e relativa. As informações foram tabuladas no Excel versão 2007 e apresentado informações absoluta e relativa A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedad 441 Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 D.O.I.: http://dx.doi.org/10.9771/cp.v10i3.23120 RESULTADOS E DISCUSSÕES Ao NIT do IF Baiano, conforme disposto em regimento (2016), compete: estabelecer uma política de propriedade intelectual e de inovação tecnológica; promover os registros, acompanhar e zelar pela manutenção e defesa dos títulos de propriedade intelectual da instituição; fomentar e fortalecer parcerias do IF Baiano com órgãos governamentais, empresas e sociedade, para a difusão de novas tecnologias; manifestar-se previamente sobre os contratos, convênios, acordos de cooperação e demais instrumentos jurídicos congêneres relacionados a projetos de pesquisa científica e tecnológica, bem como, de propriedade industrial e direitos autorais; acompanhar o andamento e efetuar os devidos pagamentos relativos aos processos de propriedade intelectual, os privilégios já concedidos, averbação e o andamento dos contratos de transferência de tecnologia;realizar outras atividades correlatas que lhes forem atribuídas pelo pró-reitor de pesquisa e inovação do Instituto Federal Baiano. Foi estudado um total de 14 grupos de pesquisa, sendo excluídos 03 grupos: 02 grupos com situação em preenchimento e um negado. Dos 14 campi existentes, há registro de 13 GP em atividade distribuídos pelos campi e reitoria: Bom Jesus da Lapa (01), Catu (01), Governador Mangabeira (01), Guanambi (02), Itapetinga (01), Santa Inês (01), Senhor do Bonfim (02), Valença (02) e Reitoria (03). Os campi de Alagoinhas, CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial. 442 Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 D.O.I.: http://dx.doi.org/10.9771/cp.v10i3.23120 Itaberaba, Serrinha, Teixeira de Freitas, Uruçuca e Xique-Xique não possuem nenhum grupo de pesquisa cadastrado na base de dados utilizados. Concentrando-se a maioria dos grupos em atividade nas localidades Guanambi, Senhor do Bonfim, Valença e na sede em Salvador. Itaberaba, Serrinha, Teixeira de Freitas, Uruçuca e Xique-Xique não possuem nenhum grupo de pesquisa cadastrado na base de dados utilizados. Concentrando-se a maioria dos grupos em atividade nas localidades Guanambi, Senhor do Bonfim, Valença e na sede em Salvador. A figura 1 apresenta a localização dos grupos de pesquisa vinculados ao IF Baiano de acordo com a localização cadastrada na base de dados do CNPq mostrando o panorama de disposição destes por campi. CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial. 443 CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedad RESULTADOS E DISCUSSÕES Figura 1 – Localização do grupo vinculado aos campi do IF Baiano Figura 1 – Localização do grupo vinculado aos campi do IF Baiano Figura 1 – Localização do grupo vinculado aos campi do IF Baiano 7% 7% 7% 15% 7% 7% 14% 14% 22% Campus Bom Jesus da Lapa campus Catu Campus Governador Mangabeira Campus Guanambi Campus Itapetinga Campus Santa Inês Campus Senhor do Bonfim campus Valença Reitoria Fonte: Chaves (2017). 7% 7% 7% 15% 7% 7% 14% 14% 22% Campus Bom Jesus da Lapa campus Catu Campus Governador Mangabeira Campus Guanambi Campus Itapetinga Campus Santa Inês Campus Senhor do Bonfim campus Valença Reitoria 7% 7% 7% 15% 7% 7% 14% 14% 22% Campus Bom Jesus da Lapa campus Catu Campus Governador Mangabeira Campus Guanambi Campus Itapetinga Campus Santa Inês Campus Senhor do Bonfim campus Valença Reitoria Fonte: Chaves (2017). Fonte: Chaves (2017). Fonte: Chaves (2017). O cadastramento de grupos de pesquisa na base de dados do CNPq apresentou aumento intermitente de 2010 a 2014 tendo ao todo 10 grupos cadastrados no período de quatro anos. Houve declínio de formação de novos grupos de pesquisa no período de 2014 a 2015, sendo cadastrados apenas 02 grupos retomando o crescimento em 2016 e tornando a declinar em 2017. Foi considerado o ano 2017 somente o primeiro semestre, período abrangido por esta pesquisa no qual houve o pedido de credenciamento de um grupo de pesquisa. Identificaram-se os anos de 2014 e 2016 como os quais se concentraram maior quantitativo de criação destes grupos de pesquisa atuantes na instituição. Importante ressaltar, que a política de governo dos ex-presidentes Luís Inácio Lula da Silva (2003 a 2011) e Dilma Rousseff (2011 a 2016) tinha como finalidade o fortalecimento da educação profissional, consolidando durante os mandatos, o programa de aceleração da rede federal de ensino tecnológico que passou por três fases abrangendo todo o território nacional e provendo investimento em ensino, pesquisa e extensão. A figura 2 apresenta a quantidade de grupos de pesquisa cadastrados no diretório do CNPq anuamente dispostos cumulativamente ao longo dos anos pesquisados. 443 vador, v. 10, n. 3 p.438-447, jul./set. 2017 D.O.I.: http://dx.doi.org/10.9771/cp.v10i3.23120 Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 D.O.I.: http://dx.doi.org/10.977 Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 D.O.I.: http://dx.doi.org/10.9771/cp.v10i3.23120 Figura 2 – Quantidade de Grupos de Pesquisa cadastrados no CNPq por ano. CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial RESULTADOS E DISCUSSÕES Figura 2 – Quantidade de Grupos de Pesquisa cadastrados no CNPq por ano. 0 2 4 6 8 10 12 14 2010 2011 2012 2013 2014 2015 2016 2017 GP Fonte: Chaves (2017). Fonte: Chaves (2017). No quesito área de predominância, dos 14 grupos em situação certificada sua maioria está inserida na área de predominância de ciências humanas em educação, o que corresponde ao total de seis grupos abordando a temática. As demais áreas de predominância dos grupos identificados são: ciências agrárias/agronomia (01), ciências agrárias/engenharia agronômica (01), ciências agrárias/medicina veterinária (01), ciências biológicas/ecológica (01), ciências exatas e da terra/química (01), e ciências exatas e da terra/geociência (02). Frente a este cenário, a afinidade dos líderes e a disposição para fomentar grupos de pesquisa e estudo proporcionam a formação dos mesmos. E se considerando a transdisciplinaridade inerente a este tipo de instituição de ensino as diversas áreas de conhecimento despertam interesses diversos de estudo científicos. A figura 3 apresenta a concentração por área de predominante dos grupos de pesquisa vinculados a instituição, criada conforme com a área de interesse dos respectivos líderes. Figura 3 – Concentração da área predominante de estudo do grupo de pesquisa. CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial. 444 Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 D.O.I.: http://dx.doi.org/10.9771/cp.v10i3.23120 7% 7% 7% 7% 7% 15% 43% 7% Ciências Agrárias( Agronomia) Ciências Agrárias( Eng Agricola) Ciências Agrárias(Med Veterinária) Ciências Biológicas(Ecologia Ciências Exatas e da Terra( Quimica) Ciências Exatas e da Terra(Geociencia) Ciências Humanas( Educação) Lingüística, Letras e Artes Fonte: Chaves (2017). Fonte: Chaves (2017). Referente aos produtos de propriedade industrial identificou-se a submissão do pedido de doze patentes em área diversas e um pedido de programa de computador. Dos pedidos de patente submetidos ao INPI quatro deles encontram-se publicados, somente, ainda em análise quanto à concessão, sendo 02 modelos de utilidade e 02 invenções. De acordo com a classificação internacional de patentes – ICP informado documentos de pedido de patente, os modelos de utilidade estão inseridos na seção necessidades humanas e operações de processamento, enquanto os documentos referentes aos depósitos de patentes de invenções pertencem às seções de física e química e metalurgia. Inferiu-se, inclusive, a partir do campus de lotação dos inventores que três deles atuam na unidade de Catu e um na unidade de Uruçuca. CONCLUSÃO A tecnologia evolui em ritmo acelerado buscando a melhoria da em suas ferramentas a fim de atender as demandas de mercado sedento por conforto e comodidade. A pesquisa é um dos instrumentos basilar para desenvolvimento de conhecimento voltado a atender as demandas mercadológicas. A rede federal de ensino tecnológico tem papel fundamental para produção do conhecimento, fomento ao empreendedorismo e desenvolvimento de mercado. Este estudo contribuiu para maior visibilidade do IF Baiano quanto propulsor de desenvolvimento de técnicas voltadas para educação, bem como, para ciências da terra no qual está concentrada a maioria dos cursos de formação técnicas ofertados. A instituição apresenta instrumentos como recurso humano qualificado, política interna de incentivo a pesquisa e inovação e núcleo de inovação tecnológica instituído para atuação em desenvolvimento de estudos e pesquisas direcionados a inovação tecnológica. Novos estudos se fazem necessário para acompanhar o desenvolvimento sistemático desta instituição enquanto integrante da estrutura científica e tecnológica do país. Deste modo, é essencial para a instituição investir em efetiva implementação do NIT para auxiliar o desenvolvimento da pesquisa institucional e transferência de tecnologia como capitação de recurso diante do cenário econômico brasileiro. RESULTADOS E DISCUSSÕES Os demais não foram encontrados documentos de publicação ou concessão disponível apenas informação de depósito de pedido de patente. Para o programa de computador, denominado K-ÁGORA, foi concedido o certificado de registro desde 04/04/2017. O desenvolvimento do software contou com a participação de alunos e pesquisadores, apoiado pelo CNPq e assessorado pelo NIT em todas as etapas para concessão de registro. Analisando a autoria dos inventos de propriedade industrial e autoria do programa de computador, pode-se evidenciar que todos pertencem a docentes efetivos do quadro de pessoal do instituto, porém não há vínculos explícitos nos registros como os recursos humanos dos grupos de pesquisa e estudos analisados. A elaboração dos documentos relativos ao pedido de patente (relatório, resumo, reivindicações) são de responsabilidade dos inventores, cabendo ao NIT realizar a interlocução junto ao INPI acompanhando desde o depósito do pedido até sua finalização, arcando com todos os custos advindos, figurando o IF Baiano como titular. CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial. 445 As submissões de pedido de patente passaram a ser realizados a partir de 2015, onde foram submetidos ao INPI cinco pedidos em 2015 apresentando crescimento quantitativo em 2016 para As submissões de pedido de patente passaram a ser realizados a partir de 2015, onde foram submetidos ao INPI cinco pedidos em 2015 apresentando crescimento quantitativo em 2016 para CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial. 445 Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 D.O.I.: http://dx.doi.org/10.9771/cp.v10i3.23120 sete pedidos. Cabe mencionar que o programa de computador K-Ágora teve seu pedido de certificação submetido em 2016. sete pedidos. Cabe mencionar que o programa de computador K-Ágora teve seu pedido de certificação submetido em 2016. Este fato pode ser justificado pela institucionalização do Núcleo de Inovação Tecnológica - NIT no ano de 2016 através da estabelecendo as diretrizes de atuação do núcleo na instituição, constantes na resolução CONSUP nº 35, de 01/09/2016, em convergência com a Portaria nº 37, de 29/10/2015 CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade REFERÊNCIA BARBOSA, S. F. F; DAL SASSO, G. T. M.; BERNS, I. Enfermagem e Tecnologia: Análise dos Grupos de Pesquisa Cadastrados na Plataforma Lattes do CNPq. Texto & Contexto Enfermagem, Florianópolis, vol.18, n. 3, p. 443-448, July/Sept. 2009. BRASIL. Lei n° 9.279, de 14 de maio de 1996. Lei de Propriedade Industrial. Dispõe sobre incentivos à inovação e à pesquisa científica e tecnológica no ambiente produtivo e dá outras providências. Disponível em: < http://www.planalto.gov.br/ccivil_03/leis/L9279.htm >. Acesso em 10/06/17. BRASIL. Lei n° 10.973, de 02 de dezembro de 2014. Lei de Inovação Tecnológica. Regula direitos e obrigações relativos a propriedade industrial. Disponível em: < http://www.planalto.gov.br/ccivil_03/_ato2004-2006/2004/lei/l10.973.htm >. Acesso em 10/06/2017. BRASIL. Lei n° 10.973, de 02 de dezembro de 2014. Lei de Inovação Tecnológica. Regula direitos e obrigações relativos a propriedade industrial. Disponível em: < http://www.planalto.gov.br/ccivil_03/_ato2004-2006/2004/lei/l10.973.htm >. Acesso em 10/06/2017. BRASIL. Lei n° 11.892, de 29 de dezembro de 2008. Lei de Criação dos Institutos Federais. Institui a Rede Federal de Educação Profissional, Científica e Tecnológica, cria os Institutos Federais de Educação, Ciência e Tecnologia, e dá outras providências. Disponível em: < http://www.planalto.gov.br/ccivil_03/_ato2007-2010/2008/lei/l11892.htm >. Acesso em 08/06/2017. BRASIL. Lei n° 11.892, de 29 de dezembro de 2008. Lei de Criação dos Institutos Federais. Institui a Rede Federal de Educação Profissional, Científica e Tecnológica, cria os Institutos Federais de Educação, Ciência e Tecnologia, e dá outras providências. Disponível em: < http://www.planalto.gov.br/ccivil_03/_ato2007-2010/2008/lei/l11892.htm >. Acesso em 08/06/2017. BRASIL. Lei n° 11.892, de 29 de dezembro de 2008. Lei de Criação dos Institutos Federais. Institui a Rede Federal de Educação Profissional, Científica e Tecnológica, cria os Institutos Federais de Educação, Ciência e Tecnologia, e dá outras providências. Disponível em: < http://www.planalto.gov.br/ccivil_03/_ato2007-2010/2008/lei/l11892.htm >. Acesso em 08/06/2017. BRASIL. Portal da Rede Federal de Educação Profissional Científica e Tecnológica. Histórico. 2016a. Disponível em: <http://redefederal.mec.gov.br/historico>. Acesso em: 03/06/2017. BRASIL. Portal da Rede Federal de Educação Profissional Científica e Tecnológica. Histórico. 2016a. Disponível em: <http://redefederal.mec.gov.br/historico>. Acesso em: 03/06/2017. 446 Cad. Prospec., Salvador, v. 10, n. 3 p.438-447, jul./set. 2017 D.O.I.: http://dx.doi.org/10.9771/cp.v10i3.23120 BRASIL. Portal da Rede Federal de Educação Profissional Científica e Tecnológica. Expansão da Rede Federal. 2016b. Disponível em: <http://redefederal.mec.gov.br/expansao-da-rede-federal>. Acesso em: 03/06/2017. Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Grupos de pesquisa: saiba mais. Disponível em: < http://lattes.cnpq.br/web/dgp/objetivos > Acesso em 07/06/2017. GODOY, Arilda Schmidt. Pesquisa Qualitativa: Tipos fundamentais. RAE – Revista de Administração de Empresas, São Paulo, v. 35, n. 3, p. 20 - 29, 1995. REFERÊNCIA Instituto Federal de Educação Ciências e Tecnologia – IF Baiano. Disponível em: <http://ifbaiano.edu.br/portal/pesquisa/nit/ > Acesso em: 20/08/2017. INSTITUTO NACIONAL DE PROPRIEDADE INTELECTUAL - INPI. Disponível em: <http://www.inpi.gov.br/sobre/estrutura > Acesso em: 07/06/2017. PERUCCHI, V. GARCIA, J. C. R. Indicadores de produção dos grupos de pesquisa do Instituto Federal de Educação, Ciência e Tecnologia da Paraíba. Revista Brasileira de Biblioteconomia e Documentação, São Paulo, v. 8, n. 1, p. 50-64, jan./jul. 2012. Disponível em: < https://rbbd.febab.org.br/rbbd/article/view/193/221 >. Acesso em: 19/06/2017. MARCONI, Marina de Andrade; LAKATOS, Eva Maria. Técnicas de pesquisa: planejamento e execução de pesquisas, amostragens e técnicas de pesquisa, elaboração, análise e interpretação de dados. São Paulo: Atlas, 1988. 205 p. MEC. Expansão da Rede Federal. 2014. Disponível em: < http://redefederal.mec.gov.br/expansao- da-rede-federal >. Acesso em: 03/06/2017. ORGANIZAÇÃO MUNDIAL DE PROPRIEDADE INTELECTUAL - OMPI. Disponível em: <http://www.wipo.int/edocs/lexdocs/laws/pt/mz/mz025pt.pdf >. Acesso em: 11/06/2017. CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial. CHAVES, A. O.; CRUZ, G. P. Eficiência do Instituto Federal Baiano: análise dos grupos de pesquisa e propriedade industrial 447
https://openalex.org/W2512221754	https://scholarworks.wm.edu/cgi/viewcontent.cgi?article=1033&context=vimsarticles	English	null	Short‐Term Habitat Use of Juvenile Atlantic Bluefin Tuna	Marine and coastal fisheries	2,016	cc-by	9,180	W&M ScholarWorks W&M ScholarWorks Virginia Institute of Marine Science Virginia Institute of Marine Science Virginia Institute of Marine Science Follow this and additional works at: https://scholarworks.wm.edu/vimsarticles Part of the Marine Biology Commons Part of the Marine Biology Commons This Article is brought to you for free and open access by the Virginia Institute of Marine Science at W&M ScholarWorks. It has been accepted for inclusion in VIMS Articles by an authorized administrator of W&M ScholarWorks. For more information, please contact scholarworks@wm.edu. Recommended Citation Recommended Citation Recommended Citation Recommended Citation Marcek, Benjamin J.; Fabrizio, Mary C.; and Graves, John, Short-Term Habitat Use of Juvenile Atlantic Bluefin Tuna (2016). MARINE AND COASTAL FISHERIES, 8(1), 395-403. 10.1080/19425120.2016.1168330 This Article is brought to you for free and open access by the Virginia Institute of Marine Science at W&M ScholarWorks. It has been accepted for inclusion in VIMS Articles by an authorized administrator of W&M ScholarWorks. For more information, please contact scholarworks@wm.edu. Marine and Coastal Fisheries Dynamics, Management, and Ecosystem Science Marine and Coastal Fisheries Dynamics, Management, and Ecosystem Science ISSN: (Print) 1942-5120 (Online) Journal homepage: http://www.tandfonline.com/loi/umcf20 Date: 08 December 2017, At: 09:17 Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=umcf20 Abstract Blueﬁn Tuna Thunnus thynnus are highly sought after in com- mercial and recreational ﬁsheries along the East Coast of North America. To appropriately assess and manage Atlantic Blueﬁn Tuna (ABT), it is necessary to understand their habitat use during multiple ontogenetic stages. We tagged 17 juvenile ABT in the northwest Atlantic Ocean with pop-up satellite archival tags (PSATs) to determine environmental factors that may affect habi- tat use. The PSATs were deployed off the coast of Massachusetts in August and September 2012. A generalized linear mixed model was applied to determine factors affecting the mean depth occu- pied by ﬁsh, and beta regression was used to understand factors affecting the proportion of time spent below the thermocline. Thermocline depth signiﬁcantly affected the mean depth occupied by juvenile ABT and the proportion of time they spent below the thermocline. Time period (dawn, day, dusk, and night) also sig- niﬁcantly affected the mean depth occupied by juvenile ABT. Additionally, the time period × lunar illumination interaction had a signiﬁcant effect on the proportion of time spent below the thermocline. This study is the ﬁrst to demonstrate that environ- mental factors such as thermocline depth, time period, and lunar illumination can signiﬁcantly impact vertical habitat use by juve- nile ABT and demonstrates the utility of generalized linear mixed models for investigating ﬁsh habitat use. Although habitat use by adult ABT is well studied (Lutcavage et al 2000; Block et al. 2001; Newlands et al. 2004; Schick et al. 2004; Stokesbury et al. 2004; Wilson et al. 2005; Teo et al. 2007; Walli et al. 2009; Druon et al. 2016), less information is available regarding habitat use by juvenile ABT (Brill et al. 2002; Galuardi and Lutcavage 2012; Druon et al. 2016). Additionally, studies of juvenile ABT habitat use have focused on temporal changes in habitat use at diel and seasonal levels (Brill et al. 2002; Galuardi and Lutcavage 2012; Druon et al. 2016) and on how horizontal habitat use changes with factors like sea surface temperature and chlorophyll concentration (Druon et al. 2016); however, the manner in which environmental factors may affect vertical habitat use by juvenile ABT has not been investigated. Downloaded by [College of William & Mary] at 09:17 08 D Downloaded by [College of William & M During summer, juvenile ABT spend the majority (>90%) of their time in the upper 30 m of the water column (Brill et al. Short-Term Habitat Use of Juvenile Atlantic Blueﬁn Tun Downloaded by [College of William & Mary] at 09:17 08 December 2017 2014). An understanding of habitat use by Atlantic Blueﬁn Tuna (ABT) at multiple ontogenetic stages is essential for accurately assessing their abundance and for determining appropriate management strategies. Corresponding author: bmarcek@vims.edu Received June 19, 2015; accepted March 13, 2016 © Benjamin J. Marcek, Mary C. Fabrizio, and John E. Graves This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. Benjamin J. Marcek, Mary C. Fabrizio & John E. Graves To cite this article: Benjamin J. Marcek, Mary C. Fabrizio & John E. Graves (2016) Short-Term Habitat Use of Juvenile Atlantic Bluefin Tuna, Marine and Coastal Fisheries, 8:1, 395-403, DOI: 10.1080/19425120.2016.1168330 To cite this article: Benjamin J. Marcek, Mary C. Fabrizio & John E. Graves (2016) Short-Term Habitat Use of Juvenile Atlantic Bluefin Tuna, Marine and Coastal Fisheries, 8:1, 395-403, DOI: 10.1080/19425120.2016.1168330 Published with license by the American Fisheries Society© Benjamin J. Marcek, Mary C. Fabrizio, and John E. Graves Published online: 12 Aug 2016. Submit your article to this journal Article views: 234 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=umcf20 Date: 08 December 2017, At: 09:17 Download by: [College of William & Mary] Marine and Coastal Fisheries: Dynamics, Management, and Ecosystem Science 8:395–403, 2016 Published with license by the American Fisheries Society ISSN: 1942-5120 online DOI: 10.1080/19425120.2016.1168330 Subject editor: Michelle Heupel, James Cook University, Queensland, Australia Abstract As with other large pelagic predators, habitat use by juvenile ABT is likely regulated by their physiology (Brill 1994; Brill et al. 2005; Galli et al. 2009; Block et al. 2011), although other factors (e.g., prey availability) may also affect habitat use (Bertrand et al. 2002; Schick and Lutcavage 2009). To investigate how environ- mental factors inﬂuence short-term habitat use by juvenile ABT, we used PSATs to gather high-frequency environmental data from individual ﬁsh (Horodysky et al. 2007; Graves et al. 2009). We examined the following null hypotheses: (1) environmental fac- tors do not affect the mean depth occupied by juvenile ABT and (2) environmental factors do not affect the proportion of time juvenile ABT spend in waters below the thermocline. Other factors, such as time of day and lunar illumination, are known to affect habitat use by adult ABT (Wilson et al. 2005), Bigeye Tuna T. obesus (Lam et al. 2014), Albacore T. alalunga (Cosgrove et al. 2014), and juvenile Southern Blueﬁn Tuna T. maccoyii (Bestley et al. 2009) and may affect habitat use by juvenile ABT as well. For Albacore, the percentage of time spent in shallow waters (<50 m) was typically greater at night (86%) than during the day (62%; Cosgrove et al. 2014). Adult ABT also were reported to exhibit diel variation in habitat use, maintaining a greater mean depth during the day than during dawn, dusk, or night (Wilson et al. 2005). No statistical differ- ences in diel habitat use have been observed for juvenile ABT (Brill et al. 2002; Galuardi and Lutcavage 2012). Increasing lunar illumination (from new moon to full moon) was asso- ciated with increasing mean depth occupied by adult ABT (Wilson et al. 2005) and was signiﬁcantly correlated with mean nighttime depth for adult Bigeye Tuna (Lam et al. 2014) and juvenile Paciﬁc Blueﬁn Tuna (Kitagawa et al. 2007a). For juvenile Southern Blueﬁn Tuna, lunar phase was also an important factor in models predicting mean depth, maximum depth, proportion of time at the surface, and propor- tion of time at depths exceeding 100 m (Bestley et al. 2009). This relationship has not yet been examined for juvenile ABT. Downloaded by [College of William & Mary] at 09:17 The PSATs were programmed to record pressure (depth), temperature, and light every 5 min and to release after a 31-d deployment. Abstract 2002); the mean depth occupied is shallower in summer (5–12 m) than in winter (41–58 m; Galuardi and Lutcavage 2012). Juvenile ﬁsh may use deep waters as well: pop-up satellite archival tags (PSATs) have recorded individuals making vertical excursions to depths up to 800 m (Galuardi and Lutcavage 2012). Due to their extensive vertical movements, juvenile ABT may encounter a wide range of temperatures in a relatively short time interval. Although juvenile ABT are known to spend the majority of their time in waters warmer than 17°C during Blueﬁn Tuna Thunnus thynnus support commercial and recreational ﬁsheries along the East Coast of North America. The commercial ﬁshery in the Atlantic Ocean targets large adult Blueﬁn Tuna, whereas the recreational ﬁshery is sup- ported by smaller ﬁsh, which are often immature (ICCAT Subject editor: Michelle Heupel, James Cook University, Queensland, Australia Corresponding author: bmarcek@vims.edu Received June 19, 2015; accepted March 13, 2016 395 396 MARCEK ET AL. (IACUC-2011-07-11-7390-jegrav) at the College of William and Mary and complied with all applicable U.S. guidelines. summer, they also use waters of 10°C or less for short periods (Brill et al. 2002; Galuardi and Lutcavage 2012; Druon 2016). Juvenile ABT may exploit these deeper, cooler waters in order to feed—similar to the observed habitat use of Paciﬁc Blueﬁn Tuna Thunnus orientalis, Skipjack Tuna Katsuwonus pelamis, and Yellowﬁn Tuna T. albacares (Marchal and Lebourges 1996; Kitagawa et al. 2007b)—or to behaviorally thermoregulate, as seen in Yellowﬁn Tuna (Block et al. 1997). Although both juvenile and adult ABT have been documented as making excursions below the thermocline (Stokesbury et al. 2004; Galuardi and Lutcavage 2012), the thermocline may act as a barrier to movement for adult ABT (Wilson et al. 2005; Walli et al. 2009). However, the potential for the thermocline to act as a barrier has not been investigated for juvenile ABT. y p pp g Tag deployment.—Juvenile ABT (91–119 cm curved fork length) were captured from coastal waters of Massachusetts (n = 17) between August 2 and September 22, 2012 (Figure 1), reﬂecting the availability of ﬁsh to the recreational ﬁshery during summer 2012. Juvenile ABT were captured via methods commonly employed in the U.S. recreational ﬁshery (i.e., rod and reel, with a lure or lure–bait combination rigged with a large “J” hook [8/0–10/0] as the terminal tackle). Abstract After release, the PSATs ﬂoated to the surface and transmitted their archived data to the Advanced Research and Global Observation Satellite system. To ensure that tags would be released from moribund ﬁsh, the PSATs were pro- grammed with two emergency release mechanisms as described by Musyl et al. (2011). The PSAT was released from a moribund ﬁsh if a constant depth was recorded for 4 d or if a maximum depth of 1,250 m was reached. Analyses.—We used the data from each PSAT to (1) model the mean depth occupied by juvenile ABT and the proportion of time they spent below the thermocline and (2) explore the effects FIGURE 1. Map of the tag deployment location and dispersal routes for the 16 juvenile Atlantic Blueﬁn Tuna (91–119 cm curved fork length) with reporting pop-up satellite archival tags deployed in August and September 2012 off the Massachusetts coast. Fish were captured using standard trolling methods. Arrows indicate dispersal direction and distance. As with other large pelagic predators, habitat use by juvenile ABT is likely regulated by their physiology (Brill 1994; Brill et al. 2005; Galli et al. 2009; Block et al. 2011), although other factors (e.g., prey availability) may also affect habitat use (Bertrand et al. 2002; Schick and Lutcavage 2009). To investigate how environ- mental factors inﬂuence short-term habitat use by juvenile ABT, we used PSATs to gather high-frequency environmental data from individual ﬁsh (Horodysky et al. 2007; Graves et al. 2009). We examined the following null hypotheses: (1) environmental fac- tors do not affect the mean depth occupied by juvenile ABT and (2) environmental factors do not affect the proportion of time juvenile ABT spend in waters below the thermocline. FIGURE 1. Map of the tag deployment location and dispersal routes for the 16 juvenile Atlantic Blueﬁn Tuna (91–119 cm curved fork length) with reporting pop-up satellite archival tags deployed in August and September 2012 off the Massachusetts coast. Fish were captured using standard trolling methods. Arrows indicate dispersal direction and distance. Abstract A minimum of 30 min elapsed between tagging events to reduce the likelihood of sampling multiple individuals from a single school. Fish were brought onto the vessel, measured for curved fork length, and tagged with a PSAT (High-Rate X-Tag; Microwave Telemetry, Columbia, Maryland). The tag anchor was inserted into the dorsal musculature at a point directly posterior and ventral to the anterior insertion of the ﬁrst dorsal ﬁn; the ﬁsh was then released. The entire tagging process was brief (~1.5 min on average). The tag anchor (3.2 cm long × 2.4 cm wide) was a hydroscopic surgical- grade nylon assembly that was attached to the PSAT with a tether consisting of 16 cm of monoﬁlament ﬁshing line (91-kg breaking strength). A more detailed description of the PSAT assembly and deployment protocol is given by Marcek and Graves (2014). Downloaded by [College of William & Mary] at 09:17 08 December 2017 Other factors, such as time of day and lunar illumination, are known to affect habitat use by adult ABT (Wilson et al. 2005), Bigeye Tuna T. obesus (Lam et al. 2014), Albacore T. alalunga (Cosgrove et al. 2014), and juvenile Southern Blueﬁn Tuna T. maccoyii (Bestley et al. 2009) and may affect habitat use by juvenile ABT as well. For Albacore, the percentage of time spent in shallow waters (<50 m) was typically greater at night (86%) than during the day (62%; Cosgrove et al. 2014). Adult ABT also were reported to exhibit diel variation in habitat use, maintaining a greater mean depth during the day than during dawn, dusk, or night (Wilson et al. 2005). No statistical differ- ences in diel habitat use have been observed for juvenile ABT (Brill et al. 2002; Galuardi and Lutcavage 2012). Increasing lunar illumination (from new moon to full moon) was asso- ciated with increasing mean depth occupied by adult ABT (Wilson et al. 2005) and was signiﬁcantly correlated with mean nighttime depth for adult Bigeye Tuna (Lam et al. 2014) and juvenile Paciﬁc Blueﬁn Tuna (Kitagawa et al. 2007a). For juvenile Southern Blueﬁn Tuna, lunar phase was also an important factor in models predicting mean depth, maximum depth, proportion of time at the surface, and propor- tion of time at depths exceeding 100 m (Bestley et al. 2009). This relationship has not yet been examined for juvenile ABT. METHODS The experimental protocols used in the present study were approved by the Institutional Animal Care and Use Committee NOTE 397 FIGURE 2. Depth and temperature proﬁles over 6 d in late August and early September 2012 for a juvenile Atlantic Blueﬁn Tuna (ﬁsh 4) that was tagged with a pop-up satellite archival tag and released off the Massachusetts coast. This individual displayed typical behavior, remaining in warmer waters near the sur- face for the majority of tag deployment but making periodic excursions to cooler, deeper waters. The majority of vertical excursions also occurred during the day, whereas the ﬁsh remained near the surface at night (gray-shaded bars). of environmental factors on habitat use by juvenile ABT. The mean depth (m) was calculated for dawn, day, dusk, and night periods, whereas the proportion of time spent below the thermocline was calculated for day and night only (as deﬁned below). The proportion of time spent below the thermocline was calculated as the time (min) during which the ﬁsh used waters below the thermocline divided by the total time (min) in the period of interest. If there was no discernible thermocline, the corresponding data were excluded from the analysis. The predictors included in the models of mean depth and proportion of time spent below the thermocline were time period (dawn, day, dusk, or night), lunar illumination, and thermocline depth. For the mean depth model, we deﬁned four discrete 1-h time periods (dawn, day, dusk, and night) from each 24-h day to investigate the effects of diel and crepuscular periods on juvenile ABT habitat use. Times of sunrise and sunset from the U.S. Naval Observatory Web site (aa.usno.navy.mil/faq/ docs/RST_defs.php) were used to deﬁne these periods. Crepuscular periods were deﬁned as sunrise and sunset ±30 min, day was deﬁned as the midpoint between sunrise and sunset ±30 min, and night was deﬁned as the midpoint between sunset and sunrise ±30 min. One-hour intervals were used for each time period to reduce the likelihood of dampening crepuscular signals by the inclusion of observa- tions from adjacent times and to reduce correlations among observations within a day. For the model examining the pro- portion of time spent below the thermocline, we deﬁned two 6-h periods (day and night): day was deﬁned as the midpoint between sunrise and sunset ±3 h, and night was deﬁned as the midpoint between sunset and sunrise ±3 h. METHODS The 6-h time intervals were used to ensure that the number of observations was sufﬁcient for calculating the proportion of time spent below the thermocline. In addition, lunar illumination data Downloaded by [College of William & Mary] at 09:17 08 December 2017 FIGURE 2. Depth and temperature proﬁles over 6 d in late August and early September 2012 for a juvenile Atlantic Blueﬁn Tuna (ﬁsh 4) that was tagged with a pop-up satellite archival tag and released off the Massachusetts coast. This individual displayed typical behavior, remaining in warmer waters near the sur- face for the majority of tag deployment but making periodic excursions to cooler, deeper waters. The majority of vertical excursions also occurred during the day, whereas the ﬁsh remained near the surface at night (gray-shaded bars). Downloaded by [College of William & Mary] at 09:17 08 D were acquired from the U.S. Naval Observatory Web site. Lunar illumination was reported as the proportion of the moon that was illuminated (0.0–1.0), where 0.0 represents the new moon and 1.0 represents the full moon. Downloaded by [College of William & M We used vertical proﬁles of depth and temperature from tagged ABT to calculate thermocline depth (Figure 2). Depth– temperature proﬁles were created for each ﬁsh by aggregating data into 5-d intervals, binning the depth data into 1-m inter- vals, and calculating the mean temperature for each bin (±SE; FIGURE 3. Vertical proﬁles of the water column occupied by juvenile Atlantic Blueﬁn Tuna (with pop-up satellite archival tags) from August to October 2012 offshore of Massachusetts. Examples of depth–temperature proﬁles for two individuals at different times during the tag deployment are shown: (A) ﬁsh 1 during late August and (B) ﬁsh 16 during mid-October. The thermocline (horizontal dotted line) was established at 15 m in August (A) and increased to 60 m in mid- October (B). The mean temperature estimate for each depth is presented with ±SE. FIGURE 3. Vertical proﬁles of the water column occupied by juvenile Atlantic Blueﬁn Tuna (with pop-up satellite archival tags) from August to October 2012 offshore of Massachusetts. Examples of depth–temperature proﬁles for two individuals at different times during the tag deployment are shown: (A) ﬁsh 1 during late August and (B) ﬁsh 16 during mid-October. The thermocline (horizontal dotted line) was established at 15 m in August (A) and increased to 60 m in mid- October (B). METHODS The mean temperature estimate for each depth is presented with ±SE. 398 MARCEK ET AL. Figure 3). Data were aggregated over 5-d intervals to ensure that there were sufﬁcient observations to construct depth– temperature proﬁles, thus allowing us to reconstruct the phy- sical conditions of the water column in areas occupied by the ﬁsh. The depth of the thermocline was identiﬁed as the depth with the maximum gradient in water temperature. Days for which the thermocline could not be deﬁned were omitted from this analysis. where Yij is the mean depth occupied by individual j in time period i; µ is the overall average mean depth occupied; α is the effect of thermocline depth; ρ is the effect of period (dawn, day, dusk, or night); γ is the effect of lunar illumination; and εij is the random unexplained error. Proportion of time spent below the thermocline.—The proportion of time that juvenile ABT were below the thermocline during the day or night period was bounded within the interval [0, 1]; furthermore, like most proportion data, the response data were characterized by skewness. These two properties violate the assumptions of normality and homogeneity of variance, which are required for the use of general linear models (Swearingen et al. 2012). We therefore used the beta distribution to model the response in a regression framework (Swearingen et al. 2012). As before, we considered and modeled the correlations between repeated observations from the same ﬁsh. Mean depth occupied by juvenile Atlantic Blueﬁn Tuna.— The mean depth occupied by juvenile ABT was analyzed by using a generalized linear mixed model with repeated measures (MIXED procedure in the Statistical Analysis System [SAS] version 9.3; SAS Institute, Cary, North Carolina). Mean depth was loge transformed to meet the assumption of homogeneity of variance (Logan 2010). To allow for this transformation, 0.01 m was added to the zero observations (mean depth = 0 m when a ﬁsh was at the surface during the entire 1-h period of observation); zero observations constituted 3 (0.002%) of the 1,812 observations. Because multiple observations of depth and temperature were collected for each ﬁsh, we assumed that consecutive observations from the same ﬁsh were correlated. Therefore, we used a mixed model with repeated measures to model the mean depth occupied by juvenile ABT, with individual ﬁsh as the subject. METHODS Because individual ABT in this study represented a random sample from the population and because substantial variation in habitat use was observed among ﬁsh, we treated individual ﬁsh as a random factor; all other factors (thermocline depth, time period, and lunar illumination) were considered ﬁxed effects. Additionally, t-tests were used to evaluate the signiﬁcance of the factors’ effects. We considered four covariance structures to describe the correlation between responses of an individual ﬁsh: variance components, compound symmetry, ﬁrst-order autoregressive, and banded Toeplitz (Littell et al. 2000). Akaike’s information criterion (AIC) was used to compare models that differed in covariance structure, and the best model was selected based on the lowest AIC value (Logan 2010). The covariance structure that best ﬁt the data (eight- banded Toeplitz) was used in the ﬁnal model. In addition, plots revealed potential two-way interactions (1) between thermocline depth and lunar illumination and (2) between time period and lunar illumination. Models that contained interactions were evaluated to determine whether the AIC value was lower (i.e., better performance) than that of the simpler model lacking the interactions (Hastie et al. 2009); we found that mean depth models lacking the interaction terms exhibited a better ﬁt to the data, and therefore the interaction terms were omitted. The ﬁnal model used in our analysis for mean occupied depth followed the form Downloaded by [College of William & Mary] at 09:17 08 December 2017 The proportion of time spent below the thermocline was analyzed using a beta regression approach with repeated mea- sures as implemented with the GLIMMIX (generalized linear mixed models) procedure in SAS. A generalized estimating equation (GEE) approach was used to estimate model para- meters due to the correlations among repeated observations. Because the GEE in GLIMMIX uses a pseudolikelihood esti- mation technique, model ﬁt could not be assessed with the typical criteria (AIC, Bayesian information criterion, etc.; Vonesh 2012). Instead, we evaluated the adequacy of the modeled covariance structure by using the variance of the Pearson residuals (Dickey 2010) and the ratio of the general- ized chi-square statistic (χ2) to its degrees of freedom (df; Schabenberger 2005). If the variance of the Pearson residuals was near 1.00, the covariance structure was considered an adequate ﬁt to the data (Dickey 2010). Additionally, a χ2:df ratio close to 1.00 indicates that the variability in the data was appropriately modeled and that there is no residual overdisper- sion (Schabenberger 2005). Yij ¼ μ þ ρi þ α þ γ þ εij; Yij ¼ μ þ ρi þ α þ γ þ εij; (1) RESULTS Tag deployment date, curved fork length (CFL) of tagged fish, number of days the tags were deployed, the percentage of archived data recovered from each tag, and the minimum straight-line distance traveled by each juvenile Atlantic Bluefin Tuna (n = 17) tagged with pop-up satellite archival tags offshore of Chatham, Massachusetts, during 2012. TABLE 1. Tag deployment date, curved fork length (CFL) of tagged fish, number of days the tags were deployed, the percentage of archived data recovered from each tag, and the minimum straight-line distance traveled by each juvenile Atlantic Bluefin Tuna (n = 17) tagged with pop-up satellite archival tags offshore of Chatham, Massachusetts, during 2012. Fish Date of tag deployment CFL (cm) Days deployed % of data recovered Minimum straight-line distance (km) 1 Aug 2 109 31 89 207.3 2 Aug 2 107 31 85 44.4 3 Aug 2 107 6 100 59.4 4 Aug 4 107 31 80 97.9 5 Aug 29 117 31 89 118.0 6 Sep 12 114 31 86 134.6 7 Sep 12 114 31 86 109.6 8 Sep 14 109 31 88 48.6 9 Sep 14 117 31 87 245.1 10 Sep 15 117 31 87 402.5 11 Sep 15 114 31 88 189.9 12 Sep 15 91 31 91 121.3 13 Sep 21 91 16 98 18.0 14 Sep 21 119 31 89 169.8 15 Sep 22 99 31 90 116.4 16 Sep 22 99 31 80 185.8 17 Sep 22 119 Did not report FIGURE 4. Mean depth occupied by juvenile Atlantic Blueﬁn Tuna (n = 16) during four time periods: dawn, day, dusk, and night. Each point represents the model-predicted mean depth (±SE) occupied during a given time period. Fish were tagged with pop-up satellite archival tags and released offshore of Massachusetts in August and September 2012. METHODS We investigated models that used either a variance components function or a ﬁrst-order autore- gressive function to describe the covariance structure; based on the variance of the Pearson residuals and the χ2:df ratio, we determined that the ﬁrst-order autoregressive structure resulted in a better model ﬁt. Because it was unlikely that repeated observations from each ﬁsh were completely independent of one another, the variance components structure—which assumes zero correlation—was deemed unreasonable for these data. Potential interactions (thermocline depth × lunar illumination; time period × lunar illumination) were consid- ered. Based on inspection of the interaction plots, the time period × lunar illumination interaction was included in the ﬁnal model, which followed the form Yij ¼ μ þ ρi þ α þ γ þ ðρi γÞ þ εij; (2) (2) where Yij is the transformed proportion of time spent below the thermocline by individual j during time period i; µ is the (1) Yij ¼ μ þ ρi þ α þ γ þ εij; 399 NOTE TABLE 2. Parameter estimates for the model describing the mean depth occupied by juvenile Atlantic Bluefin Tuna. overall mean proportion of time spent below the thermocline; and model predictors are as deﬁned for equation (1) except that the period effect ρ includes only day and night. As before, individual ﬁsh were included as a random factor. i f i d (Fi 1) Mi i i h li Factor Estimate t P Intercept 1.56 6.62 <0.01 Time period Dawn 0.21 2.05 0.04 Day 0.31 2.98 <0.01 Dusk 0.13 1.25 0.21 Night 0 – – Lunar illumination 0.19 1.88 0.06 Thermocline depth 0.04 5.35 <0.01 RESULTS Sixteen (94%) of the 17 PSATs reported between 80% and 100% of their archived data (mean ± SE = 88.3 ± 1.3%); one tag did not report (Table 1). Of the 16 reporting tags, one released prematurely at 6 d postdeployment. Additionally, one PSAT (and presumably the ﬁsh carrying it) was inferred to have been consumed by a predator 12 d after deployment (see Marcek and Graves 2014 for details). For that individual, the depth, temperature, and light proﬁles were consistent with normal behavior of juvenile ABT up to day 12 postdeploy- ment; therefore, we considered data from only the ﬁrst 11 d after tag deployment. Pooling across individuals, we observed that juvenile ABT occupied the upper 30 m of the water column for 81.1 ± 3.3% (mean ± SE) of their time, and waters between 17°C and 24°C were used 84.0 ± 3.3% of the time. Additionally, the pop-up locations of tags indicated a variety of dispersal patterns away from the tagging locations, with several ﬁsh moving in a generally southward direction toward winter foraging grounds (Figure 1). Minimum straight-line displacements ranged from 18 to 402 km (Table 1). winter foraging grounds (Figure 1). Minimum straight-line displacements ranged from 18 to 402 km (Table 1). winter foraging grounds (Figure 1). Minimum straight-line displacements ranged from 18 to 402 km (Table 1). Downloaded by [College of William & Mary] at 09:17 08 December 2017 Mean Depth Occupied by Juvenile Atlantic Blueﬁn Tuna Variation among individual ﬁsh explained 15.4% of the overall variation in mean depth occupied by juvenile ABT. Inclusion of individual ﬁsh as a random factor resulted in a lower AIC score, indicating that differences in the behavior of individuals explained a signiﬁcant portion of the variabil- ity in mean occupied depth. Fish occupied a signiﬁcantly greater mean depth during dawn (ρdawn = 0.21, t = 2.05, P = 0.04) and day (ρday = 0.31, t = 2.98, P < 0.01) than at night (Table 2; Figure 4). The mean depth occupied by juvenile ABT also increased signiﬁcantly with thermocline depth (α = 0.03, t = 5.35, P < 0.01). Although lunar illumina- tion was not a signiﬁcant factor in the model, the data Downloaded by [College of William & Mary] at 09:17 TABLE 1. Proportion of Time Spent below the Thermocline The proportion of time in which juvenile ABT used waters below the thermocline was signiﬁcantly affected by thermo- cline depth. As thermocline depth increased, the proportion of time spent below the thermocline decreased (α = –0.04, t = –2.09, P = 0.05; Table 3). Additionally, the time period × lunar illumination interaction signiﬁcantly affected the proportion of time spent below the thermocline. Regardless of lunar illumi- nation, the proportion of time juvenile ABT spent below the thermocline was greater during the day than at night (Figure 5). Additionally, there was little effect of lunar illumi- nation on the proportion of time spent below the thermocline during the day, but as lunar illumination increased, the propor- tion of time spent below the thermocline at night increased Downloaded by [College of William & Mary] at 09:17 08 De The time period × lunar illumination interaction had a signiﬁcant effect on the proportion of time spent below the thermocline by juvenile ABT. As lunar illumination increased, the proportion of time spent below the thermocline increased at night relative to day. Juvenile ABT likely spend more time below the thermocline during periods of greater lunar illumi- nation so as to detect the silhouettes of prey and to increase their feeding efﬁciency as described above. Our results sug- gested that the mean depth occupied by juvenile ABT also increased with increasing lunar illumination, although the effect was not signiﬁcant. Further investigation of this rela- tionship is necessary, as lunar illumination has been shown to impact habitat use by other tunas, such as the Bigeye Tuna (Lam et al. 2014) and Southern Blueﬁn Tuna (Bestley et al. 2009). FIGURE 5. Proportion of time for which juvenile Atlantic Blueﬁn Tuna (ABT; n = 16) occupied waters below the thermocline in relation to increasing lunar illumination. The ﬁsh were tagged with pop-up satellite archival tags and released offshore of Massachusetts in August and September 2012. Day is represented with a solid line, and night is represented with a dashed line. The curves presented here are ﬁtted to predictions from the model describing the proportion of time spent below the thermocline. Dotted lines represent the 95% conﬁdence interval surrounding the model-predicted mean response. We demonstrated that thermocline depth was a signiﬁcant factor determining both the mean depth occupied by juvenile ABT and the proportion of time they spent below the ther- mocline. RESULTS relative to the proportion spent there during the day (ρnight × γ = 2.02, t = –2.81, P < 0.01; Figure 5). suggested that the mean occupied depth increased with increasing lunar illumination (γ = 0.19, t = 1.88, P = 0.06). TABLE 3. Parameter estimates for the model describing the proportion of time spent below the thermocline by juvenile Atlantic Bluefin Tuna. Factor Estimate t P Intercept –2.13 –2.91 <0.01 Thermocline depth –0.04 –2.09 0.05 Time period × lunar illumination Day 0 Night 2.02 2.81 <0.01 TABLE 3. Parameter estimates for the model describing the proportion of time spent below the thermocline by juvenile Atlantic Bluefin Tuna. TABLE 3. Parameter estimates for the model describing the proportion of time spent below the thermocline by juvenile Atlantic Bluefin Tuna. DISCUSSION The observation that juvenile ABT occupy greater mean depths during dawn and day than at night has not been noted in previous studies of juvenile ABT habitat use (Brill et al. 2002; Galuardi and Lutcavage 2012). This behavior may be related to the feeding ecology of ABT. The retinal cell density in the eyes of tunas indicates that their best visual axis is above and in front of the ﬁsh’s direction of travel (Tamura and Wilsby 1963; Kawamura et al. 1981; Somiya et al. 2000), thus allowing ABT to detect the silhouettes of prey against downwelling light. As the sun rises, this strategy may provide a means of locating prey that have moved closer to the surface at night, thereby increasing foraging efﬁciency of juvenile ABT. They may employ a similar strategy during the day by using downwelling sunlight to detect silhouetted prey swim- ming higher in the water column. suggested that the mean occupied depth increased with increasing lunar illumination (γ = 0.19, t = 1.88, P = 0.06). suggested that the mean occupied depth increased with increasing lunar illumination (γ = 0.19, t = 1.88, P = 0.06). Downloaded by [College of William & Mary] at 09:17 08 December 2017 RESULTS Fish Date of tag deployment CFL (cm) Days deployed % of data recovered Minimum straight-line distance (km) 1 Aug 2 109 31 89 207.3 2 Aug 2 107 31 85 44.4 3 Aug 2 107 6 100 59.4 4 Aug 4 107 31 80 97.9 5 Aug 29 117 31 89 118.0 6 Sep 12 114 31 86 134.6 7 Sep 12 114 31 86 109.6 8 Sep 14 109 31 88 48.6 9 Sep 14 117 31 87 245.1 10 Sep 15 117 31 87 402.5 11 Sep 15 114 31 88 189.9 12 Sep 15 91 31 91 121.3 13 Sep 21 91 16 98 18.0 14 Sep 21 119 31 89 169.8 15 Sep 22 99 31 90 116.4 16 Sep 22 99 31 80 185.8 17 Sep 22 119 Did not report Fish Date of tag deployment CFL (cm) Days deployed % of data recovered Minimum straight-line distance (km) 1 Aug 2 109 31 89 207.3 2 Aug 2 107 31 85 44.4 3 Aug 2 107 6 100 59.4 4 Aug 4 107 31 80 97.9 5 Aug 29 117 31 89 118.0 6 Sep 12 114 31 86 134.6 7 Sep 12 114 31 86 109.6 8 Sep 14 109 31 88 48.6 9 Sep 14 117 31 87 245.1 10 Sep 15 117 31 87 402.5 11 Sep 15 114 31 88 189.9 12 Sep 15 91 31 91 121.3 13 Sep 21 91 16 98 18.0 14 Sep 21 119 31 89 169.8 15 Sep 22 99 31 90 116.4 16 Sep 22 99 31 80 185.8 17 Sep 22 119 Did not report FIGURE 4. Mean depth occupied by juvenile Atlantic Blueﬁn Tuna (n = 16) during four time periods: dawn, day, dusk, and night. Each point represents the model-predicted mean depth (±SE) occupied during a given time period. Fish were tagged with pop-up satellite archival tags and released offshore of Massachusetts in August and September 2012. FIGURE 4. Mean depth occupied by juvenile Atlantic Blueﬁn Tuna (n = 16) during four time periods: dawn, day, dusk, and night. Each point represents the model-predicted mean depth (±SE) occupied during a given time period. Fish were tagged with pop-up satellite archival tags and released offshore of Massachusetts in August and September 2012. 400 MARCEK ET AL. Proportion of Time Spent below the Thermocline 2013), indicating impaired cardiac function at low temperatures. Our results show that environmental factors, such as ther- mocline depth, lunar illumination, and time period, can have signiﬁcant effects on habitat use by juvenile ABT. However, because of the small sample size included in these analyses (n = 16) and the small spatial and temporal scales covered, additional data will help to elucidate some of the spatial and temporal dynamics of ABT habitat use that were beyond the scope of this project. Furthermore, a temporally intensive assessment of pelagic prey distribution and abundance, con- ducted simultaneously with a tagging study of juvenile ABT, would allow investigation of prey abundance effects on juve- nile ABT habitat use. A better understanding of how the distribution of juvenile ABT is affected by environmental factors and prey abundance will improve spatial and temporal estimates of catchability in the recreational ﬁshery and will result in more accurate estimates of juvenile ABT abundance. The relationships between thermocline depth and the mean depth occupied by juvenile ABT and the proportion of time spent below the thermocline may reﬂect a strategy for opti- mizing the ﬁsh’s foraging efﬁciency while allowing them to remain within their physiological tolerance. As thermocline depth increases, juvenile ABT may be able to occupy deeper waters for a longer period of time because they are not moving through the thermocline but instead remain in the relatively warm, well-mixed layer of the water column, which allows them to forage more efﬁciently. Downloaded by [College of William & Mary] at 09:17 08 December 2017 Habitat use by potential prey is likely to change with environmental factors (e.g., thermocline depth and lunar illu- mination), and juvenile ABT likely react to changes in prey depth. Incidental observations of stomach contents from juve- nile ABT indicated that some ﬁsh had recently fed on sand lances Ammodytes spp., while others fed primarily on Atlantic Herring Clupea harengus (B. J. Marcek, personal observa- tion). Juvenile ABT may alter their habitat use to maximize their co-occurrence with prey species such as sand lances and Atlantic Herring (Eggleston and Bochenek 1990; Chase 2002; Schick and Lutcavage 2009; Logan et al. 2011, 2015); such behaviors have been observed in other large pelagic ﬁshes like the Bigeye Tuna, Yellowﬁn Tuna (Grubbs and Holland 2003), and Swordﬁsh Xiphias gladius (Carey 1990). Proportion of Time Spent below the Thermocline Atlantic Herring, which are commonly found from Cape Cod to Greenland (Bigelow and Schroeder 2002), occupy depths of 0 to 200 m (Whitehead 1985) and prefer temperatures between 8°C and 12°C (Stickney 1969). Sand lances are widely distributed from inshore waters to offshore banks (Bigelow and Schroeder 2002) from Cape Hatteras to Greenland (Nizinski et al. 1990) and are abundant from New Jersey to the Gulf of Maine (Bigelow and Schroeder 2002). Sand lances are most commonly found at temperatures ranging from –2°C to 11°C (Scott 1968) and move inshore during summer, particularly at northern latitudes (Reay 1970). Differences in the distributions of sand lances and Atlantic Herring and their co-occurrence with juvenile ABT may cause those prey ﬁshes to have differ- ential predation susceptibility. Sand lances and Atlantic Herring are often found at temperatures that occur below the thermocline during summer; this may induce more frequent vertical feeding excursions by juvenile ABT, potentially lead- ing to increased mean occupied depths and more time spent below the thermocline. Downloaded by [College of William & Mary] at 09:17 Finally, we note that the beta regression technique allowed the use of proportion data to describe habitat utilization— something that was not possible with conventional linear modeling techniques without transforming the data to values outside the bounded range of [0, 1]. Beta regression could be incorporated into many habitat use studies in addition to studies that employ PSATs. For instance, implantable archival tags could yield similar data and could allow investigation of seasonal components. Beta regression models could also be applied to data from studies using passive acoustic arrays to monitor the use of a prescribed habitat or a marine protected area. Despite the fact that beta regression is new to ﬁsheries research, it clearly can be a useful tool for analyzing habitat use from temporally intensive data. ACKNOWLEDGMENTS This project was supported by the Guy Harvey Ocean Foundation and the National Science Foundation GK-12 Program (0840804). We thank the captains and crews of the Big Fish II, Big Fish III, For2na, Ocean Runner, Matador, Salty Dogs, Know Name, Game On, Sea Habit, Gina Marie, Oyster Catcher, McSeas, and Aries 55 for their participation in the study. We are also grateful to R. Brill for providing com- ments on an earlier version of this paper. This paper is Contribution 3538 of the Virginia Institute of Marine Science, College of William and Mary. Proportion of Time Spent below the Thermocline Depth–temperature proﬁles from the PSATs indi- cated that the thermocline in waters offshore of Massachusetts between late August and early September 2012 was approximately 15 m. Thermocline depth increased to about 60 m by late October, coincident with increases in the mean depth occupied by juvenile ABT and decreases in the proportion of time spent below the thermocline. Similar to the ABT observed by Brill et al. (2002) and Galuardi and Lutcavage (2012), the ﬁsh in this study made periodic excursions to depth but spent the vast majority of their time above the thermocline. Such behavior is also similar to that of juvenile Paciﬁc Blueﬁn Tuna, which are primarily found above the thermocline but make periodic excursions to depth (Kitagawa et al. 2007a). Although juvenile ABT may exploit waters below the thermocline for feeding and behavioral thermoregulation, the amount of time juvenile FIGURE 5. Proportion of time for which juvenile Atlantic Blueﬁn Tuna (ABT; n = 16) occupied waters below the thermocline in relation to increasing lunar illumination. The ﬁsh were tagged with pop-up satellite archival tags and released offshore of Massachusetts in August and September 2012. Day is represented with a solid line, and night is represented with a dashed line. The curves presented here are ﬁtted to predictions from the model describing the proportion of time spent below the thermocline. Dotted lines represent the 95% conﬁdence interval surrounding the model-predicted mean response. 401 NOTE Most of our study ﬁsh displayed vertical movement pat- terns similar to those described in previous studies of juvenile ABT (Brill et al. 2002; Galuardi and Lutcavage 2012): they spent the majority of their time in warm, shallow waters while making periodic excursions to depths well below the thermo- cline. Additionally, most of the ﬁsh in this study displayed diel differences in habitat use, as their vertical excursions took place primarily during daylight. ABT can exploit these deep, cool waters is likely limited by their physiology. Altantic Blueﬁn Tuna are known to exhibit regional endothermy, maintaining their red muscle tissue and viscera above ambient temperature (Carey and Lawson 1973); however, the heart is not maintained above ambient temperature. Therefore, the cardiac function of ABT may limit the amount of time they can spend in cold water. Cardiac data are not available for ABT, but the heart rate of juvenile Paciﬁc Blueﬁn Tuna was shown to decrease with decreasing temperature (Clark et al. REFERENCES Downloaded by [College of William & Mary] at 09:17 08 December 2017 Downloaded by [College of William & Mary] at 09:17 08 December 2017 Block, B. A., K. E. Keen, B. Castillo, H. Dewar, E. V. Freund, D. J. Marcinek, R. W. Brill, and C. Farwell. 1997. Environmental preferences of Yellowﬁn Tuna (Thunnus albacares) at the northern extent of its range. Marine Biology 130:119–132. Horodysky, A. Z., D. W. Kerstetter, R. J. Latour, and J. E. Graves. 2007. 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https://openalex.org/W3015696915	https://www.frontiersin.org/articles/10.3389/fcell.2020.00231/pdf	English	null	Abnormal Cerebellar Development Is Involved in Dystonia-Like Behaviors and Motor Dysfunction of Autistic BTBR Mice	Frontiers in cell and developmental biology	2,020	cc-by	13,178	Abbreviations: ADHD, attention-deﬁcit/hyperactivity disorder; ASD, autism spectrum disorders; BrdU, 5-bromo-2’- deoxyuridine; CB, calbindin; DAPI, 4’,6-diamidino-2-phenylindole; DCN, deep cerebellar nuclei; DEG, diﬀerential expression gene; EGL, external granule layer; FPKM, fragments per kilobase of transcript sequence per millions base pairs sequenced; GCL, granule cell layer; GCP, granule cell precursor; GFAP, glial ﬁbrillary acidic protein; GO, Gene Ontology; HE, Hematoxylin-eosin; IGL, inner granule layer; Itpr3, inositol triphosphate receptor 3; ML, molecular layer; NeuN, neuronal nuclei; P10, Postnatal day 10; PBS, phosphate buﬀered saline; PC, Purkinje cell; PCL, Purkinje cell layer; PFA, paraformaldehyde; PPI, protein–protein interaction; RT, room temperature; SD, standard deviation; TBS, Tris buﬀered saline; TRPC, transient receptor potential canonical channel; WM, white matter; WT, wild type. Keywords: autism spectrum disorders, dystonia, movement disorder, cerebella, neurodevelopment Abnormal Cerebellar Development Is Involved in Dystonia-Like Behaviors and Motor Dysfunction of Autistic BTBR Mice Rui Xiao1, Hongyu Zhong1, Xin Li1, Yuanyuan Ma1,2, Ruiyu Zhang1, Lian Wang1, Zhenle Zang1 and Xiaotang Fan1* Rui Xiao1, Hongyu Zhong1, Xin Li1, Yuanyuan Ma1,2, Ruiyu Zhang1, Lian Wang1, Zhenle Zang1 and Xiaotang Fan1* 1 Department of Military Cognitive Psychology, School of Psychology, Army Medical University, Chongqing, China, 2 Department of Basic Nursing, School of Nursing, Army Medical University, Chongqing, China Motor control and learning impairments are common complications in individuals with autism spectrum disorder (ASD). Abnormal cerebellar development during critical phases may disrupt these motor functions and lead to autistic motor dysfunction. However, the underlying mechanisms behind these impairments are not clear. Here, we utilized BTBR T+ Itprtf/J (BTBR) mice, an animal model of autism, to investigate the involvement of abnormal cerebellar development in motor performance. We found BTBR mice exhibited severe dystonia-like behavior and motor coordination or motor learning impairments. The onset of these abnormal movements coincided with the increased proliferation of granule neurons and enhanced foliation, and Purkinje cells displayed morphological hypotrophy with increased dendritic spine formation but suppressed maturation. The migration of granule neurons seemed unaffected. Transcriptional analyses conﬁrmed the differential expression of genes involved in abnormal neurogenesis and revealed TRPC as a critical regulator in proliferation and synaptic formation. Taken together, these ﬁndings indicate that abnormal cerebellar development is closely related to dystonia-like behavior and motor dysfunction of BTBR mice and that TRPC may be a novel risk gene for ASD that may participate in the pathological process of autistic movement disorders. Edited by: Jaime J. Carvajal, Andalusian Center for Development Biology (CABD), Spain Reviewed by: Yuqing Li, University of Florida, United States Ángel M. Carrión, Universidad Pablo de Olavide, Spain Correspondence: Xiaotang Fan fanxiaotang2005@163.com Edited by: Jaime J. Carvajal, Andalusian Center for Development Biology (CABD), Spain Reviewed by: Yuqing Li, University of Florida, United States Ángel M. Carrión, Universidad Pablo de Olavide, Spain Correspondence: Xiaotang Fan fanxiaotang2005@163.com Specialty section: This article was submitted to Molecular Medicine, a section of the journal Frontiers in Cell and Developmental Biology INTRODUCTION Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by persistent defects in social communication and interaction and restricted, repetitive, inﬂexible behaviors and interests (Lord et al., 2018). It is commonly diagnosed in early childhood, and the prevalence of ASD has increased dramatically throughout the last decades Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by persistent defects in social communication and interaction and restricted, repetitive, inﬂexible behaviors and interests (Lord et al., 2018). It is commonly diagnosed in early childhood, and the prevalence of ASD has increased dramatically throughout the last decades Received: 07 January 2020 Accepted: 18 March 2020 Published: 07 April 2020 ORIGINAL RESEARCH published: 07 April 2020 doi: 10.3389/fcell.2020.00231 Citation: Several brain regions have been implicated in the pathogenesis of ASD, but cerebellar abnormalities are the most reproducibly studied in this disorder. Neuropathological studies showed lower Purkinje cell (PC) numbers, missed or ectopic neurons of deeper cerebellar nuclei (DCN), cortical thickness alterations, foliation dysplasia and migration impairments in the cerebellar cortex of individuals with ASD (Amaral et al., 2008; Wegiel et al., 2010, 2014; Blatt, 2012). Indeed, cerebellar lesions are associated with increased rates of autistic behavior, and recent evidence has suggested more to be involved, like suppressed social function, restrictive or repetitive behaviors, and motor impairment symptoms such as ataxia, dystonia and tremor (Jaber, 2017). It was recently proposed that early perinatal alterations of the cerebellum are involved in ASD pathogenesis, which is supported by the ﬁnding that autism genes are frequently involved in the aberrant cerebellar development (Menashe et al., 2013). The cerebellum is characterized by a typical laminated structure consisting of a molecular layer (ML), a Purkinje cell layer (PCL) and an inner granule layer (IGL) (Xu et al., 2013). y The BTBR T+ Itpr3tf/J (BTBR) inbred strain shows a robust behavioral phenotype that mimic core symptoms of ASD patients and exhibits striking anatomical features in the cerebellum. We investigated the contribution of abnormal cerebellar development to movement disorders in BTBR mice, with the control of C57BL/6J strain (commonly used as wild- type, WT). The present study revealed distinct dystonia-like behaviors and motor learning impairments in BTBR mice that began in early postnatal days. Concomitant with the progression of behavioral impairment, a hyperplastic cerebellum with enhanced foliation was identiﬁed due to the abnormally increased proliferation of granule cell precursors (GCPs). Moreover, in the cerebellum of BTBR mice, the morphology of Purkinje neurons was altered and exhibited hypotrophy and disturbed spine formation. Evidence from RNA sequencing indicated that the nervous system development was negatively regulated, and the transient receptor potential canonical channel (TRPC) family including TRPC6, TRPC3 and TRPC4 played a key role in the signal regulation of the abnormal neurogenesis. Together, these ﬁndings suggest that abnormal cerebellar development, which may be regulated by TRPC, was involved in the pathological progression from movement disorders to autism. In addition to the core symptoms, autistic subjects frequently present with complex motor impairments, such as ataxia, dystonia, and akinesia (Cook, 2016). Behavioral Assays The cerebellum is characterized by a typical laminated structure consisting of a molecular layer (ML), a Purkinje cell layer (PCL) and an inner granule layer (IGL) (Xu et al., 2013). During cerebellar development, billions of granule neurons are produced in the external granule layer (EGL) and then descend and migrate to destinations in the IGL, leaving “T-shaped” parallel ﬁbers that are arranged in parallel along the cerebellar folia axis and synapse on the dendritic arbors of PCs (Altman and Bayer, 1978; Hampson and Blatt, 2015). The PC axons further travel to the DCN and project more broadly. As the sole eﬀerent neurons in the cerebellum, Purkinje neurons regulate all of the information transfer and are responsible for cerebellar function (Martinez et al., 2013). Notably, the loss of cerebellar PCs is one Mice were examined periodically using the tail suspension test and grid hang test from early postnatal (P) days to 5 months. Other behavioral assessments, such as horizontal ladder rung walking, rotarod and open ﬁeld tests, were initiated at 8 weeks. Animals BTBR mice were originally obtained from Jackson Laboratory (BTBR T+ Itpr3tf/J; stock number 002282) and maintained in our mouse colony at the Army Medical University. C57BL/6J mice (WT) were used as controls and provided by the Army Medical University. Only male mice were used in the experiments. After weaning at 3 weeks, mice were group-housed with 4–6 mice per cage under a controlled environment (22 ± 2◦C, 45 ± 10% humidity, 12 h light/dark cycle) with free access to water and food. The Army Medical University approved all experiments, which were performed according to the accepted standards of animal care. Eﬀorts were made to reduce animal suﬀering. Citation: Notably, a systematic research on the prevalence of movement disorders in ASD associated with speciﬁc genetic syndromes revealed 43.6–100% for ataxia and 25.0–48.3% for tremor, with additional reports for dystonia and rigidity (Bell et al., 2019). Several clinicians have proposed that these atypical movement impairments are a predictor for ASD, because these impairments often appear prior to the classical behaviors of ASD (Robledo et al., 2012; Uljarevic et al., 2017). Motor disturbances may underlie some of the behavioral core features in autism. The contribution of movements to social cognition and cascade eﬀects on social communication in individuals with autism have been reported (Cook, 2016; Baranek et al., 2018). Notably, therapeutic medicines for motor dysfunction, such as methylphenidate and atomoxetine, have improved social interaction deﬁcits and recognition memory impairment in ASD subjects (Hara et al., 2016). It is quite essential and meaningful to illuminate the relationship between autism and motor dysfunctions to provide comprehensive and precise treatment. Several hypotheses have been raised, but more evidence is needed, and the shared underlying mechanisms with autism should be further examined. y g Several brain regions have been implicated in the pathogenesis of ASD, but cerebellar abnormalities are the most reproducibly studied in this disorder. Neuropathological studies showed lower Purkinje cell (PC) numbers, missed or ectopic neurons of deeper cerebellar nuclei (DCN), cortical thickness alterations, foliation dysplasia and migration impairments in the cerebellar cortex of individuals with ASD (Amaral et al., 2008; Wegiel et al., 2010, 2014; Blatt, 2012). Indeed, cerebellar lesions are associated with increased rates of autistic behavior, and recent evidence has suggested more to be involved, like suppressed social function, restrictive or repetitive behaviors, and motor impairment symptoms such as ataxia, dystonia and tremor (Jaber, 2017). It was recently proposed that early perinatal alterations of the cerebellum are involved in ASD pathogenesis, which is supported by the ﬁnding that autism genes are frequently involved in the aberrant cerebellar development (Menashe et al., 2013). Frontiers in Cell and Developmental Biology \| www.frontiersin.org Citation: Xiao R, Zhong H, Li X, Ma Y, Zhang R, Wang L, Zang Z and Fan X (2020) Abnormal Cerebellar Development Is Involved in Dystonia-Like Behaviors and Motor Dysfunction of Autistic BTBR Mice. Front. Cell Dev. Biol. 8:231. doi: 10.3389/fcell.2020.00231 Xiao R, Zhong H, Li X, Ma Y, Zhang R, Wang L, Zang Z and Fan X (2020) Abnormal Cerebellar Development Is Involved in Dystonia-Like Behaviors and Motor Dysfunction of Autistic BTBR Mice. Front. Cell Dev. Biol. 8:231. doi: 10.3389/fcell.2020.00231 April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 1 Autism and Movement Disorders Xiao et al. (Constantino and Charman, 2016). The interaction of genetic and environmental factors was recently hypothesized to contribute to the pathogenesis of ASD (Ecker et al., 2015; Richards et al., 2015), but the etiology of the disorder remains far from clear. of the most consistent ﬁndings in postmortem studies of autistic cerebella (Fatemi et al., 2012). Therefore, a further raised question is what extended morphological aberrations of the cerebellum concomitantly occur in speciﬁc motor impairment. but the etiology of the disorder remains far from clear. In addition to the core symptoms, autistic subjects frequently present with complex motor impairments, such as ataxia, dystonia, and akinesia (Cook, 2016). Notably, a systematic research on the prevalence of movement disorders in ASD associated with speciﬁc genetic syndromes revealed 43.6–100% for ataxia and 25.0–48.3% for tremor, with additional reports for dystonia and rigidity (Bell et al., 2019). Several clinicians have proposed that these atypical movement impairments are a predictor for ASD, because these impairments often appear prior to the classical behaviors of ASD (Robledo et al., 2012; Uljarevic et al., 2017). Motor disturbances may underlie some of the behavioral core features in autism. The contribution of movements to social cognition and cascade eﬀects on social communication in individuals with autism have been reported (Cook, 2016; Baranek et al., 2018). Notably, therapeutic medicines for motor dysfunction, such as methylphenidate and atomoxetine, have improved social interaction deﬁcits and recognition memory impairment in ASD subjects (Hara et al., 2016). It is quite essential and meaningful to illuminate the relationship between autism and motor dysfunctions to provide comprehensive and precise treatment. Several hypotheses have been raised, but more evidence is needed, and the shared underlying mechanisms with autism should be further examined. Tail Suspension Test Mice were suspended by their tails for 60 s. The activity of the mice was recorded by a camera to observe the presence of dystonia-like behaviors, as references described (Liu et al., 2015; Pappas et al., 2015), including hyperﬂexion, trunk twisting, hyperextension, and forelimb or hindlimbs clasping. These phenotypes were recorded from P3 to P150. April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 2 Autism and Movement Disorders Xiao et al. trichloromethane for 20 min and covered twice with melted paraﬃn for 30 min. Cerebella were paraﬃn-embedded, and then sagittal sections (5 µm) were collected. All sections used in each mouse were taken from the same medial lateral position on the cerebellum to allow comparisons and ﬁve sections per mouse were used for each staining. Paraﬃn sections were performed dewaxing and antigen retrieval before used. For hematoxylin-eosin (HE) staining, sections were incubated in hematoxylin (ZLI-9610, ZSJQ-Bio, China) for 10 min, washed in running water, diﬀerentiated by 75% hydrochloric acid in alcohol for 1 min, washed in running water when the nucleus was black to blue, incubated in eosin (ZLI-9612, ZSJQ-Bio, China) for 10 s, dehydrated in 95% alcohol for 1 min, and mounted in DPX (06522, Sigma, United States). For immunohistochemical staining, sections were processed by washing in 0.3% Triton- X/PBS, blocking in 3% bovine serum albumin (BSA) (37◦C, 2 h), and incubation with the following primary antibodies (room temperature (RT), overnight): (1) mouse anti-5-bromo-2′- deoxyuridine (BrdU) (1:500, BD PharmingenTM, United States); (2) rabbit anti-Ki67 (1:1000, Thermo, United States); (3) rabbit anti-neuronal nuclei (NeuN) (1:200, Abcam, United States); rabbit anti-s100β (1:500, CST, United States); rabbit anti-glial ﬁbrillary acidic protein (GFAP) (1:500, Dako, Japan); and mouse anti-calbindin (CB) (1:1000, Swant, Switzerland). After washing in 0.01 M PBS for 30 min, sections were incubated with the corresponding secondary antibodies (RT, 3 h in darkness): Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:500, Jackson ImmunoResearch, United States); and cy3-conjugated donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch, United States). 4′,6-Diamidino-2-phenylindole (DAPI) (1:10000, Sigma-Aldrich, United States) was used to counterstain the nuclei in all sections. After air drying, sections were mounted with Vectashield (Vector Lab, United States). Sections for Nissl staining were incubated in a cresyl violet solution containing acetic acid (C0117, ZSJQ-Bio, China) (37◦C, 30 min), dehydrated in 95% alcohol for 1 min, and mounted in DPX (06522, Sigma, United States). Tail Suspension Test The stained sections were observed under 5X or 20X objective lens using a Zeiss Axiovert microscope (Oberkochen, Germany) with a Zeiss Axiovision 4.0 system. Accelerating Rotarod Test Motor coordination and motor learning were detected using the accelerating rotarod test. Mice were habituated to stay on the stationary drum for 3 min 1 day in advance and habituated was repeated every day for 1 min just before the session. Once stabilized, mice were put on an accelerating rod (3 cm diameter, 14 cm above the pedestal), and the speed was set to 5 rpm with a uniform acceleration to 40 rpm in 5 min. The latency of mouse falling from the rotating rod was calculated. Five trials were performed on each mouse per day for 5 consecutive days to assess motor learning in each group. Horizontal Ladder Rung Walking Test Horizontal Ladder Rung Walking Test Skilled fore- and hindlimb coordination and ﬁne motor function were assessed by the horizontal ladder rung walking test (Metz and Whishaw, 2002). To subtly test coordination, the diﬃculty of the task was divided into two patterns: pattern A used a regular rung arrangement with 2 cm intervals, and pattern B used an irregular rung pattern with randomly spaced rungs at intervals from 1 to 3 cm. Mice were put on one side of the ladder (30 cm above the ground) and allowed to freely cross it. The whole crossing process was ﬁlmed with a high-deﬁnition video camcorder (Logitech C930e), and behaviors were analyzed. The total limb falls and time to cross the ladder were recorded to assess motor function. Five trials were performed on each mouse, and the average value was calculated. Histology and Immunohistochemistry Histology and Immunohistochemistry Adult mice were completely anesthetized with sodium pentobarbital and transcardially perfused with ice-cold 0.01 M phosphate-buﬀered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Neonatal mice received BrdU injection (50 mg/kg i.p.) and then were decapitated 2 h later, and the needed tissues were dissected. Dissected cerebella were postﬁxed in 4% PFA for 48 h and dehydrated in a 50– 100% gradient alcohol series. Tissues were soaked twice in Open Field Locomotion Test Mice were put in an open ﬁeld to assess locomotor activity. The apparatus was a square plexiglass cage (40 × 40 × 30 cm) illuminated at ∼200 lux. As previously described (Cai et al., 2017), square grid lines were predeﬁned by a computer that divided the open ﬁeld chamber into a central zone and periphery. Mice were placed in the center of the apparatus, and locomotion was traced for 30 min using Ethovision 11.0 software (Noldus). The total distance in all zones and the center zone were calculated to assess the activity of mice. The apparatus was cleaned with 70% ethanol between trials. Golgi Staining Mice were decapitated immediately at P14, and brains were dissected. Cerebella were rinsed and processed for Golgi staining with the FD Rapid Golgi Stain Kit (PK401A, FD Neurotechnologies, United States) according to the manufacturer’s protocol. Sagittal sections (80 µm) were generated and mounted in DPX after drying. Slides were observed under 20X or 100X oil objective lens using a Zeiss Axiovert microscope. Sholl analysis was executed using the matched Zeiss Axiovision 4.0 system. Grid Hang Test Motor coordination and strength were detected by putting the mice on a 30 × 50 cm wire grid with 0.5 cm2 openings. The grid was inverted after the mouse grabbed the grid with its fore- and hindlimbs, and the latency to fall oﬀwas recorded. Movement on the grid and the paw placement of the mice were observed during the test. Statistical Analysis All data collection and analyses were performed randomly by experimenters blinded to the genotypes. The sample sizes are similar to those of previous publications (Huang et al., 2016, 2019; Xie et al., 2018) and listed in the ﬁgure legends. Data are represented as the means ± standard deviation (SD), and statistical analysis included the Chi squared test, non-parametric Mann–Whitney U test (for data that failed normality test), unpaired t-test, two-way ANOVA, and two-way repeated-measures ANOVA with post hoc Bonferroni multiple comparisons test were performed using SPSS 19.0 software (SPSS Inc., United States). Detailed statistical approaches and results are listed in the ﬁgure legends and Supplementary Data Sheet 1. A P-value < 0.05 was deﬁned as statistically signiﬁcant. In the graphed data ∗, ∗∗, and ∗∗∗ denote P-values less than 0.05, 0.01, and 0.001, respectively. Western Blot P14 BTBR and WT mice were decapitated, and their cerebella were dissected in ice-cold PBS. The total protein was extracted immediately, and protein concentrations were measured using a Bicinchoninic Acid Kit (Beyotime, China) as previously described (Xiao et al., 2017). The total protein (20 µg) of April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 3 Autism and Movement Disorders Xiao et al. each sample was separated by 10% SDS-polyacrylamide electrophoresis (80 V, 100 min) and then transferred to a polyvinylidene ﬂuoride (PVDF) membrane (220 mA, 60 min). The membranes were washed in 1% Tween-20/Tris-buﬀered saline (TBS) (TBS-T), blocked in 5% BSA/TBS-T (RT, 2 h), and incubated with a primary antibodies (4◦C, 12 h) (1) mouse anti-CB (1:2000, Swant, Switzerland) and (2) mouse anti-GAPDH (1:2000, Cell Cwbio, China), followed by peroxidase-conjugated goat anti-mouse secondary antibody IgG (1:1000, Santa Cruz Biotechnology, United States). Bands were visualized using the chemiluminescence detection kit (Pierce, United States) under a Gel-Pro analyzer (Bio-Rad Laboratories, United States). Band intensity was quantiﬁed in Image Lab (Bio-Rad Laboratories, United States), and calbindin protein was normalized to GAPDH. platform with a 125 bp/150 bp paired-end reads strategy. The original image data were subjected to quality control, and reads containing adapter, poly-N and low-quality reads were removed from the raw data. Clean data of high quality were used for the downstream analyses. The reads were aligned to the reference genome (10 mm) using the split read aligner TopHat v2.0.12 and Bowtie v2.2.3, and HTSeq v0.6.1 was used to estimate the abundances of mapped genes. Expected number of Fragments Per Kilobase of transcript sequence per Million base pairs sequenced (FPKM) of each gene was calculated based on the length of the gene and reads count and used as the evaluation index of gene expression levels. For diﬀerential expression gene (DEG) analysis, the DESeq R package (1.18.0) was used to perform routine statistics with a model based on negative binomial distribution. P-values < 0.05 were deemed signiﬁcant. Next, Gene Ontology (GO) enrichment pathway analysis of DEGs was conducted using the GOseq R package, which adjusts the gene length bias based on Wallenius hyper-distribution. GO pathways with P-values < 0.05 deﬁned a signiﬁcant enrichment of the DEGs. Protein-protein interaction (PPI) networks of the DEGs screened out by GO enrichment were performed in the STRING database1. Further analysis was performed in Cytoscape_v3.7.2 software. Quantiﬁcation Comparable middle sagittal sections were selected for assessments. Cerebella sagittal area (mm2) and perimeter (mm) were deﬁned as shown in Figure 2E and calculated with a Zeiss Axiovision 4.0 system. Lobes separated clearly by ﬁssures were calculated as the lobe number. BrdU- and Ki67-positive granule cell precursors were counted only in the EGL and the density was calculated as total BrdU or Ki67 cells in the EGL/EGL area, and proportion of BrdU-positive cell in the EGL was calculated as BrdU-positive cells/DAPI- stained nuclei in a high-power ﬁeld of the EGL (218 µm length) in lobe IV/V. Bergmann soma in the PCL and ﬁbers in the ML were counted under a 20X objective lens along the lobe axis (450 µm length), and the densities were calculated. Purkinje neuron density and soma size were counted in each lobe along a 500 µm-length axis in the middle of each lobe. Among these cells, 10 Purkinje neurons were randomly selected, and their soma areas were measured by a Zeiss Axiovision 4.0 system. 1https://string-db.org/ Real-Time Quantitative PCR Cerebella were collected at P14, and total RNA was extracted using Trizol (Invitrogen, United States) and reverse transcribed to cDNA, according to the manufacturer’s protocol. Real-time PCR for target genes was performed with a SYBR Green kit (Takara Company, Japan) and the CFX ConnectTM Real-time system (Bio-Rad, United States). Primers are listed in the Supplementary Table. Expression of mRNA was detected via the 11 cycle threshold-based algorithm relative to an internal control gene (GAPDH). Each sample was run in triplicate, and 6 mice from each group were used. For Golgi staining quantiﬁcation, the outer terminals of the Purkinje dendritic branches were orderly linked, and the formative closed region was deﬁned as the PC dendritic area. Primary dendrite length was measured as the primary dendrite of each Purkinje neuron from the soma up to the end at the surface of the ML. Sholl analysis was performed, as described previously (Piochon et al., 2014). Purkinje neuron branches were incised by concentric circles with 5.5 µm radius steps from the soma, and intersections in each circle were counted. Dendrite spine density and classiﬁcation were assessed referring to Risher (Risher et al., 2014). At least a 10 µm-length branch was calculated for the spine. Ten cells per mouse and 10 branches per cell were detected. BTBR Mice Exhibited Infancy-Onset Dystonia-Like Behaviors and Motor Impairments Movement disorders are widely reported in combination with autism in individuals. We found BTBR mice exhibited severe dystonia-like movements during tail suspension, when mice try to keep an upright body posture. The most prominent dystonic symptom was hyperﬂexion of one or both hyperkinetic hindlimbs during tail suspension. Hindlimb clasping or fore- and hindlimb clasping, hyperextension, and severe trunk twisting were also observed in conjunction with myodystonia (Supplementary Figure S1). BTBR mice exhibited dystonia-like behaviors as early as the tenth day (P10) (X2 = 3.902, P < 0.05), and nearly 100% of BTBR mice developed such behaviors beginning at P14 (X2 = 21.505, P < 0.001). This behavior persisted to adulthood and remained stable (Figures 1A,B). Physiological hypermyotonia was observed in WT mice between P7 and P30, but it disappeared after P30. In addition, BTBR mice also developed a defect in the ability to hang from a wire grid [Figure 1C, F(1,258) = 343.614, P < 0.001] that in some cases because of abnormal hindlimb clasping or twisting. Fine motor skill was assessed using regularly and irregularly spaced horizontal ladders at 8 weeks of age. Mice ran across the horizontal ladder in two patterns, as shown in Figure 1D. BTBR mice exhibited increased limb falls in both the regular (U = 26.000, P < 0.05) and irregular (U = 8.500, P < 0.01) patterns, which suggests a deﬁcit in ﬁne motor skill (Figure 1E). Interestingly, the time spent crossing the ladder was decreased in BTBR mice compared to WT controls (Pattern A: U = 9.000, P < 0.01; Pattern B: T20 = 6.686, P < 0.001) (Figure 1F), mainly because of the increased activity. BTBR mice were also signiﬁcantly hyperactive in the open ﬁeld, as indicated by the increased distance in all zones (T16 = 2.719, P < 0.05) and the central zone (T16 = 2.504, P < 0.05) (Figures 1I,J). Motor skill learning was assessed by means of the consecutive rotarod learning test (Figure 1H). Both BTBR and WT mice learned the task, and the time on the rod gradually increased [WT: F(4,44) = 21.867, P < 0.001; BTBR: F(4,28) = 15.306, P < 0.001]. However, learning was signiﬁcantly slower in the BTBR mice [F(1,72) = 28.232, P < 0.001] (Figure 1H). Notably, the BTBR mice showed abnormal behaviors, similar as inattention, when they were put on the rod, as shown in Figure 1G. BTBR Mice Exhibited Infancy-Onset Dystonia-Like Behaviors and Motor Impairments Instead of concentrating on the motor learning, the BTBR mice explored and ignored the unstable rotating rod under their feet. It may be an important factor for the impaired learning process. In summary, BTBR mice exhibited infancy-onset dystonia-like behaviors accompanied by severe deﬁcits in motor coordination and motor learning in addition to autistic behavior. BTBR Mice Displayed Hyperplastic Cerebella With Increased Foliation BTBR mice ﬁrstly exhibited increased foliation at P7, with multiple lobules that were not present in controls (T10 = 20.125, P < 0.001) (Figures 2F,G). Additionally, the midsagittal area (T10 = 11.234, P < 0.001) and perimeter (T10 = 20.698, P < 0.001) were increased more noticeably. At P14, when foliation patterns are established, BTBR midsagittal sections were larger (T9 = 6.739, P < 0.001) (Figure 2H), had a longer perimeter (T9 = 10.639, P < 0.001) (Figure 2I), and were considerably more foliated than controls (T9 = 22.160, P < 0.001) (Figures 2F,G). RESULTS BTBR Mice Exhibited Infancy-Onset Dystonia-Like Behaviors and Motor Impairments (Figures 2A,D). By dividing the area into three lamellas, the ML, GCL and white matter (WM), we found that the enlarged portion was mainly in the ML (T11 = 4.383, P < 0.01) and GCL (T11 = 8.880, P < 0.001) (Figure 2B). The thickness of the ML was not altered in BTBR mice (data not shown), and its increased area might have resulted from an elongated perimeter. Therefore, the enlargement of the cerebella may be due to the extension of the GCL. Another noticeable change was observed that more foliation appeared in BTBR cerebella than the WT controls (Figure 2C). To determine when BTBR mice ﬁrstly exhibited enhanced foliation, paraﬃn sections of the cerebella with HE staining were detected sequentially during the ﬁrst two postnatal weeks (Figure 2F). The initial stages of cerebellar patterning, including cardinal ﬁssure formation, were normal in BTBR mice until P3, but the average sagittal cerebellar area increased signiﬁcantly (T9 = 2.447, P < 0.05) compared to WT controls. The average sagittal cerebellar section perimeter was elongated concomitantly (T9 = 4.378, P < 0.01), which indicates that the cerebellar surface area was increased. Thus, cortical expansion and increased cross-sectional area preceded supernumerary folia in BTBR mice. (Figures 2A,D). By dividing the area into three lamellas, the ML, GCL and white matter (WM), we found that the enlarged portion was mainly in the ML (T11 = 4.383, P < 0.01) and GCL (T11 = 8.880, P < 0.001) (Figure 2B). The thickness of the ML was not altered in BTBR mice (data not shown), and its increased area might have resulted from an elongated perimeter. Therefore, the enlargement of the cerebella may be due to the extension of the GCL. Another noticeable change was observed that more foliation appeared in BTBR cerebella than the WT controls (Figure 2C). To determine when BTBR mice ﬁrstly exhibited enhanced foliation, paraﬃn sections of the cerebella with HE staining were detected sequentially during the ﬁrst two postnatal weeks (Figure 2F). The initial stages of cerebellar patterning, including cardinal ﬁssure formation, were normal in BTBR mice until P3, but the average sagittal cerebellar area increased signiﬁcantly (T9 = 2.447, P < 0.05) compared to WT controls. The average sagittal cerebellar section perimeter was elongated concomitantly (T9 = 4.378, P < 0.01), which indicates that the cerebellar surface area was increased. RESULTS BTBR Mice Exhibited Infancy-Onset Dystonia-Like Behaviors and Motor Impairments Thus, cortical expansion and increased cross-sectional area preceded supernumerary folia in BTBR mice. BTBR Cerebella Displayed Increased GCP Proliferation in the EGL Without Alteration in the Migration of Granule Neurons The foliation pattern divided by ﬁssures of diﬀerent lengths is a representative morphology of cerebella. The formation is orchestrated by multicellular anchoring centers in which granule cells are the initiating factors and provide the driving physical force (Sudarov and Joyner, 2007). During cerebella development, GCPs in the EGL proliferate and diﬀerentiate into granule cells, then gradually mature during migration through the ML to destinations in the IGL. BrdU was used to label the newborn GCPs in the EGL of P3 cerebella (Figures 3A,A1,B,B1). Co-staining of nuclei with DAPI revealed that the EGL was much thicker in BTBR cerebella compared to WT (T9 = 3.218, P < 0.05) (Figures 3A2,B2,C). Simultaneously, the density of BrdU-positive GCPs in the EGL was increased signiﬁcantly (T9 = 3.869, P < 0.01) (Figures 3A3,B3,D). The total GCPs (T9 = 3.944, P < 0.05) (Figure 3E) and proportion (T9 = 4.899, P < 0.01) (Figure 3F) were also increased in BTBR mice. To conﬁrm this result, another marker, Ki67, which is actively expressed during mitosis and degrades soon after caryomitosis, was used. Consistently, the Ki67-positive GCPs in the EGL were multiplied in BTBR RNA-seq Analyses The whole cerebella of BTBR and WT mice were collected at P14 for the mRNA sequencing assay. This experiment was performed by Novogene (Beijing, China). Libraries were generated using the NEBNext R⃝UltraTM RNA Library Prep Kit for Illumina R⃝ (NEB, United States) according to standard Illumina protocols. After clustering was performed in the cBot Cluster Generation System, the libraries were sequenced on an Illumina Hiseq 1https://string-db.org/ April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 4 Autism and Movement Disorders Xiao et al. BTBR Mice Displayed Hyperplastic Cerebella With Increased Foliation In the adult BTBR mice, the overall structure of the cerebellum was abnormal, with an obviously larger area (T11 = 7.727, P < 0.001) and more lobules (T11 = 6.826, P < 0.001) April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 5 Autism and Movement Disorders Xiao et al. Xiao et al. FIGURE 1 \| BTBR mice exhibited infancy-onset dystonia-like behavior and motor impairments. (A) Representative image of BTBR mice and WT control at each growing point in tail-suspension test. (B) Quantiﬁcation of the morbidity of dystonia at each growing point showing BTBR mice developed typical dystonic behavior and aggravated with growth (Chi square test; n = 32, 26 mice). (C) Latency to fall from the wire grid at each growing point of mice, and BTBR mice developed a weaken ability to hang since adolescence (Two-way ANOVA; n = 15-29 mice). (D) The horizontal ladder rung walking apparatus with regular arrangement (pattern A) and irregular arrangement (pattern B). (E) Average total number of limbs fall of adult mice (8 weeks) in the horizontal ladder rung walking task (non-parametric Mann–Whitney U test; n = 10, 12 mice). (F) Quantiﬁcation of the time to across the horizontal ladder (non-parametric Mann–Whitney U test, Student’s t-test; n = 10, 12 mice). (G) Representative images in the rotarod test showing inattention of BTBR mice. (H) Latency to fall from the accelerated rod of adult mice (8 weeks) showing motor and motor learning defect in BTBR mice (Two-way repeated measure test; n = 12, 8 mice). (I) Representative trace diagrams in open ﬁeld test showing hyperactivity in BTBR mice. (J) Quantiﬁcation of the distance in total and central area of adult mice (8 weeks) in the open ﬁeld. (Student’s t-test; n = 9, 9 mice). All data are displayed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. FIGURE 1 \| BTBR mice exhibited infancy-onset dystonia-like behavior and motor impairments. (A) Representative image of BTBR mice and WT control at each growing point in tail-suspension test. (B) Quantiﬁcation of the morbidity of dystonia at each growing point showing BTBR mice developed typical dystonic behavior and aggravated with growth (Chi square test; n = 32, 26 mice). BTBR Mice Displayed Hyperplastic Cerebella With Increased Foliation (C) Latency to fall from the wire grid at each growing point of mice, and BTBR mice developed a weaken ability to hang since adolescence (Two-way ANOVA; n = 15-29 mice). (D) The horizontal ladder rung walking apparatus with regular arrangement (pattern A) and irregular arrangement (pattern B). (E) Average total number of limbs fall of adult mice (8 weeks) in the horizontal ladder rung walking task (non-parametric Mann–Whitney U test; n = 10, 12 mice). (F) Quantiﬁcation of the time to across the horizontal ladder (non-parametric Mann–Whitney U test, Student’s t-test; n = 10, 12 mice). (G) Representative images in the rotarod test showing inattention of BTBR mice. (H) Latency to fall from the accelerated rod of adult mice (8 weeks) showing motor and motor learning defect in BTBR mice (Two-way repeated measure test; n = 12, 8 mice). (I) Representative trace diagrams in open ﬁeld test showing hyperactivity in BTBR mice. (J) Quantiﬁcation of the distance in total and central area of adult mice (8 weeks) in the open ﬁeld. (Student’s t-test; n = 9, 9 mice). All data are displayed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. mice compared to WT mice (T9 = 7.827, P < 0.001) (Figures 3G–I) at P3. Ki67 was further detected at P7, and it was still much more in BTBR mice than WT mice (T10 = 2.334, P < 0.05) (Figures 3J–L). These results indicate the increased proliferation of GCPs in the cerebella of BTBR mice postnatally was up to P7. glia play a vital role in granule neuron migration. The soma and ﬁbers of Bergmann glia were clearly stained with S100β and GFAP in each group (Figures 4D–H). No aberrations were found in Bergmann glia between groups, neither soma (T10 = 0.303, P = 0.768) (Figure 4I) nor ﬁbers (T10 = 0.770, P = 0.459) (Figure 4J). Furthermore, Nissl staining of 3-month old cerebella revealed conspicuous gross morphological changes in BTBR mice, but the neuron density was similar in the ML between groups (data not shown). The boundary of the IGL was well- deﬁned with no stranded cells (Figures 4K–N), which indicates that the migration of granule neurons was accomplished, in terms of results. Defect in migration of GCs might lead to increased EGL thickness in BTBR mice. BTBR Mice Displayed Hyperplastic Cerebella With Increased Foliation We observed that most of the diﬀerentiated granule neurons labeled with NeuN staining were located in the IGL in WT mice at P7, with few ectopic neurons in the EGL or ML (Figure 4A). Similarly, no ectopic mature granule neuron was found in BTBR mice (Figure 4B). Notably, the EGL thickness was comparable between the two groups (T10 = 0.856, P = 0.412) (Figure 4C), which indicates that the overproduced granule neurons in BTBR mice migrated eﬃciently. It was further conﬁrmed that migrating granule neurons identiﬁed by slim nuclei in the ML (Yang et al., 2013) were increased in BTBR mice (T10 = 5.716, P < 0.01) (Supplementary Figure S2E), but the proportion or migrating rate was comparable between groups (T10 = 0.679, P = 0.073) (Supplementary Figure S2F). Bergmann Purkinje Neurons in BTBR Cerebella Displayed Morphological Hypotrophy With Abnormal Dendritic Spine Formation Purkinje neurons are the sole eﬀerent neurons in the cerebella and play a key role in motor function. We further investigated April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 6 Autism and Movement Disorders Xiao et al. Xiao et al. FIGURE 2 \| Cerebellar cortex in BTBR mice was expanded with increased foliation since post-natal. (A) DAPI stained cerebella of WT and BTBR mice at adult stage (P90). (B) Quantiﬁcation of the sagittal area of cerebella and each component at adulthood (Student’s t-test; n = 6, 7 mice). (C) Quantiﬁcation of the average lobe number showing increased foliation of adult BTBR mice (Student’s t-test; n = 6, 7 mice). (D) Whole mount images and sagittal section of brain in WT and BTBR mice at adult stage (P90). White and black dotted line delimit the lobule outline. Black arrowheads indicate the lobule ﬁssures. (E) Schema graph illustrating the determination of perimeter and area. (F) Hematoxylin-eosin (HE) staining of middle sagittal cerebellar section at postnatal day 3, 7, and 14 (additional lobes highlighted in red in the counterdraw). (G) Quantiﬁcation of the average lobule number in WT and BTBR mice at indicated stage (Student’s t-test; P3 n = 6,5; P7 n = 6,6; P14 n = 5,6). (H) Quantiﬁcation of the average sagittal cerebellar area in WT and BTBR mice at indicated stage (Student’s t-test; P3 n = 6, 5; P7 n = 6, 6; P14 n = 5, 6). (I) Quantiﬁcation of the average sagittal cerebellar section perimeter in WT and BTBR mice at indicated stage (Student’s t-test; P3 n = 6,5; P7 n = 6,6; P14 n = 5,6). All data are displayed as mean ± SD. P < 0.05, P < 0.01, *P < 0.001. Scale bar: (A,F) 200 µm; (D) 1 mm. FIGURE 2 \| Cerebellar cortex in BTBR mice was expanded with increased foliation since post-natal. (A) DAPI stained cerebella of WT and BTBR mice at adult stage (P90). (B) Quantiﬁcation of the sagittal area of cerebella and each component at adulthood (Student’s t-test; n = 6, 7 mice). (C) Quantiﬁcation of the average lobe number showing increased foliation of adult BTBR mice (Student’s t-test; n = 6, 7 mice). (D) Whole mount images and sagittal section of brain in WT and BTBR mice at adult stage (P90). White and black dotted line delimit the lobule outline. Purkinje Neurons in BTBR Cerebella Displayed Morphological Hypotrophy With Abnormal Dendritic Spine Formation Black arrowheads indicate the lobule ﬁssures. (E) Schema graph illustrating the determination of perimeter and area. (F) Hematoxylin-eosin (HE) staining of middle sagittal cerebellar section at postnatal day 3, 7, and 14 (additional lobes highlighted in red in the counterdraw). (G) Quantiﬁcation of the average lobule number in WT and BTBR mice at indicated stage (Student’s t-test; P3 n = 6,5; P7 n = 6,6; P14 n = 5,6). (H) Quantiﬁcation of the average sagittal cerebellar area in WT and BTBR mice at indicated stage (Student’s t-test; P3 n = 6, 5; P7 n = 6, 6; P14 n = 5, 6). (I) Quantiﬁcation of the average sagittal cerebellar section perimeter in WT and BTBR mice at indicated stage (Student’s t-test; P3 n = 6,5; P7 n = 6,6; P14 n = 5,6). All data are displayed as mean ± SD. P < 0.05, P < 0.01, P < 0.001. Scale bar: (A,F) 200 µm; (D) 1 mm. ML. The Purkinje neuron density in each lobe was comparable between groups (Figure 5E). However, Purkinje neurons in BTBR cerebella exhibited signiﬁcant soma hypotrophy compared to WT, especially in the posterolateral lobes, from lobe IV to X (lobe IV/V: T10 = 3.217, P < 0.01; love VI/VII: T10 = 5.470, P < 0.001; lobe VIII: T10 = 4.142, P < 0.01; lobe IX: T10 = 2.972, whether the Purkinje neurons in the cerebellum of BTBR mice were aﬀected during the critical time when the dystonia- like behavior reached the fastigium at P14. The Purkinje neurons were labeled with the speciﬁc marker of calbindin (CB) (Figures 5A–D,A’–D’). At P14, Purkinje neurons were arranged in a monolayer, and the bushy dendrites grew into the April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 7 Autism and Movement Disorders Xiao et al. FIGURE 3 \| Granule cell precursor proliferation was increased in BTBR mice postnatally. (A,B) BrdU staining (green) of sagittal section of cerebellar vermis at P3. Dotted line in (A1–A3,B1–B3) delimit external granular layer (EGL) where proliferative granule cells originate. Nucleus was counterstained with DAPI (blue). (C) Quantiﬁcation of the EGL thickness showing thicker EGL in BTBR mice at P3 (Student’s t-test; n = 6,5). (D) Quantiﬁcation of BrdU positive cells per mm2 in EGL of each group at P3 (Student’s t-test; n = 6,5). Purkinje Neurons in BTBR Cerebella Displayed Morphological Hypotrophy With Abnormal Dendritic Spine Formation (E) Quantiﬁcation of total BrdU positive cells in EGL of sagittal section at P3 (Student’s t-test; n = 6,5). (F) Quantiﬁcation of the percentage of BrdU positive cells in EGL at P3 (Student’s t-test; n = 6,5). (G,H) Ki67 staining (red) of sagittal section of cerebellar vermis at P3 Nucleus was counterstained with DAPI (blue). White panels in (G,H) are magniﬁed in (G1,H1). (I) Quantiﬁcation of Ki67 positive cells per mm2 in EGL of sagittal section at P3 (Student’s t-test; n = 6,5). (J,K) Ki67 staining (red) of sagittal section of cerebellar vermis at P7. Nucleus was counterstained with DAPI (blue). White panels in (J,K) are magniﬁed in (J1,K1). (L) Quantiﬁcation of Ki67 positive cells per mm2 in EGL of sagittal section at P3 (Student’s t-test; n = 6,6). All data are displayed as mean ± SD. P < 0.05, P < 0.01, P < 0.001. Scale bar: (A,B,G,H,J,K) 200 µm; (A1–A3,B1–B3) 10 µm; (G1,H1,J1,K1) 20 µm. FIGURE 3 \| Granule cell precursor proliferation was increased in BTBR mice postnatally. (A,B) BrdU staining (green) of sagittal section of cerebellar vermis at P3. Dotted line in (A1–A3,B1–B3) delimit external granular layer (EGL) where proliferative granule cells originate. Nucleus was counterstained with DAPI (blue). (C) Quantiﬁcation of the EGL thickness showing thicker EGL in BTBR mice at P3 (Student’s t-test; n = 6,5). (D) Quantiﬁcation of BrdU positive cells per mm2 in EGL of each group at P3 (Student’s t-test; n = 6,5). (E) Quantiﬁcation of total BrdU positive cells in EGL of sagittal section at P3 (Student’s t-test; n = 6,5). (F) Quantiﬁcation of the percentage of BrdU positive cells in EGL at P3 (Student’s t-test; n = 6,5). (G,H) Ki67 staining (red) of sagittal section of cerebellar vermis at P3 Nucleus was counterstained with DAPI (blue). White panels in (G,H) are magniﬁed in (G1,H1). (I) Quantiﬁcation of Ki67 positive cells per mm2 in EGL of sagittal section at P3 (Student’s t-test; n = 6,5). (J,K) Ki67 staining (red) of sagittal section of cerebellar vermis at P7. Nucleus was counterstained with DAPI (blue). White panels in (J,K) are magniﬁed in (J1,K1). (L) Quantiﬁcation of Ki67 positive cells per mm2 in EGL of sagittal section at P3 (Student’s t-test; n = 6,6). All data are displayed as mean ± SD. P < 0.05, P < 0.01, P < 0.001. Scale bar: (A,B,G,H,J,K) 200 µm; (A1–A3,B1–B3) 10 µm; (G1,H1,J1,K1) 20 µm. Purkinje Neurons in BTBR Cerebella Displayed Morphological Hypotrophy With Abnormal Dendritic Spine Formation P < 0.05; lobe X: T10 = 2.649, P < 0.05) (Figures 5A1– D1,A1’–D1’,F). Western blotting was used to conﬁrm this result (Figure 5G), and calbindin protein expression was decreased in BTBR cerebella (T10 = 2.416, P < 0.05) (Figure 5H), in accordance with the decrease in soma size. We also detected the PCs in adulthood and found a sparse cell distribution with signiﬁcant cell loss in the posterior lobes in BTBR mice, including lobes VIII (T6 = 3.019, P < 0.05), IX (T6 = 4.971, P < 0.001) and X (T6 = 2.662, P < 0.05) (Supplementary Figure S3). These results indicate that the hypotrophy of Purkinje neurons in postnatal cerebellum of BTBR mice may be a signal of cell death in adulthood. Golgi staining was used to examine the dendrites of PCs at P14 (Figures 6A,B,A’,B’). Representative images showed no diﬀerences in gross morphology of Purkinje neurons between groups. There were no abnormalities in extended areas of dendrites (T6 = 2.315, P = 0.060) (Figure 6C), primary dendrite length (T6 = 0.951, P = 0.378) (Figure 6D), or the complexity April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 8 Autism and Movement Disorders Xiao et al. FIGURE 4 \| Radial migration of granule neurons in cerebella was not altered in BTBR mice. (A,B) NeuN stained (red) granule cells showing mature neurons were all distributed in inner granular layer (IGL) both in WT and BTBR mice at P7 Nucleus was counterstained with DAPI (blue). (C) Quantiﬁcation of EGL thickness in each group at P7. (Student’s t-test; n = 6,6). (D) Schema graph of sagittal cerebella illustrating the observed region (black panel) in ﬁgure (E–H). (E–F) S100β positive Bergman glia somas in Purkinje cell layer (PCL) at P7. (G,H) GFAP positive Bergmann glia ﬁbers in molecular layer (ML) at P7. (I) Quantiﬁcation of Bergman glia somas in PCL per mm (Student’s t-test; n = 6,6). (J) Quantiﬁcation of Bergman glia ﬁbers in ML per mm. (Student’s t-test; n = 6,6). (K–N) Nissl staining of middle sagittal cerebellar section in adult (P90) WT and BTBR mice. (L,N) Are Magniﬁed images of black panels in (K,M). Dotted line in (L,N) indicate the boundary between ML and PCL. All data are displayed as mean ± SD. Scale bar: (A,B,K,M) 200 µm; (E–H,L,N) 10 µm. Purkinje Neurons in BTBR Cerebella Displayed Morphological Hypotrophy With Abnormal Dendritic Spine Formation FIGURE 4 \| Radial migration of granule neurons in cerebella was not altered in BTBR mice. (A,B) NeuN stained (red) granule cells showing mature neurons were all distributed in inner granular layer (IGL) both in WT and BTBR mice at P7 Nucleus was counterstained with DAPI (blue). (C) Quantiﬁcation of EGL thickness in each group at P7. (Student’s t-test; n = 6,6). (D) Schema graph of sagittal cerebella illustrating the observed region (black panel) in ﬁgure (E–H). (E–F) S100β positive Bergman glia somas in Purkinje cell layer (PCL) at P7. (G,H) GFAP positive Bergmann glia ﬁbers in molecular layer (ML) at P7. (I) Quantiﬁcation of Bergman glia somas in PCL per mm (Student’s t-test; n = 6,6). (J) Quantiﬁcation of Bergman glia ﬁbers in ML per mm. (Student’s t-test; n = 6,6). (K–N) Nissl staining of middle sagittal cerebellar section in adult (P90) WT and BTBR mice. (L,N) Are Magniﬁed images of black panels in (K,M). Dotted line in (L,N) indicate the boundary between ML and PCL. All data are displayed as mean ± SD. Scale bar: (A,B,K,M) 200 µm; (E–H,L,N) 10 µm. of the dendrite arborization as assessed by Sholl analysis [F(1,204) = 0.105, P = 0.757] (Figures 6E,F) of BTBR mice. However, spine density of PCs in BTBR cerebella was signiﬁcantly increased (T6 = 5.793, P < 0.01) compared to WT controls, with a close array in dendritic branches (Figures 6A’1-B’2,H). The Dendritic spines exhibit a transformed morphology during their development and maturation, which reﬂects diﬀerent synaptic function at diﬀerent stages (Nimchinsky et al., 2002). April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 9 Xiao et al. Autism and Movement Disorders URE 5 \| Purkinje neurons’ density did not variate but was signiﬁcantly hypotrophy in BTBR mice at postnatal day 14. (A–D) Calbindin staining Purkinje neurons in ebellar lobe I/II, IV/V, VIII, X of WT (A–D) and BTBR (A’–D’) mice at P14. Purkinje neuron somas in white panels are magniﬁed in (A1-D1,A’1–D’1). Quantiﬁcation of Purkinje neurons number per 500µm in lobe I-X (Student’s t-test; n = 6,6). (F) Quantiﬁcation of Purkinje neurons soma size showing hypotrophy be VI-X of BTBR mice. (Student’s t-test; n = 6,6). (G) Representative image of western blotting for calbindin protein in WT and BTBR mice at P14. Densitometric quantiﬁcation of calbindin showing decreased expression in BTBR mice cerebella (Student’s t-test; n = 6,6). Purkinje Neurons in BTBR Cerebella Displayed Morphological Hypotrophy With Abnormal Dendritic Spine Formation All data are displayed as mean ± SD. < 0.05, P < 0.01, *P < 0.001. Scale bar: (A–D) 25 µm. FIGURE 5 \| Purkinje neurons’ density did not variate but was signiﬁcantly hypotrophy in BTBR mice at postnatal day 14. (A–D) Calbindin staining Purkinje neurons in cerebellar lobe I/II, IV/V, VIII, X of WT (A–D) and BTBR (A’–D’) mice at P14. Purkinje neuron somas in white panels are magniﬁed in (A1-D1,A’1–D’1). (E) Quantiﬁcation of Purkinje neurons number per 500µm in lobe I-X (Student’s t-test; n = 6,6). (F) Quantiﬁcation of Purkinje neurons soma size showing hypotrophy in lobe VI-X of BTBR mice. (Student’s t-test; n = 6,6). (G) Representative image of western blotting for calbindin protein in WT and BTBR mice at P14. (H) Densitometric quantiﬁcation of calbindin showing decreased expression in BTBR mice cerebella (Student’s t-test; n = 6,6). All data are displayed as mean ± SD. P < 0.05, P < 0.01, P < 0.001. Scale bar: (A–D) 25 µm. FIGURE 5 \| Purkinje neurons’ density did not variate but was signiﬁcantly hypotrophy in BTBR mice at postnatal day 14. (A–D) Calbindin staining Purkinje neurons in cerebellar lobe I/II, IV/V, VIII, X of WT (A–D) and BTBR (A’–D’) mice at P14. Purkinje neuron somas in white panels are magniﬁed in (A1-D1,A’1–D’1). (E) Quantiﬁcation of Purkinje neurons number per 500µm in lobe I-X (Student’s t-test; n = 6,6). (F) Quantiﬁcation of Purkinje neurons soma size showing hypotrophy in lobe VI-X of BTBR mice. (Student’s t-test; n = 6,6). (G) Representative image of western blotting for calbindin protein in WT and BTBR mice at P14. (H) Densitometric quantiﬁcation of calbindin showing decreased expression in BTBR mice cerebella (Student’s t-test; n = 6,6). All data are displayed as mean ± SD. P < 0.05, P < 0.01, P < 0.001. Scale bar: (A–D) 25 µm. TRPC Genes Were Involved in the Impaired Cerebellar Development in BTBR Mice The spines are classiﬁed into three subtypes, thin, stubby and mushroom (Figure 6G), and the maturation of the spines was generally assessed. The proportion of the immature long, thin subtype was signiﬁcantly increased (T6 = 6.147, P < 0.01) in BTBR mice compared to WT mice, and the transitional stubby subtype (T6 = 2.617, P < 0.05) and mature wide-headed mushroom spines (T6 = 7.738, P < 0.001) were decreased (Figures 6A’1-B’2,I). These results demonstrate that the dendrite spine formation of PCs was strongly promoted but the mature process was retarded in BTBR mice. To examine the underlying mechanism of the abnormal cerebellar development in BTBR mice, we performed RNA- seq in whole cerebella tissue of WT and BTBR mice at P14. We identiﬁed 3992 diﬀerentially expressed genes (P < 0.05) in BTBR mice, with 1858 upregulated and 2134 downregulated (Figure 7A). For functional annotation, April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 10 Autism and Movement Disorders Xiao et al. FIGURE 6 \| Dendritic spines of Purkinje neurons in BTBR mice were signiﬁcantly increased with disturbed maturation at postnatal day 14. (A,B) Golgi-stained Purkinje neurons in cerebella of WT and BTBR mice. Dendritic branches in white panels are magniﬁed in (A’,B’) and furtherly magniﬁed in (A’1,A’2,B’1,B’2) showing increased and immature dendritic spines in BTBR mice. Yellow, blue, and red arrowheads indicate thin, stubby and mushroom spine types respectively. (C) Quantiﬁcation of Purkinje neuron dendritic proﬁle area in each group. (Student’s t-test; n = 4,4). (D) Quantiﬁcation of the length of the Purkinje neuron’s primary dendrite in each group (Student’s t-test; n = 4,4). (E) Schema graph showing the method of Sholl analysis. Purkinje neuron’s branches are incised by concentric circles with 5.5 µm radius steps from the soma. (F) Quantiﬁcation of intersections of branches and circles at different radius showing similar level of dendrite arborization in WT and BTBR mice (Two-way repeated measure test; n = 4, 4 mice). (G) Schema graph illustrating the spine maturity progresses (up to down) from long thin structure (yellow) to transitional stubby (blue) and mushroom mature form (red). (H) Quantiﬁcation of dendritic spines of Purkinje neurons per 10 µm branch showing increased spine density in BTBR mice (Student’s t-test; n = 4,4). (I) Quantiﬁcation of the percentage of each spine type showing immature development trend in BTBR mice (Student’s t-test; n = 4,4). TRPC Genes Were Involved in the Impaired Cerebellar Development in BTBR Mice All data are displayed as mean ± SD. P < 0.05, P < 0.01, P < 0.001. Scale bar: (A,B) 25 µm; (A’,B’) 5µm; (A’1,A’2,B’1,B’2) 2 µm. FIGURE 6 \| Dendritic spines of Purkinje neurons in BTBR mice were signiﬁcantly increased with disturbed maturation at postnatal day 14. (A,B) Golgi-stained Purkinje neurons in cerebella of WT and BTBR mice. Dendritic branches in white panels are magniﬁed in (A’,B’) and furtherly magniﬁed in (A’1,A’2,B’1,B’2) showing increased and immature dendritic spines in BTBR mice. Yellow, blue, and red arrowheads indicate thin, stubby and mushroom spine types respectively. (C) Quantiﬁcation of Purkinje neuron dendritic proﬁle area in each group. (Student’s t-test; n = 4,4). (D) Quantiﬁcation of the length of the Purkinje neuron’s primary dendrite in each group (Student’s t-test; n = 4,4). (E) Schema graph showing the method of Sholl analysis. Purkinje neuron’s branches are incised by concentric circles with 5.5 µm radius steps from the soma. (F) Quantiﬁcation of intersections of branches and circles at different radius showing similar level of dendrite arborization in WT and BTBR mice (Two-way repeated measure test; n = 4, 4 mice). (G) Schema graph illustrating the spine maturity progresses (up to down) from long thin structure (yellow) to transitional stubby (blue) and mushroom mature form (red). (H) Quantiﬁcation of dendritic spines of Purkinje neurons per 10 µm branch showing increased spine density in BTBR mice (Student’s t-test; n = 4,4). (I) Quantiﬁcation of the percentage of each spine type showing immature development trend in BTBR mice (Student’s t-test; n = 4,4). All data are displayed as mean ± SD. P < 0.05, P < 0.01, *P < 0.001. Scale bar: (A,B) 25 µm; (A’,B’) 5µm; (A’1,A’2,B’1,B’2) 2 µm. the GO term enrichment of diﬀerentially expressed genes was analyzed in biological processes. The top-ranking pathways were primarily involved in central nervous system development, neurogenesis, diﬀerentiation, cell development and morphogenesis (Figure 7B), which are highly consistent with the abnormal development of the cerebella in BTBR mice. The pathway of negative regulation of nervous system development was noticeable, which are prominently listed and comprehensive in cerebellar development. After further screening using protein- protein interaction (PPI) networks analysis (Supplementary Figure S4), the critical genes were identiﬁed, and signiﬁcantly increased TRPC6 was a highly suspicious candidate in BTBR mice (Figure 7C). TRPC Genes Were Involved in the Impaired Cerebellar Development in BTBR Mice TRPC6 is especially expressed during cerebellar development (Huang et al., 2007), and it regulates neurogenesis and synaptic formation (Zhou et al., 2008; Xu et al., 2012). PPI networks (Supplementary Figure S4) indicated the direct interaction of TRPC6 to the changed allele, Itpr3, of BTBR mice, suggesting its critical role. The expression was veriﬁed using RT-PCR (Figure 7D). We simultaneously detected CAMK IV gene expression, which acts downstream of TRPC6, and found that it was upregulated as expected (Figure 7E). TRPC3 and 4 were also detected and exhibited decreased and increased expression, respectively, consistent with the RNA-seq results (Figure 7E). Therefore, the RNA-seq suggests that dysregulated TRPC expression might contribute to the impaired cerebellar development and might result in more serious disorders over time. DISCUSSION The abnormal hindlimb clasping or twisting in BTBR mice also caused a defect to hang from a wire grid. Additionally, impaired ﬁne motor skills in ASD patients are highly linked with social symptomatology (LeBarton and Iverson, 2013). In agreement with these ﬁndings, we report here that BTBR mice exhibited skilled walking pattern deﬁcits using the regularly and irregularly spaced horizontal ladder test. The rotarod task is often regarded as a test of cerebellar coordination and motor ability. WT mice showed better performance on the rotarod task across the consecutive learning test, while BTBR mice exhibited slower learning which might due to the inattention. g Postmortem and functional imaging studies widely identiﬁed the cerebellum as one of the most important brain regions associated with motor deﬁcits in ASD patients (Wang et al., 2014; Hampson and Blatt, 2015). The onset of motor deﬁcits in BTBR mice coincided with the critical period of cerebellar development, suggesting abnormalities in the cerebellum as the neural substrate of motor impairments. Haijie Yang reported an increase in cerebellar foliation and larger gross brain volume of BTBR mice (Yang et al., 2015). In agreement with this ﬁnding, we found that increased cerebellar size and IGL area were obvious compared to the WT mice, with microscopic enhanced foliation. The phenotype was meaningful, for that folia in the cerebellum serve as a broad platform for organizing cerebellar circuits and be critical in sensory- motor tasks (Sudarov and Joyner, 2007). Welker suggested it as the fundamental unit of sensorimotor integration (Welker, 1990), and disturbed foliation was involved in defects of motor coordination (Le Roy-Duﬂos, 2001; Chen et al., 2005). Inward accumulation of proliferated GCPs is a pivotal driving force in the cerebellar foliation, and the existing mouse model of disturbed foliation demonstrated an aberrant proliferation of GCPs (Corrales et al., 2006; Wefers et al., 2017). The present study detected increased GCP proliferation and IGL expansion in the BTBR mouse cerebellum. Impaired radial migration was also observed in rodents with increased foliation (Hosaka et al., 2012; Ryan et al., 2017), and a dramatic increase in foliation was credited with a prolonged period of migration of GCPs in human cerebella (Sillitoe and Joyner, 2007). However, Bergmann glia guided migration was not altered in BTBR mice from early postnatal days to adulthood. DISCUSSION Besides, Purkinje neurons in BTBR mice were found to have somatic hypotrophy, with increased dendritic spine formation but suppressed spine maturation. TRPC was suggested to be responsible for the impaired neurodevelopment and further motor dysfunction. Our observations demonstrated that disturbed cerebellar neurogenesis occurred during the comorbidities of ASD and movement disorders, and attention should be paid to the key role played by TRPC protein family for further study and future approaches of therapy. p Other reports suggest that PCs also participate in cerebellar foliation (Altman, 1997; Sudarov and Joyner, 2007). PCs anchor the outline of the cortex via axonal projections to the underlying WM at positions that deﬁne the base of the ﬁssures. Cerebella of BTBR mouse displayed hypotrophic Purkinje neurons at an early developmental period. The abnormal development of granule cells could ultimately regulate the growth of PCs (Salinas et al., 1994; Shimada et al., 1998; Sadakata et al., 2004), and we inferred that the disrupted patterning of Purkinje cells may be secondary to abnormal GC development. We cannot ignore the fact that PCs are the sole eﬀerent neurons in cerebellum which connect to the outer brain and participate in more complicated neural activity. Abnormal PC development determined the dysfunction of cerebella. Parallel ﬁbers extended by granule neurons in the EGL traveled up and stretched to both sides, being parallel to the pial surface in the ML to connect the Purkinje dendrite. Considering the multiplying granule neurons and invariable even decreased PCs, superabundant incoming of information to an individual PC was predictable. Moreover, the synaptic structure identiﬁed by dendritic spines in Purkinje neurons was signiﬁcantly aﬀected because much more spines existed in a lower matured proportion. Synapses were likely overproduced, but the maturation was suppressed. Immature spines commonly aid in the initiation of synaptic contact (Dunaevsky et al., 1999), and mature spines containing an abundance of neurotransmitter receptors are truly to support synaptic activity (Matsuzaki et al., 2001; Nimchinsky et al., 2002). Abnormal spine formation and maturation would impact the neural circuit and disturb the allomeric function of the brain. Additionally, increasing aﬀerent signal to PCs would inevitably break the physiological balance in transduction, which indicates the critical role played by disturbed information transfer in cerebellar dysfunction. Motor dysfunction represents a heterogeneous array of non- diagnostic symptoms in ASD. In this study, we identiﬁed dystonia-like behavior, such as hyperﬂexion, clasping and twisting, eliciting by tail suspension in BTBR mice. DISCUSSION We demonstrated that BTBR mice exhibited severe infancy- onset dystonia-like behaviors with signiﬁcant impairments in motor coordination and motor learning, which were also observed in patients with ASD. These motor dysfunctions were highly linked to the abnormal development of the April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 11 Autism and Movement Disorders Xiao et al. FIGURE 7 \| Trpc gene differential expression disturbed cerebellar development in BTBR mice at postnatal day 14. (A) Hierarchical clustering of differential expression genes in cerebella of C57BL/6J and BTBR mice at postnatal day 14. (B) Enriched top ﬁve GO pathway in biological process. The former signiﬁcant pathway involved in cerebella development is highlighted in red frame. Number of differentially expressed genes in each pathway is list side to the bar. (C) Heat map depicting 6 signiﬁcant genes identiﬁed from highlighted pathway in B with PPI by STRING (Supplementary Figure S2). Trpc6 is highlighted in red frame. (D) Validation of gene expression in control and BTBR mice by real time PCR. (E) Validation of Trpc3, Trcp4 and Camk4 genes expression in control and BTBR mice by real time PCR. FIGURE 7 \| Trpc gene differential expression disturbed cerebellar development in BTBR mice at postnatal day 14. (A) Hierarchical clustering of differential expression genes in cerebella of C57BL/6J and BTBR mice at postnatal day 14. (B) Enriched top ﬁve GO pathway in biological process. The former signiﬁcant pathway involved in cerebella development is highlighted in red frame. Number of differentially expressed genes in each pathway is list side to the bar. (C) Heat map depicting 6 signiﬁcant genes identiﬁed from highlighted pathway in B with PPI by STRING (Supplementary Figure S2). Trpc6 is highlighted in red frame. (D) Validation of gene expression in control and BTBR mice by real time PCR. (E) Validation of Trpc3, Trcp4 and Camk4 genes expression in control and BTBR mice by real time PCR. April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 12 Autism and Movement Disorders Xiao et al. extra lobes of cerebella in BTBR mice are likely arose from over-produced GCPs. cerebellum. The emerging of dystonia-like behavior in BTBR mice coincided with an increasing proliferation of GCPs, which gave rise to enlargement of the EGL in cerebella and enhanced foliation. REFERENCES orphan nuclear receptor 4. Mol. Cell. Biol. 25, 2722–2732. doi: 10.1128/mcb.25. 7.2722-2732.2005 Altman, J., and Bayer, S. A. (1978). Prenatal development of the cerebellar system in the rat. I. Cytogenesis and histogenesis of the deep nuclei and the cortex of the cerebellum. J. Comp. Neurol. 179, 23–48. doi: 10.1002/cne.901790104 Constantino, J. N., and Charman, T. (2016). Diagnosis of autism spectrum disorder: reconciling the syndrome, its diverse origins, and variation in expression. Lancet Neurol. 15, 279–291. doi: 10.1016/s1474-4422(15)00151-9 Cook, J. (2016). From movement kinematics to social cognition: the case of autism. Altman, J. B. S. (1997). Development of the Cerebellar System in Relation to its Evolution, Structure, and Functions. New York, NY: CRC Press. Cook, J. (2016). From movement kinematics to social cognition: the case of autism. Philos. Trans. R. Soc. Lond. B Biol. Sci. 371:20150372. doi: 10.1098/rstb.2015. 0372 Philos. Trans. R. Soc. Lond. B Biol. Sci. 371:20150372. doi: 10.1098/rstb.2015. 0372 Amaral, D. G., Schumann, C. M., and Nordahl, C. W. (2008). Neuroanatomy of autism. Trends Neurosci. 31, 137–145. doi: 10.1016/j.tins.2007.12.005 Corrales, J. D., Blaess, S., Mahoney, E. M., and Joyner, A. L. (2006). The level of sonic hedgehog signaling regulates the complexity of cerebellar foliation. 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Neuroimaging in autism spectrum disorder: brain structure and function across the lifespan. Lancet Neurol. 14, 1121–1134. doi: 10.1016/s1474-4422(15)00050-2 Blatt, G. J. (2012). The neuropathology of autism. Scientiﬁca 2012:703675. doi: 10.6064/2012/703675 Cai, R., Ding, X., Zhou, K., Shi, Y., Ge, R., Ren, G., et al. (2009). Blockade of TRPC6 channels induced G2/M phase arrest and suppressed growth in human gastric cancer cells. Int. J. Cancer 125, 2281–2287. DISCUSSION Thus, the y TRPC is a non-selective cation channel that dominantly modulates the Ca2+ entry pathway and the release of intracellular Ca2+ (Dietrich and Gudermann, 2007). TRPC3, 4 and 6 belong to the TRPC protein family are particularly expressed in cerebella during the ﬁrst 6 weeks after birth, at the critical neurogenesis period of the cerebellum, to regulate cerebellar development (Huang et al., 2007).TRPC3 expression reﬂects development of the Purkinje cells and TRPC4 expression is restricted to granule neurons and their precursor. TRPC6 plays an essential role in G2/M phase transition (Shi et al., 2009), and inhibition or activation of TRPC6 expression suppresses or accelerates cell growth (Cai et al., 2009; Shi et al., 2009), respectively. Moreover, TRPC6 participates in the development of dendritic spines and regulates the formation of excitatory synapses in the hippocampus (Zhou et al., 2008), and inhibition of TRPC6 reduces dendritic arborization and spine density (Tai et al., 2008). The results of RNA-seq analysis indicated that TRPC family might be an important regulator involved in the abnormality of cerebellar development of BTBR mice. Moreover, other studies suggest the relationship between TRPC and ASD. Wei Li found that TRPC signaling was impaired in hippocampal neurons of Mecp2 mutant mice, another ASD mouse model, April 2020 \| Volume 8 \| Article 231 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 13 Autism and Movement Disorders Xiao et al. care and use of Laboratory animals (NIH Publications No. 8023, revised 1987) and approved by Animal Experiment Committee of Laboratory Animal Center of Army Medical University. which led to activity-dependent BDNF release disturbances that further accounted for sensory and motor abnormalities (Li et al., 2012). Later, Griesi-Oliveira K demonstrated a reduction or haploinsuﬃciency of the TRPC6 gene in ASD individuals, which led to impaired neuronal development, morphology, and function (Griesi-Oliveira et al., 2015). These ﬁndings imply TRPC genes could be novel predisposing genes for ASD to elucidate the underlying pathophysiology mechanism. ETHICS STATEMENT The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2020.00231/ full#supplementary-material All of the animal breed and experiments are performed accordance with the National Institutes of Health guide for the DATA AVAILABILITY STATEMENT We thank Prof. Haiwei Xu for his good suggestion and excellent technical assistance. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualiﬁed researcher. AUTHOR CONTRIBUTIONS RX carried out the experiments, collected and analyzed the data, and wrote the manuscript. HZ, RZ, LW, and ZZ maintained the mice and collected the samples. XL and YM contributed to the quantiﬁcation and data analysis. XF provided resources and funding, performed the experiments planning, supervised the project, and revised the manuscript. CONCLUSION We demonstrated that abnormal neurogenesis of cerebella in BTBR mice primarily aﬀected foliation and disturbed synaptic formation, which possibly lead to dystonia-like behavior and motor dysfunction. The TRPC family was highly indicated as responsible for the impaired cerebellar development and as a novel predisposing gene for ASD. Therefore, TRPC should receive more attention and be further explored to elucidate the pathological process of ASD and possible novel treatments. FUNDING This work was ﬁnancially supported by National Natural Science Foundation of China, project #: 31871043. REFERENCES Neurobiol. 53, 1031–1044. doi: 10.1007/s12035-014-9052-7 Salinas, P. C., Fletcher, C., Copeland, N. G., Jenkins, N. A., and Nusse, R. (1994). Maintenance of Wnt-3 expression in Purkinje cells of the mouse cerebellum depends on interactions with granule cells. Development 120, 1277–1286. Huang, T.-N., Yen, T.-L., Qiu, L. R., Chuang, H.-C., Lerch, J. P., and Hsueh, Y.-P. (2019). 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https://openalex.org/W4386430598	https://www.e3s-conferences.org/articles/e3sconf/pdf/2023/57/e3sconf_ebwff2023_06045.pdf	English	null	Current trends in language ecosystem for a sustainable world	E3S web of conferences	2,023	cc-by	3,790	 Corresponding author: murugovaelena@yandex.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). Current trends in language ecosystem for a sustainable world Elena Murugova1  and Svetlana Evtushenko1 1Don State Technical University, Gagarin Sq., 1, Rostov-on-Don, 344003, Russia Elena Murugova1  and Svetlana Evtushenko1 1Don State Technical University, Gagarin Sq., 1, Rostov-on-Don, 344003, Russia Abstract. The article touches upon the issues of neology and ecolinguistics that are relevant for modern linguistics. From the point of view of ecolinguistics, language is an ecosystem capable of constantly changing and self-regulating. In this ecosystem, new words are constantly being formed, the meanings of old words are changing, which reflects the most significant processes and changes taking place in society at the moment of its development. The aim of this article is to identify current trends in language development reflecting the sustainable development of modern society: the features of the functioning of derived neologisms in modern English, the role of borrowings in replenishing the vocabulary of the language, as well as the study and determination of the specifics of their use in various spheres of life. The studied neologisms reflect almost all areas of life of modern English-speaking society, while most of the new lexical units are vocabulary related to everyday life, household needs, medicine and science caused by the coronavirus pandemic. E3S Web of Conferences 420, 06045 (2023) EBWFF 2023 E3S Web of Conferences 420, 06045 (2023) EBWFF 2023 https://doi.org/10.1051/e3sconf/202342006045 1 Introduction The lexical composition of the language is the most flexible, dynamic and movable side of the language, which directly reflects what is happening in the world. All changes in the life of society find their expression in language and the language is constantly updated with new words. An example is the events related to the coronavirus pandemic and the rapid social changes in the lives of people and society as a whole. So, there was a need to designate many new concepts and a huge number of new words appeared in the language: word-formation and semantic neologisms, borrowings, etc. A large number of different studies have been devoted to this problem [1-5] and others. In recent decades, the increasing globalization and digitalization of all spheres of public life, as well as the active development of science, the global information environment and modern digital technologies, contributes to the emergence of new ways of communication, since new technologies lead to a change in society and the formation of a new picture of the world. One of these ways is the Internet, which accelerates the pace of globalization. Thanks to information technologies and the Internet, new opportunities for communication and quick and free access to information have appeared. D. Crystal notes, "The Internet is enabling a dramatic expansion to take place in the range and variety of language“[6]. The E3S Web of Conferences 420, 06045 (2023) EBWFF 2023 https://doi.org/10.1051/e3sconf/202342006045 Internet is becoming an integral tool of business, communication and popular culture in the world [7]. Internet is becoming an integral tool of business, communication and popular culture in the world [7]. Thus, social changes, scientific and technical achievements are the cause of the emergence of neologisms. But the word is considered a neologism only for a very short period of time. At the beginning of entering the language, new lexemes function in narrow groups. Over time, the phenomenon that new words denote becomes known to an increasing number of native speakers and gradually neologisms become common. The study of the linguistic features of neologisms is an actual trend in modern linguistics to which many studies have been devoted [8-14]. and others. There are many different promising areas of linguistic research devoted to the study of neologisms, one of which is the study of neologisms from the point of view of ecolinguistics. 1 Introduction The ecology of language studies the influence of languages on each other and their interaction with external factors and explores the role of language in the possible solution of environmental problems. The main task of language ecology is to preserve the identity of each individual language and maintain linguistic diversity. Language ecology issues are currently gaining special importance due to the relevance of modern problems of ecology, environment, climate, the study of the ecologization of modern knowledge. The term ecology of language (linguistic ecology) for the interpretation of ecology in a linguistic context was most widely used in linguistics thanks to the famous American linguist E. Haugen. As the linguist notes, "the ecology of language is the science of interactions between any single language and the society that uses this language. The ecology of a language depends on the people who learn it, use it and pass it on to other people" [15]. In E. Haugen's concept, language appears as "part of an ecosystem in which it is formed and evolves, like any living organism, while satisfying all the needs of its "users"[15]. Later, the concept of ecolinguistics as a science began to be developed in the works of other linguists. p g g p g Ecolinguistics considers language in terms of the relationship between other languages, well as languages and the society that speaks these languages. According to linguists, ecolinguistics considers language as an ecosystem that change or form new words, change the meanings of existing words, or regulate itself [16] The ecolinguistic approach requires an analysis of extralinguistic factors (the emergence of new realities, the processes of globalization, the development of technology), as well as intra-linguistic factors (laws of consistency, analogies, traditions, trends towards saving language resources, semantic changes) that affect the language ecosystem and contribute to the emergence of new words. The ecolinguistic approach can be used in the study of the conditions for the appearance of neologisms, conceptual areas of concentration of neologisms in the language, as well as in identifying ways to enrich the language with new words and determining the number of words that have been borrowed from other languages. 1 Introduction The ecolinguistic approach reflects the trend towards building a sustainable society, makes it possible to detect harmful phenomena in the language, possible contamination of the language by various borrowings of other languages, which poses a threat to the preservation of the purity and cultural identity of the language. 3 Results The distribution of the studied neologisms, considering the method of vocabulary replenishment, is presented as follows: p p 1. The most common way to replenish the lexical composition of the language is word formation - 61% of all studied words 1. The most common way to replenish the lexical composition of the language is word formation - 61% of all studied words 2. Semantic neologisms make up 21% of all studied words. 2. Semantic neologisms make up 21% of all studied words. 3. In the course of our research, it turned out that borrowings make up a fairly large proportion of all neologisms - about 18% 3. In the course of our research, it turned out that borrowings make up a fairly large proportion of all neologisms - about 18% The lexicon of a language takes inventory of the material and spiritual culture of the respective society, and it is in the vocabulary that cultural difference is most prominently manifested. Neologisms and realities can not only be borrowed from other languages, but also appear in society when there is such a need. The basis for the emergence of new linguistic units is the need to name new realities, as well as the need to designate phenomena that were previously present, but did not have a corresponding designation or changed their social role. New words contribute to the enrichment of the language system and reflect the diversity of accumulated knowledge and the progress of civilization. In this regard, the method of describing the new vocabulary by the so-called thematic groups is of interest. New lexical units are divided into groups depending on the sphere of human activity to which they relate and denote the most significant aspects of society. The study of thematic groups helps to determine the directions of socio-cultural development of a modern sustainable ecosystem society and trends in the development of the lexical system as a whole. The communicative sphere affects the following areas of research, which are presented in Figure 1. The communicative sphere affects the following areas of research, which are presented in Figure 1. 18,5% 0,6% 0,4% everyday life, household needs medicine and science anthropocentric characteristics socio-political, financial relations, religion leisure, hobbies ethnonyms, toponyms, languages human actions reduced vocabulary industry, labor plants, animals, nature politically correct vocabulary psychoactive substances emotional sphere characteristics of speech activity interpersonal relationships Fig. 1. 2 Materials and methods The aim of this article is to identify the features of the functioning of derived neologisms in modern English, the role of borrowings in replenishing the vocabulary of the language, as well as the study and determination of the specifics of their use in various spheres of life. 2 2 E3S Web of Conferences 420, 06045 (2023) EBWFF 2023 https://doi.org/10.1051/e3sconf/202342006045 In this paper, we analyzed 1333 lexical units from the "New word entries" section of the Oxford English Dictionary (OED) within the period of 2021-2022 years [17]. The following methods were used in the study: the method of analyzing dictionary definitions, the method of representative decreasing of the material, and elements of statistical calculation. 3 Results Composition of the new vocabulary, considering thematic groups Fig. 1. Composition of the new vocabulary, considering thematic groups 3 E3S Web of Conferences 420, 06045 (2023) EBWFF 2023 https://doi.org/10.1051/e3sconf/202342006045 The studied neologisms reflect almost all areas of life of modern English-speaking sustainable ecosystem society, while most of the new lexical units are vocabulary related to everyday life, household needs -18.5% (Baff, “A slipper; = baffie n. Usually in plural.”). The second most productive is vocabulary related to medicine and science -12.2% (nanoplastic, n.: "A hypothetical material produced by nanotechnology, capable of changing its shape and other properties as required.”). At the same time, a variety of disciplines served as sources for vocabulary replenishment: from botany to philosophy. This group includes neologisms whose appearance is caused by the coronavirus pandemic (CV, n. “«A coronavirus; (now) esp. the coronavirus which causes the disease Covid-19. Also: the disease Covid-19.”). The third most productive is the group, which includes vocabulary describing people and their needs, character, mental qualities, etc. -12% (oversharer, n.: “A person who reveals an inappropriate amount of detail about his or her personal life”"). A significant number of words denotes social and political relations, religious affiliation 11.5% (deliverology, n.: “A target-driven process designed to ensure the successful implementation of reforms or achievement of policy goals within government or the public...”). 8% of words represent leisure, games, hobbies, music, sports, art, etc., and constitute 8% of the studied non - lexemes (game day, n.: “A day of sporting competition; a day dedicated to playing sports or games.”). In the communicative environment there are neologisms-ethnonyms, toponyms - 7.5% (chicagoese, adj. and n.: “relating to, or characteristic of the city of Chicago, its inhabitants, or the speech or language used there”), words describing industry, machinery, tools, labor activity (7%) (Chicken factory, n.: “A building where chickens are hatched in temperature- controlled conditions in large numbers.”), as well as neologisms describing human actions (6%) (sprit, n.4: "A sudden quick movement; a spring, jump, leap.””). A number of words represent stylistically reduced vocabulary (5%) (Candy-ass, n. and adj.: “A cowardly, timid, effeminate, ineffectual man or a wimp, a sissy.”). y Politically correct language units were also identified (Doitered, adj.: “Having diminished mental faculties, esp. as a result of old age; confused, muddled. Also: physically debilitated by old age”") - 2%. 3 Results Lexemes denoting the production, distribution and use of psychoactive substances have been discovered (Junked out, adj.: "Incapacitated by drugs; addicted to heroin.”) - 0,8%. Characteristics of speech activity (Mumbly, adj.: "Characterized by mumbling; included to mumble.”) and interpersonal relationships (Sprunt, v.2: "intransitive. To pay romantic or amorous attention to a person.”) made up an insignificant part of the new lexical units: 0.6% and 0.4%, respectively. The study of thematic groups of neologisms confirmed the fact that the language always adapts to the environment and people who speak it. A vivid illustration of the responsiveness of language to current events is the presence of vocabulary related to the coronavirus pandemic among the neologisms. The group includes words denoting both the disease itself and the virus causing it, as well as the nominations of restrictions caused by the fight against the disease ("social distancing", "social isolation", "physical distancing"). The neolexemes "Novichok" (a family of toxic substances) and "Kompromat", borrowed from the Russian language, attract attention. The inclusion of these neonominations in the dictionary can serve as an illustration of the political situation impact on the world, on the emergence of new words. 4 Discussion In the course of our research, it was found out that borrowings composed a sufficiently large proportion of all neologisms - 18%. We have raised the question - in which thematic 4 E3S Web of Conferences 420, 06045 (2023) E3S Web of Conferences 420, 06045 (2023) EBWFF 2023 https://doi.org/10.1051/e3sconf/202342006045 groups the role of borrowing is most significant. We compared what proportion in each thematic group are neologisms-borrowings, and what are neologisms obtained at the expense of internal resources of the English language (word formation and reinterpretation). The results are presented in Figure 2 75 83 91 72 82 52 87 90 100 75 91 100 100 100 100 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 9 25 10 13 48 18 28 9 17 25 0 0 0 0 0 word formation, semantic neologisms neologisms-borrowings . Fig. 2 Comparison of the spheres of functioning of neologisms 0 Fig. 2 Comparison of the spheres of functioning of neologisms Fig. 2 Comparison of the spheres of functioning of neologisms Socially significant euphemisms (based on health restrictions); 3. Ethnonyms; 4. Censoring abusive language; 5. Sexual orientation; 6. General vocabulary. The distribution of politically correct neologisms, considering thematic groups, is presented in figure 3. Gender-neutral vocabulary Socially significant euphemisms (health restriction) Ethnonyms Censoring abusive language Sexual orientation General vocabulary 43% Fig. 3 Composition of politically correct vocabulary, considering thematic groups Fig. 3 Composition of politically correct vocabulary, considering thematic groups Fig. 3 Composition of politically correct vocabulary, considering thematic groups The second most productive is the group that includes socially significant euphemisms describing various health limitations - 25% (Neurodivergence, n.: "Divergence in mental or neurological functioning from what is considered typical or normal, esp. where this falls on the autism spectrum”). Ethnonyms make up 18% of analyzed words (Multicultural London English, n.: “A variety of English spoken mostly by young people in the multicultural neighborhoods of central."). Profanity from the point of view of ecolinguistics is represented by the following words, such as Yes-girl, n. “«A young woman who assents to romantic or sexual proposals; a promiscuous woman”"). Fig. 2 Comparison of the spheres of functioning of neologisms Borrowings function most productively in the creation of ethnonyms and toponyms, i.e. vocabulary used to nominate villages, reservoirs, etc., nationality, nations etc. There are 99 words in this group, and 48 of them are borrowings (48%). That is, the number of neolexemes created with the help of internal resources of English and lexeme-borrowings is approximately the same. There is no such correlation in other thematic groups of neologisms. Thus, the group "socio-political relations, religion" contains only 28% of borrowings. Groups "Life", "Nature" - 25% of borrowings. "Medicine, science" - 17% of borrowings. 13% of borrowings are among the vocabulary on the topic "Industry, labor". 10% - in the group "Human actions". 9% of borrowings are among neologisms describing people and their needs, character traits, mental qualities, etc. and descriptions of emotional life. No borrowings were found among the politically correct groups of neolexemes "Psychoactive substances", "Speech activity", "Interpersonal relations". It is worth noting a certain correlation between some thematic groups of neologisms and the languages from which they were borrowed. Thus, the language that became the source of the largest number of new words turned out to be Latin, and the second largest thematic group was the thematic group "science". Latin is the language of science, which in turn is confirmed by the predominance among the analyzed neologisms of new linguistic units 5 E3S Web of Conferences 420, 06045 (2023) E3S Web of Conferences 420, 06045 (2023) EBWFF 2023 https://doi.org/10.1051/e3sconf/202342006045 related to the topic of science. And also, the predominant number of Latin borrowings out of the total number of borrowings. related to the topic of science. And also, the predominant number of Latin borrowings out of the total number of borrowings. In 2021-2022, politically correct language units began to be actively used, while most of them are gender-neutral vocabulary. The main way to create politically correct neolexemes is multiplication: in order to avoid gender concretization, plural pronouns are used instead of using a third-person singular pronoun. (They, pron. meaning 2c: "Used to refer to a person whose sense of gender identity does not correspond to generally accepted sex and gender differences, and who usually asked to refer to the use of them (instead of he or she.”). ) Among the studied politically correct words, six thematic groups were identified: ) Among the studied politically correct words, six thematic groups were identified: 1. Gender-neutral vocabulary; 2. 5 Conclusion and recommendation Thus, grounded on the material of neologisms (2021 – 2022), the following trends in the development of the modern English language can be identified: - the role of borrowings in the formation of new words is increasing, due to the increasing influence of multiculturalism policy, migration, interest in the cultural 6 6 E3S Web of Conferences 420, 06045 (2023) E3S Web of Conferences 420, 06045 (2023) EBWFF 2023 https://doi.org/10.1051/e3sconf/202342006045 peculiarities of different countries, the expansion of intercultural communication between countries; peculiarities of different countries, the expansion of intercultural communication between countries; ; - we found a large number of borrowings among ethnonyms and toponyms; ; - we found a large number of borrowings among ethnonyms and toponyms; - the growing influence of extralinguistic factors on the formation of new words: the processes of globalization, the development of technology and the Internet, - the establishment of the principles of political correctness and tolerance in speech communication, which contributes to the sustainable development of society - introduction of politically correct vocabulary, most often represented by genderonyms formed by multiplication. Thus, neologisms indicate the flexibility of language, its ability to develop and adapt to constant changes to the surrounding reality, reflect current trends in the development of a sustainable society. New words denote the emergence of new realities, and semantic neologisms give an idea of transformations in the understanding of reality, of a new interpretation by native speakers of already existing phenomena. To summarize, we would like to note that at the present stage of language development, the formation of new nominative units (neologisms) is directly dependent on the socio- cultural situation, since the language expresses the culture of the people speaking it. Neologisms reflect all the most important and urgent problems occurring in society and affect the sustainable development of this society. Whether neolexems will become commonly used depends to a greater extent not on linguistic factors, but on how deep the need of native speakers for the designation of new phenomena is, how much native speakers will need this word. Nowadays, due to globalization, environmental issues have gone beyond the original interpretation and have become a distinctive characteristic of many modern sciences, including linguistics and the problem of studying neologisms from the point of view of ecolinguistics has become very relevant and important. 5 Conclusion and recommendation Further study of the ways of formation of new words and their meanings, their functioning in society, will reduce the negative impact of globalization processes on the language; raise the level of national consciousness and preservation of linguistic identity. References 1. Plag, Ingo. Word-formation in English (second). Cambridge University Press. 258 p. (2018) 1. Plag, Ingo. Word-formation in English (second). Cambridge University Press. 258 p. (2018) 2. Thorne, Tony. 2020. #CORONASPEAK – the language of Covid-19 goes viral – 2. Language and Innovation (2020). 2. Thorne, Tony. 2020. #CORONASPEAK – the language of Covid-19 goes viral – 2. Language and Innovation (2020). 3. Lei, Siyu, Ruiying Yang, and Chu-Ren Huang. Emergent neologism: A study of an emerging meaning with competing forms based on the first six months of COVID-19. Lingua 258 (2021) 4. Mulyono, and Agus Subiyanto. Productivity of New Indonesian Vocabulary in the Pandemic Time of Covid-19. E3S Web Conf. In The 6th International Conference on Energy, Environment, Epidemiology, and Information System (ICENIS 2021) 317 (02029) (2021). 5. Roig-Marín, Amanda. A. English-based coroneologisms: A short survey of our Covid- 19-related vocabulary. English Today 37 (4): 193 - 195. (2021) 6. Crystal, D. Frontmatter. Language and the Internet, i-iv. Cambridge: Cambridge University Press (2001). 7 E3S Web of Conferences 420, 06045 (2023) EBWFF 2023 https://doi.org/10.1051/e3sconf/202342006045 7. Brignall III, Thomas Wells and Thomas Van Valey. The Impact of Internet Communications on Social Interaction. Sociological Spectrum. Mid-South Sociological Association 25 (3): 335-348. (2005) 8. Buckingham, Hugh W. “Chapter 3: Where Do Neologisms Come From?" Jargonaphasia, ed. Jason W. Brown, 39-62. Academic Press (1981). 9. Ahmad, Khurshid. Neologisms, Nonces and Word Formation. Proceedings of the 9th EURALEX Int. Congress, ed. Heid, U., Evert, S., Lehmann, E., Rohrer, C. Munich, vol. II: 711–730. Universitat Stuttgart, Munich (2000). 10. Lehrer, Adrienne. Understanding trendy neologisms. Rivista di Linguistica 15 (2): 369–382. (2003) 11. Lehrer, Adrienne. 2006. Neologisms. Encyclopedia of Language & Linguistics (second), ed. K. Brown: 590-593. Elsevier. (2006) 12. Liu, Wei, and Wenyu Liu. Analysis on the Word-formation of English Netspeak Neologism. Journal of Arts and Humanities 3 (12): 22—30. (2014) 13. Paterson, Alasdair. New words and new world order: the vocabulary of global Warfare. Procedia - Social and Behavioral Sciences 154: 144-147 (2014) 14. Casado, Laura Llanos, and Milka Villayandre Llamazares. An Analysis of Neologism Creation Processes in Texts from Spain and Latin America Included in CORPES XXI. Procedia - Social and Behavioral Sciences 198: 278-286 (2015) 15. Haugen E. The Ecology of Language, in: Fill / A. Muhlhausler, P. The Ecolinguistics Reader. Language, Ecology and Environment. – London, New York (2001). 16. Shamne N. L., Rets I. V. 17. Oxford English Dictionary [Electronic resource]. – URL: https://www.oed.com/ References The problem of studying neologisms and their influence on the ecology of language. Vestnik Volgogradskogo gosudarstvennogo universiteta. Seriya 2: Yazykoznaniye – Science Journal of Volgograd State University. Series. 2: Linguistics, no. 1 (25), pp. 72–77. (2015) 17. Oxford English Dictionary [Electronic resource]. – URL: https://www.oed.com/ 17. Oxford English Dictionary [Electronic resource]. – URL: https://www.oed.com/ 8 8
https://openalex.org/W4283070417	https://ejfa.me/index.php/journal/article/download/2798/1576	English	null	Asymmetrical relationship between COVID-19 global fear index and agricultural commodity prices	Emirates journal of food and agriculture	2,022	cc-by	7,970	A B S T R A C T Grains and oilseed crops are widely used as key input elements for global food safety in food and livestock sub-sectors, as well as in other sectors such as energy, services and industry. In addition, they play an active role in the international agricultural markets. Therefore, price structure in the grain and oilseed market is important for agriculture and many other sectors. This study was designed to reveal how the fear of COVID-19 has globally affected grain prices. The study covered the one-year period from March 11, 2020, when COVID-19 was first recognized as a pandemic, to March 11, 2021. The Global Fear Index (GFI) and the price sub-indices created by the Grain Oil Council were used to determine the impact of the fear caused by COVID-19 on grain prices. Assuming an asymmetrical relationship between variables, the Nonlinear Autoregressive Distributed Lag model was used to determine this relationship. According to the model results, it was found that in the long term, agricultural commodity prices gave an increase (decrease) response to the positive (negative) effects in the GFI, and that the effect of an increase in the GFI on agricultural commodity prices was greater than the effect of a decrease. Accordingly, it is thought that the analysis and predictions which take into account the asymmetrical effect would give more realistic results and thus contribute considerably to the market regulations. It will also help policymakers make more rational decisions in their search for solutions to the problems in the cereal market. Keywords: COVID-19; Agricultural Commodities Prices; NARDL model; Asymmetric Effects Asymmetrical relationship between COVID-19 global fear index and agricultural commodity prices Merve Ayyildiz* Yozgat Bozok University, Faculty of Agriculture, Department of Agricultural Economics, 66900, Yozgat, Turkey Emirates Journal of Food and Agriculture. 2022. 34(3): 239-247 doi: 10.9755/ejfa.2022.v34.i3.2798 http://www.ejfa.me/ Emirates Journal of Food and Agriculture. 2022. 34(3): 239-247 doi: 10.9755/ejfa.2022.v34.i3.2798 http://www.ejfa.me/ R E S E A R C H A R R E S E A R C H A R T I C L E Corresponding author: Merve Ayyildiz, Yozgat Bozok University, Faculty of Agriculture, Department of Agricultural Economics, 66900, Yozgat, Turkey. E-mail: merve.ayyildiz@yobu.edu.tr Emir. J. Food Agric ● Vol 34 ● Issue 3 ● 2022 Corresponding author: Merve Ayyildiz, Yozgat Bozok University, Faculty of Agriculture, Department of Agricultural Economics, 66900, Yozgat, Turkey. E-mail: merve.ayyildiz@yobu.edu.tr Received: 23 August 2021; Accepted: 24 February 2022 LITERATURE REVIEW Reaching of COVID-19 to pandemic status has raised health concerns as well as global economic problems. This has caused stagnation and uncertainty in the markets on a national and global scale and, like many sectors, had considerable effect on the agricultural sector. Considering the fact that the structural changes in the markets have a direct effect on price changes especially in times of crisis, research on price structure in agricultural markets has also been of great interest during COVID-19. Research to assess the impact of COVID-19 on agricultural commodity prices basically focus on the correlation between energy and agricultural markets, price changes before and during COVID-19, and the impact of concern and panic brought on by COVID-19 on agricultural commodity prices. In the early days of the pandemic, the prices of important crops such as barley, corn, wheat and rice tended to fall slightly. However, due to the increase in the rate of spread of the pandemic, quotas for trade and continued demand increase, prices have risen rapidly since the second half of 2020. Indeed, international prices for wheat, corn and barley increased by 22.8, 45.6 and 32.2%, respectively, over the one-year period starting from the date when the World Health Organization declared COVID-19 a pandemic. According to Ezeaku et al. (2021), although the grain market has shown a resilient structure to the epidemic and the price volatility has remained low, the COVID-19 pandemic continues to be a source of uncertainty in the market. As a matter of fact, it is predicted that the COVID-19 pandemic in the long term may lead to a major change in grain market due to a decrease in biofuel demand, increased concerns about food safety, changes in consumer behavior, increased investments in digital supply chains, a decrease in global feed demand, a return to the globalization trend in supply chains and an increase in government interventions (Skuratovic, 2021). In recent years, the increase in demand for energy plants and the fact that oil is one of the main inputs in agricultural production have further strengthened the relationship between energy prices and agricultural commodity prices. Therefore, it is inevitable that changes in energy prices will cause changes and fluctuations in agricultural commodity prices. This interaction becomes more pronounced especially in times of uncertainty and crisis. INTRODUCTION led to major changes in the balance of unemployment, growth and supply and demand with restrictions on both national and international movement of people and goods (Beckman et al., 2021; Ceylan et al., 2020). Thus, the rapid spread of COVID-19 has brought a global economic problem along with a global health problem. Corona virus was declared a pandemic by the World Health Organization on March 11, 2020, less than three months after its first report on December 31, 2019 in Wuhan town of Hubei province in China. During the one-year period, the total number of cases exceeded 130 million and there were nearly 3 million deaths (World Health Organization, 2021). Compared to the previous Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), new corona virus has a quite high rate of spread. In just a few months, it has become one of the world’s greatest health problems, triggering global fear (Erokhin and Gao, 2020; Daglis et al., 2020). The transformation of COVID-19 into an economic crisis has had impact in many sectors (Bai et al., 2020). This has brought the focus of many researchers to COVID-19 impact assessments on a sectoral basis (Ozturk, 2020; Sharma and Nicolau, 2020; Fana et al., 2020; Eroglu, 2021; Milani, 2021). In the early stages of the pandemic, the agricultural sector did not receive as much attention as other sectors of the economy. However, the pressure on supply and demand and the concerns that food security may be at risk and that a global food crisis may occur in the future have taken the sector to a strategic position over time (Clapp and Moseley, 2020; Schmidhuber, 2020; Musa et al., 2020; Ramakumar, 2020; Poudel et al., 2020; Salisu et al., 2020; Elleby et al., 2020). On the other hand, due to The temporary policies developed by many countries around the world focusing on social distancing to slow the spread of the pandemic seem to be effective in reducing the spread of the disease while causing constrictions in the economies of the countries (Hart et al., 2020; Poudel et al., 2020). Also due to globalization, COVID-19 has Received: 23 August 2021; Accepted: 24 February 2022 Received: 23 August 2021; Accepted: 24 February 2022 239 Emir. J. INTRODUCTION Food Agric ● Vol 34 ● Issue 3 ● 2022 Merve Ayyildiz the interrelationship of agriculture with many sectors, the possible effect of pandemic in the agricultural sector has led to a domino effect in sectors closely related to agriculture (Hart et al., 2020). The pandemic has directly and indirectly caused economic shocks and social costs in the agricultural sector through macroeconomic factors, energy and credit markets, and input and output prices in agricultural factor markets (Stephens et al., 2020; Schmidhuber, 2020). As a result of measures to control the spread of the disease, food demand has been reshaped, the workforce has experienced severe contractions, and agriculture and food systems experienced disruptions (Elleby et al., 2020; Laborde et al., 2021; Daglis et al., 2020). services and industry sectors, and play an active role in the international agricultural products trade. Therefore, price structure in the grain market is important for many agricultural and non-agricultural sectors. The present study aimed to determine how the fear of COVID-19 globally affects grain prices asymmetrically. In the literature, the impact of the crisis on food prices and their volatility was investigated, but no study was found that addressed the asymmetrical relationship. In addition, the fact that evaluating the prices in grain sector, which is related to the food safety, livestock, nutrition and energy sectors, has a critical value in this period adds to the importance of the study. In the study, the global fear index (GFI) developed by Salisu and Akanni (2020) to measure the fear/panic associated with the COVID-19 outbreak was used. This allowed us to evaluate the impact of COVID-19 case and death numbers as a whole on agricultural commodity prices. International trade restrictions have led to a loss of revenue in exporter countries due to the disruption in global food supply chain while the countries whose food supply is largely based on imports had concerns about food security (Siche, 2020; Laborde et al., 2020; Vickers et al., 2020; Beckman and Countryman, 2021; Mouloudj et al., 2020; Daglis et al., 2020). On the other hand, the bottlenecks in the economy and the global health concerns created by COVID-19 are putting pressure on input and output prices in the agricultural sector (Borgards et al., 2021; Karagol et al., 2021). LITERATURE REVIEW For this reason, studies examining the relationship between energy prices and agricultural commodity prices have been of great interest in the literature during the COVID-19 pandemic. Using the daily data in the period 2002-2020, Umar et al. (2021) stated that the change in oil prices led to changes and volatility in agricultural commodity prices during the Global Financial Crisis, the European sovereign debt crisis and the COVID-19 pandemic crisis. Wang et al. (2020), on the The focus of the present study was on the impact of COVID-19 on grain and oilseed markets. Grain and oilseed products are widely used as critical inputs in food and livestock sub-sectors which are of paramount importance in global food safety, as well as in energy Emir. J. Food Agric ● Vol 34 ● Issue 3 ● 2022 240 Merve Ayyildiz in the long-term wheat prices. In contrast, Kumar et al. (2020) statistically demonstrated that COVID-19 had an in-depth effect on the spot and future prices of wheat in the National Commodity and Derivatives Exchange [NCDEX], one of India’s major stock exchanges, in the early stages of the pandemic (January-April 2020 period) and that volatility in the commodity market increased further during this period. Sun et al. (2021) examined the causality relationship between trade policy uncertainties and agricultural commodity markets to investigate whether agricultural trade policy uncertainty (TPU) was important for agricultural commodity prices (ACP) from a Chinese perspective in the period from 2005:M1 to 2020:M10. As a result of this study, which examined four different periods, they found a positive relationship at 10% level of significance from TPU to ACP in the periods of 2008: M7-2008: M12, 2020: M5-2020: M9, which also covered the COVID-19 process. In their study, they concluded that in general, the COVID-19 pandemic disrupted the trade flow of agricultural commodities, reduced Chinese imports and significantly increased ACP. Gutierrez and Pierre (2020) evaluated the global response of grain prices to oil prices, stock-use ratio and export shocks using the Global Vector Automatic Regression (GVAR) model, a multi-country time series model that is independently modeled on each market and linked to trade-based compound variables, based on leading countries in wheat, barley and corn exports. LITERATURE REVIEW They stated that despite the concerns about disruption in the supply chain the oil market may have contributed to the stability of global grain prices in early 2020, that export restrictions in the first half of 2020 could significantly increase global prices, and that such restrictions could affect more than the target commodity through cross- commodity price links. other hand, found a strong long-term positive correlation between crude oil prices and agricultural commodity prices. Ezeaku et al. (2021) stated that the impact of the shocks in the oil market during the COVID-19 process varied in different crops. They found that corn and wheat prices had a considerable and positive reaction to oil market shocks, but soybean and rice prices reacted negatively to the oil shock. On the other hand, the effect of COVID-19 on agricultural commodity prices is evaluated independently from other sectors. In the literature, there are studies that examine the changes in agricultural commodity prices by taking into account the number of cases and deaths caused by COVID-19. There are also studies that considered COVID-19 as a period and observed the price changes by comparing it with other periods. Since the pandemic has led to the global economic crisis, the impact of this process on prices has been specifically investigated. The pandemic has led to reductions in the growth rates of the national economies, contractions in domestic demands and similar deteriorations in world trade. National and international measures to slow down the spread of the disease have also greatly affected the agricultural sector. Tough differed on regional, national and global scales, prices have changed markedly in the process. Singh et al. (2020) examined the changes in food prices in different regions of Nepal by addressing two separate periods before and during the COVID-19 period. They found that there was a significant increase in all food prices except animal products during COVID-19, but these price changes differed by region. Daglis et al. (2020) examined the impact of global COVID-19 case numbers on oat and wheat prices through multiple impact-response analysis. They concluded that there was a cointegration relationship between global case numbers and oat and wheat prices, and that the spread of COVID-19 increased wheat and oat prices. Salisu et al. Emir. J. Food Agric ● Vol 34 ● Issue 3 ● 2022 1 IGC GOI Barley sub-index was calculated using Argentina Up River, Australia Port Adelaide, Black Sea Fob, EU (France) Rouen and EU (Germany) Hamburg. IGC GOI Maize sub-Index was calculated from Argentina Rosario (Up River), Black Sea, Brazil Paranagua, US Gulf. IGC GOI Wheat Sub-Index was calculated from Argentina Up River, Australia Port Adelaide, Black Sea, Canada St. Lawrence and Vancouver, EU (France) Rouen, US Gulf and PNW. All indices were calculated by IGC. Unit root test The Global Fear Index (GFI), which aims to measure daily concerns and feelings about the spread and severity of COVID-19, is a composite index of two factors based on reported cases and deaths. This index, which has a score range of 0-100, indicates the absence or existence of fear. GFI is composed of The Reported Cases Index (RCI) which measures how much the reported cases deviate from the expected ones over a 14-day period, and The Reported Death Index (RDI) which measures how much the reported deaths deviate from the expected ones over the same 14- day period. RCI is calculated as follows: Many methods are applied for the estimation of asymmetric relationship in the time series, and these methods are preferred based on the stationary condition of the series. Augmented Dickey-Fuller (ADF) and Phillips-Perron (PP) unit root tests were used to test the stationary ratings of the series. The stationary ratings of the series were examined depending on constant and constant and trend- containing. Accordingly, based on both unit root test result, it was observed that the variables other than the lngfi were stationary when the first-degree difference was taken (Table 2). lngfi variable was found to get stationary when the first-degree difference (I(1)) was received according to PP statistic but it was stationary at the level (I(0)) according to ADF statistic. 0.5 ( ) t t t GFI RCI RDI = + 0.5 ( ) t t t GFI RCI RDI = + On the other hand, the sub-indices created on the basis of 1 January 2000 were revised based on the date of March 11, 2020, when COVID-19 was announced as a pandemic by the World Health Organization. All variables in the study were used in logarithmic form to facilitate interpretation of the results of the analysis. Unlike Engle-Granger cointegration (1987) and Johansen Cointegration (1988 and 1990) models, the Autoregressive Distributed Lag (ARDL) model allows both to study cointegration between series with varying degrees of stationary condition and to model long and short-term dynamics at the same time (Pesaran et al., 2001). In this context, taking into account the unit root test results, Nonlinear Autoregressive Distributed Lag (NARDL) boundary test, which is an extension of the linear ARDL model, was selected as the cointegration model (Shin et al., 2014). LITERATURE REVIEW (2020) aimed to demonstrate the predictive power of the Global Fear Index in the predictability of commodity prices by using a data set of commodity prices and global fear indices of 24 globally traded products, including agricultural products such as cocoa, coffee, oats, rice, wheat, sugar, soybean. The results confirmed that there was a positive relationship between commodity price returns and the global fear index, and that commodity returns increased along with the fear about COVID-19. Cariappa et al. (2020) examined the volatility in retail and wholesale wheat prices for five different regions of India during this period using the GARCH model. The findings suggested that wheat prices increased after the lockdown, but this increase was not immense overall. In other words, they argued that the effect of the lockdown was not large enough to cause a structural breakdown and volatility The change in agricultural commodity prices during the COVID-19 crisis has been studied on a linear scale in recent literature. However, there are no linear approaches to how positive and negative shocks related to COVID-19 would affect agricultural commodity prices. Examining the models which may involve asymmetrical relationship among variables using symmetrical methods can lead to misleading of policymakers. In this respect, it is considered important to take into account the asymmetrical relationship in determining policies to ensure stability. Assuming that the targeting the asymmetrical relationships may yield more effective results, how the positive and negative shocks in the global fear index (GFI) developed by Salisu and Akanni (2020) affect agricultural commodity prices was examined using the Nonlinear Autoregressive Distributed Lag (NARDL) model in the present study. Emir. J. Food Agric ● Vol 34 ● Issue 3 ● 2022 241 Merve Ayyildiz Descriptive analysis Understanding the characteristics of the variables examined is critical in determining the econometric technique. The mean, standard deviation, skewness, kurtosis and distribution normality of variables were tested under descriptive statistics (Table 1). It was observed that the means of price indices (lnbarley, lnmaize, lnrice, lnsoybean, lnwheat) had similar values, and lnmaize had the highest average. The skewness values of the price index series were determined as left skewed, and the kurtosis values were determined as platykurtic distribution. The mean global fear index (LNGFI) series was 3.975, the skewness value was close to 2, while the kurtosis value had leptokurtic distribution. According to Jargue-Bera test statistics, not all series were distributed normally. Data The data set consisted of the Global Fear Index and the Grain and Oilseed Sub-Index1. Daily series of each sub- index price of grain and oilseed index (GOI) developed by the International Grain Council (IGC) was taken from www.igc.int/en/default.aspx. The GFI series were obtained from Salisu and Akanni (2020). The starting period of the study was taken as the date on which COVID-19 was declared as a pandemic by the World Health Organization (WHO). Accordingly, daily data was collected between March 11, 2020 and March 11, 2021. MATERIAL AND METHOD Table 1: Descriptive statistical analysis Table 1: Descriptive statistical analysis Table 1: Descriptive statistical analysis lnbarley lnmaize lnrice lnsoy bean lnwheat lngfi Mean 4.69 4.73 4.67 4.83 4.68 3.98 Median 4.63 4.66 4.67 4.79 4.65 3.94 Maximum 4.91 5.03 4.74 5.13 4.84 4.52 Minimum 4.54 4.47 4.61 4.55 4.54 3.68 Std. Dev. 0.11 0.19 0.03 0.19 0.09 0.16 Skewness 0.53 0.27 0.17 0.13 0.36 1.92 Kurtosis 1.87 1.47 2.25 1.46 1.74 6.95 Jarque‑ Bera 25.72 28.31 7.41 26.32 22.86 28.26 Probability 0.00 0.00 0.02 0.00 0.00 0.00 RESULTS AND DISCUSSION NARDL bound cointegration test results are given in Table 3. According to these results, the significance levels vary and there was a long-term relationship between each Crop Price Index and the Global Fear Index. After fulfilling the cointegration specification, we continued with the estimated short-run and long-run coefficients which are presented in Table 4. AIC (Akaike Information Criteria) was used to determine the optimal lagging length during the analysis phase. Asymmetrical error correction model, on the other hand, is given below (Equation 2): Model I-a: Model I-a: ( ) 1 1 1 1 0 t t t t p q j t j t t j t t j t j j lnY lnY lngfi lngfi lnY lngfi lngfi         + + − − − − − + + − − − − − = = ∆ = + + + + ∆ + ∆ + ∆ + ∑ ∑ (2) When creating the NARDL model for each agricultural commodity price index, the maximum lagging length of 4 was used for dependent and dynamic regressors and optimal models were determined using the Akaike Information Criterion (AIC). After this stage, four diagnostic tests were used to verify the validity of the predicted models: Durbin-Watson for the first- degree autocorrelation detection, Breusch-Pagan-Godfrey for determining heterogeneity, Breusch-Godfrey LM for serial correlation detection and Ramsey RESET for determining the functionality of the model. The long-term relationship between variables was previously demonstrated by the bound test and supported by the negative and significant error correction coefficient (ECTt-1) given in Table 4. WLR test results showing the existence of a long-term asymmetrical relationship for each model were statistically significant. However, it was shown that the model established for lnsoybean had the problem (2) Where   + + = and   − − = , t+ and t−are the short- run adjustments towards positive and negative changes in , , , , t t t t t lnbarley lnmaize lnrice lnsoybean lnwheat . The NARDL model follows the same pathway for testing the null hypothesis ( 0    + − = = = ) of no cointegration against the alternative hypothesis ( 0    + − ≠ ≠ ≠) of cointegration. Model estimation b For FPSS the critical upper (lower) values are 5.00 (4.13), 3.87 (3.1), 3.35 (2.63) for 1, 5, and 10%, respectively. Table 3: Bound cointegration test s h o c k i n , , , , t t t t t lnbarley lnmaize lnrice lnsoybean lnwheat using the asymmetric dynamic multiplier effect on , , , , t t t t t lnbarley lnmaize lnrice lnsoybean lnwheat w i t h a percentage change in t lngfi + and t lngfi − expressed as (Equation 3): Model I-b: ( ) ( 0) 0 , , 0,1 ( ) h t j t j h h j h j t t lnY lnY m m h lngfi lngfi + + + − = + − = ∂ ∂ = ∑ = = ∂ ∂ ∑ (3) (3) Model I: t t t t lnY lngfi lngfi u   + + − − = + + (1) (1) As h→∞, h m+ → β+ and – h m → β⁻, where β+ and β⁻ represent the positive and negative long-run asymmetric coefficients. As h→∞, h m+ → β+ and – h m → β⁻, where β+ and β⁻ represent the positive and negative long-run asymmetric coefficients. where β+and β⁻ are the asymmetric long-run parameters to be estimated, and utdenotes the error process (i.e., deviations from the long run equilibrium relationship with a stationary zero-mean). Model estimation Unlike the Autoregressive Distributed Lag model (ARDL), which assumes that all external data series have a symmetrical effect on the dependent variable, the NARDL model suggests that there may be an asymmetric effect. Therefore, in the present study, the NARDL model developed by Shin et al. (2014) was used to examine the short- and long-term asymmetrical effect of COVID-19 on major staple crops prices. NARDL model predictions: t lnY lvariable denotes lnbarley, lnmaize, lnrice, lnsoybean and lnwheat series (Equation 1). Emir. J. Food Agric ● Vol 34 ● Issue 3 ● 2022 Emir. J. Food Agric ● Vol 34 ● Issue 3 ● 2022 242 Merve Ayyildiz Table 2: Unit root tests Table 2: Unit root tests Table 2: Unit root tests Level I (0) First difference I (1) ADF (C) ADF (C/T) PP (C) PP (C/T) ADF (C) ADF (C/T) PP (C) PP (C/T) lnbarley 0.57 ‑1.67 0.65 ‑1.92 ‑12.91 ‑13.02 ‑13.05 ‑13.14 lnmaize 0.08 ‑2.60 0.17 ‑2.66 ‑12.17 ‑12.21 ‑12.21 ‑12.17 lnrice ‑2.52 ‑2.56 ‑2.45 ‑2.38 ‑6.46 ‑6.47 ‑13.93 ‑13.94 lnsoybean ‑0.31 ‑2.18 ‑0.36 ‑2.39 ‑14.57 ‑14.54 ‑14.59 ‑14.56 lnwheat ‑0.04 ‑1.64 ‑0.18 ‑1.77 ‑14.93 ‑14.92 ‑14.96 ‑14.95 lngfi ‑4.76 ‑4.67 ‑3.42 ‑3.95 ‑‑‑ ‑‑‑ ‑30.19 ‑27.31 a At 1% significance level, when Augmented Dickey‑Fuller (ADF) and Phillips‑Perron unit root test statistics contained constant (C) and constant and trend (C/T), MacKinnon (1996) critical levels were ‑3.46 and ‑3.99, respectively. b Lagging lengths were determined automatically using Akaike Information Criterion for ADF and using Bartlett Kernel for PP. a At 1% significance level, when Augmented Dickey‑Fuller (ADF) and Phillips‑Perron unit root test statistics contained constant (C) and constant and trend (C/T), MacKinnon (1996) critical levels were ‑3.46 and ‑3.99, respectively. b Lagging lengths were determined automatically using Akaike Information Criterion for ADF and using Bartlett Kernel for PP a At 1% significance level, when Augmented Dickey‑Fuller (ADF) and Phillips‑Perron unit root test statistics contained constan MacKinnon (1996) critical levels were ‑3.46 and ‑3.99, respectively. ( ) , p y Lagging lengths were determined automatically using Akaike Information Criterion for ADF and using Bartlett Kernel for PP. Table 3: Bound cointegration test FPPS lnbarley=f (lngfı+, lngfı‑) 3.35* lnmaize=f (lngfı+, lngfı‑) 4.38* lnrice=f (lngfı+, lngfı‑) 3.32 lnsoybean=f (lngfı+, lngfı‑) 3.58 lnwheat=f (lngfı+, lngfı‑) 2.88* a*, , * denotes significance at 1, 5 and 10% level, respectively. RESULTS AND DISCUSSION The next step utilizes the Wald test to examine the long-run asymmetry (  + − = ) and short-run asymmetry ( ( 1) ( 1) ( 0) ( , ) ( 0) ( , ) q q j k i j k i   − + − − = = ∑ = ∑ ). Finally, we test the disequilibrium following a positive or negative 243 Emir. J. Food Agric ● Vol 34 ● Issue 3 ● 2022 Merve Ayyildiz Fig 1. Rubber stem girth of rubber as affected by sweet corn-okra intercropping a b c Table 4: Dynamic asymmetric estimates of Global Fear index effects with NARDL Y (lnbarley) Y (lnmaize) Y (lnrice) Y (lnsoybean) Y (lnwheat) lnYt‑1 ‑0.02 (0.01) ‑0.05* (0.01) ‑0.01 (0.01) ‑0.05 (0.02) ‑0.025 (0.01) lngfi+ 0.01 (0.00) 0.01 (0.01) 0.01 (0.00) 0.01 (0.01) 0.01 (0.00) lngfi‑ 0.01 (0.01) 0.01 (0.002) 0.01 (0.01) 0.01* (0.00) lngfi‑ t‑1 0.01 (0.00) ∆lnYt‑1 0.19* (0.06) 0.24*** (0.06) 0.09 (0.06) 0.11* (0.06) 0.12* (0.01) ∆lnYt‑2 0.15 (0.06) ∆lnYt‑3 0.11* (0.06) ∆lngfi+ ∆lngfi‑ ‑0.02* (0.01) c 0.09 (0.04) 0.20* (0.06) 0.24 (0.09) 0.12 (0.05) Ectt‑1 ‑0.02* (0.01) ‑0.05 * (0.01) ‑0.01* (0.00) ‑0.051* (0.01) ‑0.03* (‑0.03) Long‑run Asymmetric Effects lngfi+ 0.38 (0.16) 0.32* (0.09) 0.47 (0.53) 0.17 (0.08) 0.28* (0.15) lngfi‑ 0.30 (0.15) 0.21 (0.09) 0.45 (0.52) 0.07 (0.08) 0.23 (0.14) c 4.61* (0.05) 4.53 * (0.04) 4.84* (0.22) 4.60* (0.03) 4.64* (0.06) Error Metrics R2 0.99 0.99 0.97 0.99 0.99 Adj. R2 0.99 0.99 0.97 0.99 0.99 DW 2.04 1.94 1.99 1.98 1.84 Merve Ayyildiz Fig 1. Rubber stem girth of rubber as affected by sweet corn-okra intercropping a b c Merve Ayyildiz Fig 1. Rubber stem girth of rubber as affected by sweet corn-okra intercropping a b c Merve Ayyildiz Merve Ayyildiz a b a c Fig 1. gi , , p y b ( ) is the standard error, χ2 H B‑P‑G refers to Heteroskedasticity Test: Breusch‑Pagan‑Godfrey; χ2 SCLM represents Breusch‑Godfrey Serial Correlation LM Test; χ2 FF means Ramsey RESET Test; and WLR refers to Wald test of the additive Long‑term symmetry condition. [ ] values represent probability values of diagnostic tests RESULTS AND DISCUSSION Rubber stem girth of rubber as affected by sweet corn-okra intercropping 244 Emir J Food Agric ●Vol 34 ●Issue 3 ●2022 Table 4: Dynamic asymmetric estimates of Global Fear index effects with NARDL Y (lnbarley) Y (lnmaize) Y (lnrice) Y (lnsoybean) Y (lnwheat) lnYt‑1 ‑0.02 (0.01) ‑0.05* (0.01) ‑0.01 (0.01) ‑0.05 (0.02) ‑0.025 (0.01) lngfi+ 0.01 (0.00) 0.01 (0.01) 0.01 (0.00) 0.01 (0.01) 0.01 (0.00) lngfi‑ 0.01 (0.01) 0.01** (0.002) 0.01 (0.01) 0.01* (0.00) lngfi‑ t‑1 0.01 (0.00) ∆lnYt‑1 0.19* (0.06) 0.24*** (0.06) 0.09 (0.06) 0.11* (0.06) 0.12* (0.01) ∆lnYt‑2 0.15 (0.06) ∆lnYt‑3 0.11* (0.06) ∆lngfi+ ∆lngfi‑ ‑0.02* (0.01) c 0.09 (0.04) 0.20* (0.06) 0.24 (0.09) 0.12 (0.05) Ectt‑1 ‑0.02* (0.01) ‑0.05 * (0.01) ‑0.01* (0.00) ‑0.051* (0.01) ‑0.03* (‑0.03) Long‑run Asymmetric Effects lngfi+ 0.38 (0.16) 0.32* (0.09) 0.47 (0.53) 0.17 (0.08) 0.28* (0.15) lngfi‑ 0.30 (0.15) 0.21 (0.09) 0.45 (0.52) 0.07 (0.08) 0.23 (0.14) c 4.61* (0.05) 4.53 * (0.04) 4.84* (0.22) 4.60* (0.03) 4.64* (0.06) Error Metrics R2 0.99 0.99 0.97 0.99 0.99 Adj. R2 0.99 0.99 0.97 0.99 0.99 DW 2.04 1.94 1.99 1.98 1.84 AIC ‑7.51 ‑6.60 ‑8.04 ‑6.38 ‑7.10 Diagnostic Tests χ2 H B‑P‑G 0.98[0.43] 1.19[0.32] 6.45[0.00] 4.10[0.01] 1.25[0.29] χ2 SCLM 0.84[0.43] 0.67[0.51] 0.03[0.98] 1.99[0.14] 1.37[0.26] χ2 FF 0.38[0.70] 0.92[0.36] 0.11[0.91] 1.76[0.08] 1.26[0.21] WLR 3.24* 3.66*** 1.70* 2.45 2.69 a*, , * denotes significance at 1, 5 and 10%, respectively. b ( ) is the standard error, χ2 H B‑P‑G refers to Heteroskedasticity Test: Breusch‑Pagan‑Godfrey; χ2 SCLM represents Breusch‑Godfrey Serial Correlation LM Test; χ2 FF means Ramsey RESET Test; and WLR refers to Wald test of the additive Long‑term symmetry condition. [ ] values represent probability values of diagnostic tests. Table 4: Dynamic asymmetric estimates of Global Fear index effects with NARDL Emir. J. Food Agric ● Vol 34 ● Issue 3 ● 2022 244 Merve Ayyildiz is another issue that significantly affected market prices of corn (FAO, 2020). On the other hand, corn prices on a global scale varied depending on high production expectations and rapid stock increases in the U.S. and Brazil, and the quota and tax policies of exporting countries. The effect of oil prices on corn prices was also very clear during this period. Corn, which is widely used in ethanol production, reacts quickly to the change in energy prices arising from energy demand. RESULTS AND DISCUSSION In other words, barley prices reacted to the positive (negative) effects in the global fear index by increasing (decreasing), and it can be stated that the effect of an increase in the global fear index on prices was greater than the that of decrease (lngfi+ = 0.376 > lngfi- = 0.297). As Lock (2021) pointed out, global barley prices during this period were shaped largely by export controls imposed by the Russian Federation and Argentina and as a result of China›s high demand. Therefore, it could be stated that the price volatility in the barley market was a result of the uncertainty caused by COVID-19. Accordingly, the findings that similar price fluctuations could occur in global markets during periods of increased or decreased fear and panic were consistent with the literature. It was shown that the global fear index affected corn prices asymmetrically in the long term. Accordingly, it was concluded that a 1% increase in the global fear index (lngfi+) represented by COVID-19 could increase corn prices by 0.32%, while a decrease of 1% (lngfi-) could reduce corn prices (lnmaize) by 0.21%. This indicated that corn prices reacted to the positive (negative) effects in the global fear index by increasing (decreasing). Similar to barley prices, the effect of an increase in the global fear index on corn prices was larger than the effect of a decrease effect. In recent years, approximately 90% of the world’s corn exports have been made by Argentina, Brazil, the USA and Ukraine, and changes in the supply, demand and trade of these countries have significantly affected the world’s corn prices. In the early stages of the pandemic, restriction measures taken in countries and around the world caused structural shocks in demand. The recession in the services sector due to restrictions such as quarantine led to contractions in the livestock and feed sectors, directly affecting corn prices in many countries (Neroba, 2020). As in barley, the fact that China is a major importer of corn It is known that uncertainty in economies is a significant pressure on prices, which was clearly shown with global wheat, barley and corn prices in the present study. In fact, the results of the present study were in line with the predictions put forward by Daglis et al. (2020), Karagöl et al. (2021) Laborde et al. RESULTS AND DISCUSSION During COVID-19 process, the contraction in ethanol demand, especially in the United States, continued to put pressure on corn prices (Elleby, 2020; Mizik et al., 2020; Neroba, 2021; Sun et al., 2021). of heterogeneity, and the hypothesis that the functionality of the model is valid was rejected at 10% significance level. At the same time, in the model established for lnrice, due to the heterogeneity in the model, the hypothesis that there is no heterogeneity between the variables was rejected. Considering the descriptive tests, the validity of the NARDL models established for lnrice and lnsoybean was found to be questionable and no evaluation was made. In addition, Figure 1(a), 1(b), 1(c) describes cumulative summary tests for NARDL models established for lnbarley, lnmaize and lnwheat, respectively. Accordingly, CUSUM plots show the stability of models estimated at the 95% confidence interval. Unlike barley and corn prices, the effect of the decrease in the global fear index (lngfi-) on wheat prices (lnwheat) was not statistically significant. Therefore, it can only be stated that a 1% increase in the global fear index (lngfi+) had an increasing effect of 0.28% on wheat prices (lnwheat). Wheat export leader Russia appeared to have intervention power in the market, and its tax and quota policies and customs controls on exports resulted in price increases. But the growing demand for Australian and Argentine wheat increased the competition in the markets and balanced the rapid price increases. On the other hand, the increase in wheat demand in global markets, especially the high wheat demands of China and Pakistan in the last 15 years, has played an important role in the price increases. With the pandemic process, the increase in wheat demand has caused many countries to increase their wheat stocks under the current uncertainty and risk conditions. This has created the perception that prices will remain high in international markets (Anonymous, 2021). It can also be stated that strong price increases in corn has contributed to upward movement as wheat becomes more attractive economically for feed rations (Agricultural Market Information System (AMIS), 2021). In the long-term asymmetric relationship between the global fear index and the barley price, it was found that a 1% increase in the fear index (lngfi+) could lead to a 0.38% increase in the barley price (lnbarley) and a 1% decrease could lead to a 0.30% decrease in the price of barley (lngfi-). REFERENCES AMIS. 2021. AMIS Market Monitor, No. 88. Agricultural Market Information System. Anonymous. 2021. Urun Masalari Bugday Bulteni. Available from: https:// www.tarimorman.gov.tr/BUGEM/Belgeler/%C3%BCltenler/ OCAK%202021/Bu%C4%9Fday%20Ocak%20B%C3%BClteni. pdf [Last accessed on 2021 May 04]. Bai, L., Y. Wei, G. Wei, X. Li and S. Zhang. 2021. Infectious disease pandemic and permanent volatility of international stock markets: A long-term perspective. Finance Res. Lett. 40: 101709. Beckman, J. and A. M. Countryman. 2021. The importance of agriculture in the economy: Impacts from COVID-19. Am. J. Agric. Econ. 103: 1595-1611. In its production forecasts report released in 2021, the International Grain Council stressed that not many problems appear in the supply side but that demand pressure will greatly affect the course of the prices. The trend of many countries to increase stocks due to uncertainty created by the pandemic indicated possible long-term volatility in the global wheat, corn and barley trade. The fact that importers increased their imports considerably through tax breaks while exporter countries made major changes in export quotas and taxation and the signal of a decrease in export amounts fueled the trend of price increases. This led to increased speculative activity in the stock markets and strengthened the commitment of the financial and agricultural markets. Beckman, J., F. Baquedano and A. Countryman. 2021. The impacts of COVID-19 on GDP, food prices, and food security. Q Open. 1: 1-17. Borgards, O., R. L. Czudaj and T. H. Van Hoang. 2021. Price overreactions in the commodity futures market: An intraday analysis of the covid-19 pandemic impact. Res. Policy. 71: 101966. Cariappa, A. G. A., K. K. Acharya, C. Adhav, R. Sendhil and P. Ramasundaram. 2021. Impact of COVID-19 induced lockdown on wheat prices: Empirical evidence from interrupted time series analysis. Ceylan, R. F., B. Ozkan and E. Mulazimogullari. 2021. Historical evidence for economic efects of COVID‑19. Eur. J. Health Econ. 21: 817-823. Clapp, J. and W. G. Moseley. 2020. This food crisis is different: COVID-19 and the fragility of the neoliberal food security order. J. Peasant Stud. 47: 1393-1417. The pressure of the COVID-19 process on cereal prices remains to be strong. It was found in the present study that the changes in COVID-19 related cases and deaths affected barley, corn and wheat prices asymmetrically in the long term. The effect of an increase in the Global fear index on prices was higher than that of a decrease. Daglis, T., K. N. Konstantakis and P. G. Michaelides. RESULTS AND DISCUSSION (2021) that the increasing spread rate of the pandemic may lead to an increase in agricultural commodity prices. In addition, the findings of the present study also lent support to the conclusion that export restrictions may affect more than targeted commodities through cross-commodity price links for wheat, barley and corn prices reached by Gutierrez and Pierre (2020) using the Global Vector Auto Regression (GVAR) model. Generally, the number of leading exporter countries in wheat, barley and corn exports in world trade is small, but their total share in the market exceeds 80%. The findings of the present study clearly indicate that olypolistic structure has the Emir. J. Food Agric ● Vol 34 ● Issue 3 ● 2022 245 Merve Ayyildiz power to determine world prices. The fact that COVID-19 uncertainty has led to asymmetrical effect on prices in the long term and that the effect of an increase in the fear index on prices is higher than the effect of a decrease could be explained by the fact that negative shocks are frequently more influential than positive shocks in oligopoly markets. the sudden changes in the prices of these agricultural commodities, which are important for food security, can cause major problems in the economies of importing countries, especially the low-income ones. In this context, it is very important to ensure price stability in the market. Accordingly, the analysis and predictions taking into account the asymmetrical effect could help reduce the volatility in cereal prices. In addition, the uncertainty brought about by COVID-19 pandemic is thought to have a greater impact on prices than supply and demand shocks. Therefore, trade policies to be developed taking into account the asymmetrical effect revealed by this study which would guarantee the dynamic circulation of grains, financing methods to facilitate trade for developing countries, and the search for solutions to adapt climate and environmental risks to the grain supply chain are predicted to have considerable, stabilizing effects on the prices in global grain markets. CONCLUSION The COVID-19 pandemic has had a significant impact on agricultural markets on a global scale. In this process, decreased energy prices as a result of lower energy demand led to major changes in the grain sector through reasons such as increased concerns about food safety, changes in consumer behavior, increase in investments in digital supply chains, decrease in global feed demand, return to the globalization trend in supply chains and increase in government interventions, changes in quotas and tax policies in foreign trade. This puts pressure on cereal prices. Therefore, the change in prices and the direction of this change are closely related to the fear and panic brought about by the pandemic. It can be concluded that exporting countries in particular are very effective in guiding the world prices. 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https://openalex.org/W4304127990	https://repozitorij.uni-lj.si/Dokument.php?id=163287&dn=	English	null	Effectiveness of the Overtaking Ban for Heavy Vehicles on the Four-Lane Divided Highway in Different Weather Conditions	Applied sciences	2,022	cc-by	11,459	applied sciences applied sciences applied sciences Citation: Rijavec, R.; Marsetiˇc, R.; Strnad, I. Effectiveness of the Overtaking Ban for Heavy Vehicles on the Four-Lane Divided Highway in Different Weather Conditions. Appl. Sci. 2022, 12, 10169. https:// doi.org/10.3390/app121910169 Academic Editors: Javier Alonso Ruiz and Angel Llamazares Keywords: highway trafﬁc management; heavy vehicles trafﬁc control; vehicle exclusion; lane ﬂow distribution; lane speed distribution; weather conditions 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. Robert Rijavec * , Rok Marsetiˇc and Irena Strnad * Trafﬁc Technical Institute, Faculty of Civil and Geodetic Engineering, University of Ljubljana, 1000 Ljubljana, Slovenia * Correspondence: robert.rijavec@fgg.uni-lj.si (R.R.); irena.strnad@fgg.uni-lj.si (I.S.); Tel.: +386-1-476-8570 (R.R. Abstract: In many European countries and also in Slovenia, the highway network was rapidly built in order to reduce congestion and to increase the level of trafﬁc safety on congested sections of the road network, thus enabling a higher level of service and accelerating polycentric development. Unfortunately, trafﬁc demand is growing over all limits, be it tourist car trafﬁc or transit-heavy vehicle trafﬁc. Thus, countries are forced to actively manage road freight trafﬁc, which is present all year round. Accordingly, in Slovenia, permanent and timed restrictions were introduced for trucks regarding overtaking on highways. Overtaking is prohibited during the day but trucks are allowed to change lanes at night. It should be noted, however, that there may be circumstances that can restrict the normal travel of heavy vehicles in all lanes in one way or another, whether at night or during the day. We would like to convince highway trafﬁc managers that weather-responsive adaptive trafﬁc control could be more efﬁcient when weather conditions are considered. This article presents an approach to simulate trafﬁc ﬂow on a short section of a two-lane unidirectional carriageway under various weather conditions. Using two scenarios for lane trafﬁc control, i.e., with and without a truck overtaking ban, as examples, we show that knowledge of the trafﬁc characteristics of each lane in different weather conditions is important for decision-making and for the timeliness of trafﬁc management. We found that under certain trafﬁc and weather conditions, prohibiting vehicles from overtaking with limited speed limits on four-lane divided highways or proper trafﬁc lane control has a positive effect on the trafﬁc ﬂuency or available conditional capacity of the highway. To some extent, this conﬁrms that the decision of the operator of the Slovenian highway system regarding the driving regime for heavy vehicles was correct. Through our research, we found that dynamic bans can be more effective when we include the dynamics of trafﬁc demand, and environmental and weather conditions. Article Effectiveness of the Overtaking Ban for Heavy Vehicles on the Four-Lane Divided Highway in Different Weather Conditions Robert Rijavec * , Rok Marsetiˇc and Irena Strnad * 1. Introduction and Background Due to its geostrategic location, Slovenia is one of the European transit countries. Two important trans-European corridors (road and rail) run through Slovenian territory: the Mediterranean corridor (Seville–Barcelona–Venice–Ljubljana–Budapest) and the Baltic– Adriatic corridor (Ravenna–Venice–Graz–Brno–Gdansk). Thus, signiﬁcant incidents and high trafﬁc densities along these corridors affect not only trafﬁc ﬂows in the country where they occur, but also trafﬁc ﬂows in neighbouring EU countries: Italy, Austria, Hungary, and Croatia. 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/). Driver behaviour cannot be predicted with certainty, although drivers do behave predictably to some degree, with certain patterns that can be experientially identiﬁed and used in a feedback loop to modify input data to generate trips. In this case, trafﬁc, also called trafﬁc demand, encounters various forms of resistance that determine how it is distributed across the road network [1,2]. https://www.mdpi.com/journal/applsci Appl. Sci. 2022, 12, 10169. https://doi.org/10.3390/app121910169 Appl. Sci. 2022, 12, 10169 2 of 17 2 of 17 It is understandable that AHTMSs (Active Highway Trafﬁc Management Systems) are effective, but not always as desired or intended by the trafﬁc control theory [3,4]. Therefore, it is necessary to look for new approaches to encourage drivers to adopt consistent and safe trafﬁc behaviour [5]. Signiﬁcant changes are expected in this area in the near future, as the use of Advanced Vehicle Assistance Systems (ADASs) will enable drivers to more easily understand what the road infrastructure and other vehicles are telling them at any vehicle location and not just at the location of Variable Message Signs (VMSs). Indeed, the next generation of vehicle data collection technologies will be able to provide information about the lanes vehicles are using, which is currently only a wish of the systems mentioned above. Various factors inﬂuence the capacity utilisation of the highway network. The choice of lane is one of the trafﬁc factors that affect the capacity utilisation of the directional lane of a highway. Lane change areas and the modelling of this phenomenon are a challenge for trafﬁc analysts as well as for autonomous vehicle developers [6–8]. 1. Introduction and Background Generally, drivers change lanes due to differences in speed, such as to overtake slower vehicles, due to obstacles in the lane they are using, or the selﬁshness of certain drivers who “keep the left lane for themselves” [9]. Lane-changing trafﬁc ﬂow can be characterised by lane-changing models [10,11]. The empirical models described by Wu [12] are more or less based on Daganzo’s model of road trafﬁc behaviour [13]. Considering trafﬁc ﬂow rate q and density k, the lane distribution of the two characteristics, i.e., LFRi and LDRi, is the ratio between the corresponding value qi and ki on the trafﬁc lane i and the total for the overall carriageway qtot and ktot [14]. Mathematical models usually specify the number of lane changes [15]; however, in practice, we are not very good at imagining the latter because we cannot yet track individual vehicles. A higher frequency of lane changes results in a greater general heterogeneity of highway trafﬁc ﬂow. When we talk about AHTMS and real-time road trafﬁc information systems, the reliability of the data and the corresponding robust short-term forecast play a very important role [16]. Prediction is usually based on models that learn on the basis of the characteristic patterns of trafﬁc conditions given by various characteristics of trafﬁc ﬂow in time and space. Their importance is related to typical events in the past, which remain relevant until the trafﬁc demand is known in advance and this will be determined only by the announcement of the arrival of an individual vehicle in the future. The usual presence of different categories of vehicles is reﬂected in the heterogeneity of the trafﬁc ﬂow structure. The degree of heterogeneity in Slovenia is currently expressed by the share of heavy vehicles in the trafﬁc ﬂow in an individual trafﬁc lane of a directional carriageway. Regardless of how the trafﬁc detection sensors classify vehicles into different categories, such as based on vehicle type, number of axles, or gross weight, all types of heavy vehicles with a Maximum Authorised Mass (MAM) exceeding 3.5 t are classiﬁed as heavy vehicles. In some other countries of the world, heterogeneity is complemented by the presence of a share of tourist or recreational vehicles, e.g., vehicles with tourist trailers. Greater heterogeneity results in worse conditions for the movement of vehicles in the trafﬁc ﬂow, which is particularly noticeable on uphill and downhill slopes [17]. 1. Introduction and Background Different vehicles have different driving and dynamic capabilities; therefore, they can be treated separately within trafﬁc control. Changing lanes is also inﬂuenced by weather or environmental conditions [18,19]. Effective operation of AHTMSs at the strategic and operational levels requires knowledge of the effects of different weather conditions on trafﬁc ﬂow [20]. Weather phenomena, such as rain and snow, fog, and other adverse environmental factors that affect the visibility and stability of vehicles, alter conditions that affect highway driving dynamics and capacity [17,21,22]. Event forecasts, including weather and trafﬁc advisories, can affect trafﬁc demand [16,17,23], resulting in reduced trafﬁc ﬂow and improved road capacity utilisation. p p y The inﬂuence of weather and meteorological conditions on the roadway on trafﬁc is also a well-researched area in Slovenia, especially in terms of the type and intensity of precipitation (rain or snow), temperature, and visibility conditions [24]. Somewhat less well- researched are wind and combinations of weather phenomena, as well as other phenomena Appl. Sci. 2022, 12, 10169 3 of 17 3 of 17 that are very difﬁcult to measure or monitor regionally such as hail and extreme winds. The impact of forecasts or other trafﬁc and travel information is even less researched [25,26]. We do not know for sure whether the impacts are the result of a lower trafﬁc ﬂow because vehicle speeds are reduced and drivers travel with a greater safety distance, whether they affect trafﬁc demand (e.g., drivers do not make the trip), or whether it is a coincidence due to an extraordinary event, perhaps a road closure for a particular vehicle type at a different location. We know from experience that meteorological conditions on the road and weather phenomena affect the speed of trafﬁc ﬂow. Regardless of trafﬁc conditions and the mutual inﬂuence of vehicles, they affect trafﬁc demand and road capacity. Various studies report that the trafﬁc ﬂow rate on highways is lower due to poor weather conditions than under good conditions, i.e., dry weather and good visibility [22,24,27–29]: in light precipitation (rain, snow) from 5% to 10%; in heavier precipitation up to 15%; in heavy snowfall from 30% to 65%. 1. Introduction and Background At free-ﬂow trafﬁc and in light precipitation (light rain or snow), the vehicle speed is reduced by up to 5% compared to up to 10% in medium precipitation intensity (rain), and up to 15% in heavy rain; in limited visibility, speed is reduced between 10% and 15%, and by up to 60% in exceptional cases; in heavy snowfall, it is reduced by up to 30%, and by up to 60% in some exceptional cases. These are general research results, usually related to a heterogeneous trafﬁc ﬂow, where all vehicles are included in the trafﬁc characterisation. In the mass of research conducted, there are not many ﬁndings on the impacts on heavy vehicles, the impacts that are local in nature and related to the geometric characteristics of the road, and the impacts on the characteristics of trafﬁc ﬂow along individual trafﬁc lanes. Therefore, as a contribution to the empirical research, this study evaluated the impact of weather conditions on the speed of trafﬁc ﬂow of light and heavy vehicles in the case of Slovenia. Many researchers have attempted to answer the question of how trafﬁc ﬂow structure and ambient illumination affect the distribution of trafﬁc across lanes. In particular, the approach to determine the probability that a lane change occurs due to an overtaking manoeuvre on the highway, which can be a conﬂict manoeuvre before a trafﬁc accident, was investigated [30]. This manoeuvre is also inﬂuenced by other trafﬁc conditions [31,32], as well as the environment [18]. These researchers all determined how the factors of day and night affect the distribution of vehicle ﬂow in lanes or, as one could also say, the overtaking of vehicles on a unidirectional multi-lane carriageway. They found that, at night, drivers use only one lane, which is understandable and mostly related to the low trafﬁc volume (trafﬁc demand). In adverse weather conditions, drivers are signiﬁcantly more reluctant and careful when changing lanes. They take more time to make decisions [33], which can have a corresponding positive or negative effect on the capacity utilisation of the carriageway. The AHTMS currently determines the occurrence of trafﬁc instability through a cal- culation of the standard deviation of vehicle speeds at a given equivalent trafﬁc ﬂow rate within the measuring time interval in the carriageway and overtaking lane—also referred to as the “left lane” or “lane 2” [34–36]. 1. Introduction and Background • Advise drivers to drive according to the driving rules, ﬁrst in the right lane and then in the overtaking lane, when such a manoeuvre is required (e.g., message “left lane: overtaking only” for the case of the right-hand trafﬁc rules). Common problems related to efﬁciency in some EU countries have already been addressed [35,38], but Slovenia and some other similar countries in the region are charac- terised by heterogeneity and multiculturalism of drivers and rapidly changing weather conditions. There is a research gap in understanding how lane trafﬁc control is affected by different weather conditions. A summary of the empirical research is presented in Section 2. On the basis of the data from the test sites, the trafﬁc ﬂow characteristics were determined by trafﬁc lanes. We performed pre-processing and veriﬁcation of input data collected from various trafﬁc sensors and Road Weather Stations (RWS). Since we were interested in the lane distribution of trafﬁc ﬂows during the period when the trafﬁc ﬂow was not yet condensed, we excluded some data from the analysis. Thus, we excluded all data from the periods of peak load and the periods when congestion occurred. Similar pre-processing was also performed on RWS environmental data. Following the time code, we joined trafﬁc data and road weather data into a common database that formed the basis for mathematical modelling of the effects of trafﬁc lane control. We assert that, with adequate and timely knowledge of the lane distribution trafﬁc ﬂow characteristics, we can detect trafﬁc instabilities that may cause trafﬁc conﬂicts and collisions. Then, we act ac- cordingly, e.g., with overtaking bans for trucks. With appropriate measures of active trafﬁc control of HGV in different weather conditions, we can improve the efﬁciency of trafﬁc management. As we can inﬂuence trafﬁc ﬂow in this way, we can achieve higher trafﬁc ﬂow rates or better lane utilisation, thus reducing delays, which we demonstrated by using a microsimulation trafﬁc model of a two-lane unidirectional carriageway highway section. This is discussed in Section 3. The results of the impact of heavy vehicle trafﬁc control are presented in Section 4. This section also includes a discussion comparing the delays in trafﬁc ﬂow in the case of a truck overtaking ban or, if this measure is not implemented, in different weather conditions. 1. Introduction and Background Lastly, Section 5 offers conclusions and discusses the ﬁndings of the study by presenting some directions for further research on this subject. 1. Introduction and Background This happens when there are vehicles in the overtaking lane with high speeds that are well above the time mean speed. Another reason for the occurrence of such a trafﬁc situation relates to slower vehicles, usually Heavy Goods Vehicles (HGV). In European countries, the maximum speed on highways is set administratively and is different for heavy vehicles than for light vehicles. On multi-lane highways, this phenomenon is manifested by a constant change of trafﬁc lanes or a change in the lane distribution of trafﬁc ﬂow rates, as well as a change in the density and speed distribution across the lanes [15]. To prevent or mitigate the consequences of such trafﬁc ﬂow, the following trafﬁc control measures in trafﬁc lanes can be used: • Prohibit the driving of slow vehicles (usually heavy trucks and buses) in the overtaking lane, introduce a combination of “minimum speed” trafﬁc signs in individual lanes (e.g., 110 km/h in the left lane—lane 2 and 80 km/h in the right lane—lane 1), or install “no overtaking by HGVs” trafﬁc signalisation [37]; • Prohibit the driving of slow vehicles (usually heavy trucks and buses) in the overtaking lane, introduce a combination of “minimum speed” trafﬁc signs in individual lanes (e.g., 110 km/h in the left lane—lane 2 and 80 km/h in the right lane—lane 1), or install “no overtaking by HGVs” trafﬁc signalisation [37]; Appl. Sci. 2022, 12, 10169 4 of 17 • Additionally, display the limit of the generally permitted speed as a preventive mea- sure or limit the speed on the carriageway by one level; • Additionally, display the limit of the generally permitted speed as a preventive mea- sure or limit the speed on the carriageway by one level; • Advise drivers to adjust their speed to the trafﬁc ﬂow; the AHTMS uses arrow signals on VMSs to encourage the same direction of travel without changing lanes and, if necessary, set a minimum speed limit for each lane; • Advise drivers to drive according to the driving rules, ﬁrst in the right lane and then in the overtaking lane, when such a manoeuvre is required (e.g., message “left lane: overtaking only” for the case of the right-hand trafﬁc rules). 2. Trafﬁc Characteristics and Weather in Slovenia Slovenia is a very geographically diverse country, located in Central Europe, part of the EU and part of the Schengen area [39]. In addition to the high share of transit freight trafﬁc, during the tourist season, there is a large ﬂow rate of passenger cars from Central Europe towards the Adriatic and vice versa, especially in recent times following changes in travel habits due to the world facing the COVID-19 pandemic [40]. The highway network runs both through the mountainous region, where the roadway is most exposed to snowfall and ice, and through the Mediterranean belt, where a strong wind called Burja has the strongest inﬂuence. Due to its geographical diversity, it is difﬁcult to accurately predict weather conditions in Slovenia. Different weather phenomena dictate adapted driving, but problems most often arise in the trafﬁc control of trucks and buses in transit. Trafﬁc safety is particularly at risk on road sections where weather conditions change very rapidly or where combined adverse weather phenomena occur frequently (e.g., big storms with snow drifts, heavy precipitation with poor visibility, and fog). Trafﬁc control operators generally advise drivers to drive more carefully. At the moment, for a greater level of safety in bad weather conditions, the following trafﬁc management measures are implemented: restrictions, detours and exclusion of trucks and buses, restrictions of sudden changes of direction when changing lanes or restrictions on overtaking, speed control, and information on alternative routes. Most highways are Appl. Sci. 2022, 12, 10169 5 of 17 5 of 17 four-lane with an AADT of up to 90,000 vehicles/day, of which up to 9000 are heavy vehicles/day [41]; however, on certain highways, trafﬁc volumes are often 2–3 times higher during peak seasons, and the same is true for HGVs on peak days such as Mondays and Thursdays. The trafﬁc manager, the concessionaire The Motorway Company in the Republic of Slovenia (Slovenian: Družba za avtoceste v Republiki Sloveniji, DARS), has set up an AHTMS with a network of trafﬁc sensors providing aggregated trafﬁc data in trafﬁc lanes with a measurement interval of T = 1 min and T = 5 min. Similarly, data on road weather conditions are available from the Road Weather Information System (RWIS). Environmental weather data from the RWIS were recorded in the time interval of T = 5 min. 2. Trafﬁc Characteristics and Weather in Slovenia We focused on test site 0178 with a high trafﬁc ﬂow density near Ljubljana (N46.035, E14.452) and test site 0865 in the Vipava Valley (N45.855, E13.945), where strong winds often occur. To investigate the characteristics of trafﬁc ﬂow, we joined trafﬁc ﬂow and environmental records into a single database and presented them in time intervals of T = 5 min as a part of a separate survey [19], i.e., before the COVID-19 pandemic. We should point out that the pandemic situation in the year 2020 signiﬁcantly changed trafﬁc ﬂow patterns in Slovenia. Labels of groups of time-related environmental data were assigned to the selected characteristics of trafﬁc ﬂow, i.e., afﬁxes to labels of _d for dry road, no precipitation, and good visibility, _w1, _w2, and _w3 for wet road and rain at the same time (of three levels), _lv for low visibility, but no precipitation, and _s for snowfall and winter road conditions. Group _d included data based on measurements of ideal or prevailing weather conditions, deﬁned according to [42]. Trafﬁc ﬂow characteristics were classiﬁed into groups _w1, _w2, and _w3 according to the rainfall intensity and road wetness [36]. In group _lv, we classiﬁed trafﬁc characteristics at the time when visibility was lower than 250 m, regardless of meteorological conditions on the roadway. Group _s was deﬁned on the basis of data from the RWS, which uses a speciﬁc algorithm to classify precipitation and detect light, moderate, and heavy snowfall and determines conditions on the roadway surface using built-in road surface sensors [25]. In winter conditions, we ﬁltered the data from the database that correspond to slippery, icy, or snowy road conditions. Thus, on a corresponding homogeneous short carriageway section, we calculated trafﬁc ﬂow density ki,c in trafﬁc lane i = {1,2} under weather conditions c = {_d, _w1, _w2, _w3, _lv, _s} and analysed the “classic” relationships of density–speed and ﬂow–speed. An example of the analysis at test site 0178 on highway A1 near Ljubljana is presented in Figures 1 and 2. Figure 1. Example of scatter graph of time mean speed vs. trafﬁc density on the carriageway near Ljubljana in different trafﬁc lanes: (a) lane 2—left (overtaking) lane; (b) lane 1—right lane. Figure 1. Example of scatter graph of time mean speed vs. trafﬁc density on the carriageway near Ljubljana in different trafﬁc lanes: (a) lane 2—left (overtaking) lane; (b) lane 1—right lane. Appl. Sci. 2. Trafﬁc Characteristics and Weather in Slovenia 2022, 12, 10169 6 of 17 Figure 2. Example of scatter graph of time mean speed vs. trafﬁc ﬂow rate on the carriageway near Ljubljana in different trafﬁc lanes: (a) lane 2—left (overtaking) lane; (b) lane 1—right lane. Figure 2. Example of scatter graph of time mean speed vs. trafﬁc ﬂow rate on the carriageway near Ljubljana in different trafﬁc lanes: (a) lane 2—left (overtaking) lane; (b) lane 1—right lane. In any case, we found out from the literature review and experience with known events in unstable trafﬁc ﬂow that, at an increased total trafﬁc ﬂow rate qtot,c, the ﬂow rate in the left lane q2,c is higher than in the right lane q1,c. This conﬁrms the ﬁnding about the unused capacity of lane 1 (LFR2 > LFR1), where the speeds are lower [12,14,18]. It is also interesting to note that this difference varies depending on weather conditions [19], which means that it makes sense to consider it as a decision-making criterion in active trafﬁc lane control. Differences in the trafﬁc ﬂow density ∆kc occurred at the maximum measured ﬂows. In good weather conditions, the density was lower in lane 1 (k1 < k2), while it was lower in the overtaking lane (k2 < k1) in adverse weather conditions. In addition to the above-mentioned trafﬁc ﬂow characteristics, we empirically analysed the remaining characteristics on the basis of the ﬁndings [15,43], which allowed us to perform a microsimulation in the next step to substantiate the appropriateness of the decision to not permit HGV to change trafﬁc lanes. Trafﬁc Flow Speed Analysis In order to further analyse the results and evaluate the measures taken to control HGVs, we also determined the empirical speed distribution functions of light and heavy vehicles at mentioned test sites near Ljubljana. We considered the vehicle categorisation of the current AHTMS, which groups vehicles into light and heavy vehicles [36]. At the same time, in the years before the COVID-19 pandemic, there were no special administrative limits or overtaking bans for HGVs. Figure 3a shows the results of the speed analysis for the example of test site 0178 for lane 2 for passenger cars (_pc), and Figure 3b for lane 1. Similar results are shown in Figure 3c,d for lanes 1 and 2 for heavy vehicles (_hv). For test site 0865 in the Vipava Valley, we also investigated the inﬂuence of wind on the speed V of all vehicles (Figure 4). We determined four empirical distribution functions of speed V as a function of four types of AHTMS measures with respect to wind strength for trafﬁc lane i. Wind strength was expressed not by the magnitude of some other statistical characteristic value (e.g., the maximum speed of wind gusts), but by the trafﬁc management measure Zi for a speciﬁc vehicle category [36]. Z0 means that the wind does not require any special trafﬁc management measures. An example of speed analysis for test point 0865 is shown in Figure 4. Nevertheless, in brackets, we still present the informative values of maximum wind gust speeds for individual bans or trafﬁc management measures [44]: • Z1: exclusion for camper vans, refrigerator vehicles, and sheeted vehicles with the MAM of 8 t (wind from 80 km/h to 99 km/h); • Z2: exclusion for camper vans, all sheeted vehicles, and refrigerator vehicles (wind from 100 km/h to 129 km/h); Appl. Sci. 2022, 12, 10169 7 of 17 • Z3: exclusion for camper vans, all Z2 level vehicles, and buses (wind from 130 km/h to 149 km/h); • Z4: ban for all vehicles—road closure (wind over 150 km/h). Figure 3. Empirical speed distribution functions for different weather conditions at test site 0178: (a) passenger cars in lane 2; (b) passenger cars in lane 1; (c) heavy vehicles in lane 2; (d) heavy vehicles in lane 1. 3. Modelling the Efﬁciency of Trafﬁc Lane Control and Weather Conditions A hypothetical simulation model was created in the PTV Vissim software environment. Using the Wiedemann 99 trafﬁc model, we set up a 2 km long unidirectional highway carriageway link. The scheme is shown in Figure 5, where the positions of the cross- sections P1 and P2 are marked, along with the virtual simulation test points, such as sensor loops {K1, K2, K3, K4} on the individual trafﬁc lanes corresponding to the individual trafﬁc lane i = {1—right lane; 2—left (overtaking) lane} and measuring points {K5, K6} on the carriageway. The measuring points at the cross-sections were used to observe the lane distribution of trafﬁc ﬂow. The ﬁrst 600 m of the model was intended to establish a stable trafﬁc ﬂow, the next 1200 m was intended for measurements, and another 200 m was intended for exit vehicles from the simulation model of the road network. In the section between P1 and P2, there were link control sensors T1 and T2 on each lane, from which we obtained the values of trafﬁc ﬂow characteristics on the 1000 m long section, separately by trafﬁc lanes and for the “average” lane. The “average” lane represents the average value of the simulation results of both trafﬁc lanes. In the selected microsimulation model, trafﬁc ﬂow is distributed among trafﬁc lanes on the basis of parameters describing the behaviour of drivers and the characteristics of vehicles entering the simulation model. In order to use the model in a real environment, one should calibrate the model and make a comparison between the actual characteristics of the trafﬁc ﬂow and the modelled characteristics. Figure 5. Schema of unidirectional carriageway in the simulation trafﬁc model. The trafﬁc model simulation time was Ts = 60 min and we gradually loaded the model in 10 min time intervals with different values of the ﬂow rate of all vehicles on the carriage- way. The total trafﬁc ﬂow rate increased uniformly at each interval qtot,c = {1500 veh/h, 2000 veh/h, 2500 veh/h, 3000 veh/h, 3500 veh/h, 4000 veh/h}. We modelled the situa- tion in which LFR2,c ≈LFR1,c occurs in the trafﬁc ﬂow, i.e., in the case in which vehicles change lanes intensively and both trafﬁc lanes are approximately equally loaded. This Figure 5. Schema of unidirectional carriageway in the simulation trafﬁc model. Figure 5. Schema of unidirectional carriageway in the simulation trafﬁc model. Trafﬁc Flow Speed Analysis • Z3: exclusion for camper vans, all Z2 level vehicles, and buses (wind from 130 km/h to 149 km/h); • Z3: exclusion for camper vans, all Z2 level vehicles, and buses (wind from 130 km/h to 149 km/h); Z b f ll hi l d l ( i d 150 k /h) ); • Z4: ban for all vehicles—road closure (wind over 150 km/h). Figure 3. Empirical speed distribution functions for different weather conditions at test site 0178: (a) passenger cars in lane 2; (b) passenger cars in lane 1; (c) heavy vehicles in lane 2; (d) heavy vehicles in lane 1. Figure 4. Empirical speed distribution functions with different trafﬁc management measures in the case of wind at test point 0865: (a) lane 2—left (overtaking) lane; (b) lane 1—right lane. Figure 4. Empirical speed distribution functions with different trafﬁc management measures in the case of wind at test point 0865: (a) lane 2—left (overtaking) lane; (b) lane 1—right lane. Appl. Sci. 2022, 12, 10169 8 of 17 8 of 17 In the case of measure Z4, the highway is closed for all vehicles, which means that only light passenger cars that leave the trafﬁc network and are not yet familiar with trafﬁc control measures are subject to speed measurements. If wind gust speeds exceed a certain level with a simultaneous increase in wind strength and vice versa, highway or expressway trafﬁc is regulated in accordance with the decision of the supervisory group of the trafﬁc operators. If a negative trend occurs, the ban is lifted. Each such turn takes up to 2 h. p g p In the case of extreme winds, heavy trafﬁc is excluded or the road is closed; hence, this generally does not affect the highway capacity utilisation. Thus, we did not consider the inﬂuence of wind on a possible measure of the truck overtaking ban. 3. Modelling the Efﬁciency of Trafﬁc Lane Control and Weather Conditions The trafﬁc model simulation time was Ts = 60 min and we gradually loaded the model in 10 min time intervals with different values of the ﬂow rate of all vehicles on the carriage- way. The total trafﬁc ﬂow rate increased uniformly at each interval qtot,c = {1500 veh/h, 2000 veh/h, 2500 veh/h, 3000 veh/h, 3500 veh/h, 4000 veh/h}. We modelled the situa- tion in which LFR2,c ≈LFR1,c occurs in the trafﬁc ﬂow, i.e., in the case in which vehicles change lanes intensively and both trafﬁc lanes are approximately equally loaded. This Appl. Sci. 2022, 12, 10169 9 of 17 9 of 17 was observed through the viewer interface and control points (Figures 5 and 6a,b). In the simulation, we considered the constant share of heavy vehicles Phv = 10%. was observed through the viewer interface and control points (Figures 5 and 6a,b). In the simulation, we considered the constant share of heavy vehicles Phv = 10%. Figure 6. Example of uniform trafﬁc ﬂow distribution across trafﬁc lanes: (a) NB_ scenario; (b) WB_ scenario. Figure 6. Example of uniform trafﬁc ﬂow distribution across trafﬁc lanes: (a) NB_ scenario; (b) WB_ scenario. We modelled three scenarios, two of them for six weather conditions: 1. Without trafﬁc control restrictions and without separate consideration of weather con- ditions c; “ALL” weather conditions are considered—NB_ALL simulations (Figure 6a); 2. Without trafﬁc control restrictions, but with weather conditions c considered—NB_c simulations (Figure 6a): a. NB_d simulation taking into account the empirical speed distribution func- tions of light (F-0178,Vpc,i,d) and heavy vehicles (F-0178,Vhv,i,d), in trafﬁc lanes i = {1, 2}, in “dry” and fair weather (Figure 3); b. NB_w1-, NB_w2-, NB_w3 simulation by taking into account the empirical speed distribution functions of light (F-0178,Vpc,i,wi) and heavy vehicles (F- 0178,Vhv,i,wi), in trafﬁc lanes with different precipitation intensities and wetness of road surface (Figure 3); c. NB_lv simulation by taking into account the empirical speed distribution func- tion of light (F-0178,Vpc,i,lv) and heavy vehicles (F-0178,Vhv,i,lv) in trafﬁc lanes with “poor visibility” (Figure 3); d. NB_s simulation by taking into account the empirical speed distribution func- tion of light (F-0178,Vpc,i,s) and heavy vehicles (F-0178,Vhv,i,s) in trafﬁc lanes under “winter conditions” (Figure 3); 3. With the trafﬁc control of “overtaking ban for HGVs”, but with consideration of road-weather conditions c—WB_c simulations (Figure 6b): {WB_d, WB_w1, WB_w2, WB_w3, WB_lv, WB_s}. 3. Modelling the Efﬁciency of Trafﬁc Lane Control and Weather Conditions The road-weather conditions were determined parametrically using the corresponding empirical speed distribution functions of light and heavy vehicles as presented in the previous section. In total, we performed 13 different simulation cases and each case included ﬁve iterations, yielding a total of 65 simulated cases. While there were many iterations of different scenarios simulations, the average values of the results are discussed in Section 4. 4. Results and Discussion This section presents the results of trafﬁc ﬂow simulation using a microscopic trafﬁc model for a case of three scenarios of trafﬁc lane control with various weather and trafﬁc conditions. The simulation results of the ﬁrst scenario represent a reference value without considering weather conditions and considering the speed distribution function for the “average” lane. Then, two scenarios follow with knowledge of the lane speed distribution function of different vehicles in different weather conditions, and the third with the measure of “overtaking ban for HGVs”, which can be used to reduce the instability of the trafﬁc ﬂow. In this way, faster vehicles are separated from slower ones, resulting in greater speed homogeneity of trafﬁc ﬂow in each trafﬁc lane. For each simulated case, the relative delays rD,Sc were determined [45]. For the speciﬁc simulation scenario Sc = {NB_ALL, NB_c, WB_c}, the line graphs in Figure 7a,b show the total relative delay rD,Sc as a function of trafﬁc ﬂow density kSc, which we ensured to be nearly identical for all compared cases. Appl. Sci. 2022, 12, 10169 10 of 17 10 of 17 Figure 7. Total relative delays of vehicles in the “average” lane depending on the trafﬁc ﬂow density for simulation scenarios: (a) NB_ALL, NB_c; (b) NB_ALL, WB_c. Figure 7. Total relative delays of vehicles in the “average” lane depending on the trafﬁc ﬂow density for simulation scenarios: (a) NB_ALL, NB_c; (b) NB_ALL, WB_c. In Table 1, the relative delay differences between rD,NB_ALL and rD,NB_c (rD,NB_ALL − rD,NB_c) and between rD,NB_d and rD,NB_c (rD,NB_d −rD,NO_c) were added to delays rD,Sc. Table 2 shows the results of relative delays and differences between rD,NB_ALL and rD,WB_c (rD,NO_ALL −rD,WB_c) and between rD,WB_d and rD,WB_r (rD,WB_d −rD,WB_c). In Table 1, we compared the difference in total relative delays between the scenario that assumes no special trafﬁc lane control and considers the input parameters determined without considering the weather conditions (reference simulation NB_ALL) and the simulation scenarios that consider different weather conditions (simulations NB_c). In Table 2, we compared the difference with the scenario that assumes a trafﬁc management measure. Both tables also show the relative delay difference with respect to dry weather conditions rD,NB_d and rD,WB_d (simulations NB_d and WB_d). Table 1. Relative delays of vehicles depending on trafﬁc density for simulation scenarios NB_ALL and NB_c, along with relative differences for the “average” lane. 4. Results and Discussion kSc rD,NB_ALL kSc rD,NB_d kSc rD,NB_lv kSc rD,NB_s kSc rD,NB_w1 kSc rD,NB_w2 kSc rD,NB_w3 (veh/km) (veh/km) (veh/km) (veh/km) (veh/km) (veh/km) (veh/km) 6.0 4.1% 6.0 4.2% 6.0 4.5% 6.0 4.6% 6.0 4.2% 6.0 4.4% 6.0 4.5% 8.7 5.4% 8.6 6.0% 8.6 7.0% 8.6 7.1% 8.7 5.9% 8.7 6.4% 8.6 7.0% 11.4 9.3% 11.4 9.2% 11.4 9.6% 11.3 9.7% 11.4 9.2% 11.4 9.4% 11.3 9.5% 14.3 12.9% 14.2 12.9% 14.4 13.5% 14.4 13.7% 14.3 13.0% 14.4 13.4% 14.3 13.2% 17.3 15.6% 17.3 16.0% 17.2 17.2% 17.7 18.3% 17.4 16.5% 17.4 16.8% 17.4 16.8% 20.7 20.9% 20.8 21.1% 20.6 22.5% 20.8 23.3% 20.8 21.5% 20.7 21.7% 20.7 22.0% 13.1 11.4% 13.1 11.6% 13.0 12.4% 13.1 12.7% 13.1 11.7% 13.1 12.0% 13.1 12.1% kSc rD,NB_ALL −rD,NB_d rD,NB_ALL −rD,NB_lv rD,NB_ALL −rD,NB_s rD,NB_ALL − rD,NB_w1 rD,NB_ALL − rD,NB_w2 rD,NB_ALL − rD,NB_w3 (veh/km) 6.0 2% 10% 13% 2% 7% 9% 8.7 11% 28% 30% 8% 19% 28% 11.4 −1% 3% 4% −1% 1% 1% 14.3 0% 5% 7% 1% 4% 3% 17.3 3% 10% 17% 6% 7% 8% 20.7 1% 8% 12% 3% 4% 5% 13.1 2% 9% 12% 3% 6% 7% kSc rD,NB_d −rD,NB_lv rD,NB_d −rD,NB_s rD,NB_d −rD,NB_w1 rD,NB_d −rD,NB_w2 rD,NB_d −rD,NB_w3 (veh/km) 6.0 7% 10% 0% 5% 6% 8.6 16% 18% −3% 7% 16% 11.4 4% 5% 0% 2% 2% 14.2 4% 6% 1% 3% 2% 17.3 7% 14% 3% 5% 5% 20.8 7% 10% 2% 3% 4% 13.1 7% 10% 1% 4% 5% Appl. Sci. 2022, 12, 10169 11 of 17 11 of 17 Table 2. Relative delays of vehicles depending on trafﬁc density for simulation scenarios NB_ALL and WB_c, along with relative differences for the “average” lane. 4. Results and Discussion kSc rD,NB_ALL kSc rD,WB_d kSc rD,WB_lv kSc rD,WB_s kSc rD,WB_w1 kSc rD,WB_w2 kSc rD,WB_w3 (veh/km) (veh/km) (veh/km) (veh/km) (veh/km) (veh/km) (veh/km) 6.0 4.1% 6.0 3.8% 6.0 3.9% 6.0 3.9% 6.0 3.8% 6.0 3.9% 6.0 3.8% 8.7 5.4% 8.6 5.2% 8.7 5.2% 8.7 5.3% 8.7 5.2% 8.7 5.3% 8.7 5.4% 11.4 9.3% 11.4 7.1% 11.3 7.3% 11.3 7.3% 11.3 7.1% 11.4 7.5% 11.4 7.6% 14.3 12.9% 14.1 10.4% 14.1 10.6% 14.1 10.9% 14.1 10.4% 14.1 10.5% 14.1 10.6% 17.3 15.6% 17.1 14.0% 17.2 15.1% 17.4 15.2% 17.2 14.3% 17.3 14.7% 17.3 14.9% 20.7 20.9% 20.6 18.9% 20.6 19.2% 20.8 19.8% 20.7 18.9% 20.6 19.1% 20.6 19.2% 13.1 11.4% 13.0 9.9% 13.0 10.2% 13.1 10.4% 13.0 10.0% 13.0 10.2% 13.0 10.2% kSc rD,NB_ALL −rD,WB_d rD,NB_ALL −rD,WB_lv rD,NB_ALL −rD,WB_s rD,NB_ALL − rD,WB_w1 rD,NB_ALL − rD,WB_w2 rD,NB_ALL − rD,WB_w3 (veh/km) 6.0 −7% −5% −4% −7% −4% −8% 8.7 −4% −4% −2% −4% −2% −1% 11.4 −24% −21% −22% −24% −20% −19% 14.3 −19% −18% −16% −19% −18% −18% 17.3 −10% −3% −3% −8% −6% −5% 20.7 −9% −8% −5% −9% −8% −8% 13.1 −13% −10% −9% −12% −10% −10% kSc rD,NB_d −rD,WB_d rD,NB_lv −rD,WB_lv rD,NB_s −rD,WB_s rD,NB_w1 −rD,WB_w1 rD,NB_w2 −rD,WB_w2 rD,NB_w3 −rD,WB_w3 (veh/km) 6.0 −10% −14% −15% −9% −10% −15% 8.6 −13% −25% −25% −11% −18% −22% 11.4 −23% −24% −25% −23% −20% −20% 14.2 −20% −21% −21% −20% −21% −20% 17.3 −12% −12% −17% −13% −12% −11% 20.8 −10% −15% −15% −12% −12% −12% 13.1 −15% −17% −18% −15% −15% −16% kSc rD,WB_d −rD,WB_lv rD,WB_d −rD,WB_s rD,WB_d −rD,WB_w1 rD,NB_d −rD,WB_w2 rD,NB_d −rD,WB_w3 (veh/km) 6.0 2% 4% 0% 4% 0% 8.6 0% 2% 0% 2% 4% 11.4 3% 2% 0% 6% 7% 14.2 2% 5% 0% 1% 2% 17.3 8% 8% 2% 5% 6% 20.8 1% 4% 0% 1% 2% 13.1 3% 5% 1% 3% 3% Table 2. Relative delays of vehicles depending on trafﬁc density for simulation scenarios NB_ALL and WB_c, along with relative differences for the “average” lane. The graph in Figure 7a shows that the total relative delay rD,Sc was higher than the reference value when considering weather conditions and knowing the distribution of the trafﬁc ﬂow rate in lanes. When comparing the average values (kSc ≈13.0 veh/km), the relative delays were 2% to 12% higher in all cases (Table 1); however, at the approximate value of trafﬁc ﬂow density of 12 veh/km, this difference decreased again. 4. Results and Discussion The differences were similar for all simulation scenarios in the same size class up to a density of 14 veh/km. At maximum trafﬁc ﬂow rate and density of just over 20 veh/km, which we simulated in the sixth time interval, relative delays increased again to 1% in dry weather conditions, from 3% to 5% in wet conditions, to 8% in poor visibility, and up to 12% in snowy conditions. Generally, results of the paired t-test indicated that there is a non-signiﬁcant medium difference between rD,NB_ALL (µ = 11.4, σ = 6.4) and rD,NB_d (µ = 11.6, σ = 6.4), t = 1.9, p = 0.060. Otherwise there is a signiﬁcantly large difference between rD,NB_ALL and non rD,NB_d, e.g., rD,NB_S (µ = 12.7, σ = 7.1), t = 3.5, p = 0.009. , y g y , 3% to 5% in wet conditions, to 8% in poor visibility, and up to 12% in snowy conditions. Generally, results of the paired t-test indicated that there is a non-signiﬁcant medium difference between rD,NB_ALL (µ = 11.4, σ = 6.4) and rD,NB_d (µ = 11.6, σ = 6.4), t = 1.9, p = 0.060. Otherwise there is a signiﬁcantly large difference between rD,NB_ALL and non rD,NB d, e.g., rD,NB S (µ = 12.7, σ = 7.1), t = 3.5, p = 0.009. , _ g , _ µ p The graph in Figure 7b presents rD,Sc as a function of trafﬁc ﬂow density for the scenario without special trafﬁc management measures (NB_ALL) and with the measure “overtaking ban for HGVs” (WB_c), but with knowledge of the lane speed distribution functions for different weather conditions and different vehicles. Regardless of trafﬁc ﬂow density, trafﬁc control reduced rD,Sc by an average of 9% to 13% considering the weather conditions (Table 2). Differences in relative delays between different weather conditions appeared with increasing trafﬁc density and with the same trafﬁc control measure. It is interesting to note that rD,Sc did not deviate signiﬁcantly from the reference value up to the value of the trafﬁc ﬂow density kSc ≤9 veh/km, which we interpreted as a lower efﬁciency of the trafﬁc management measure. For the cases kSc > 9 veh/km, the differences Appl. Sci. 2022, 12, 10169 12 of 17 12 of 17 increased and reached values from −19% to −24%, with the greatest impact in dry weather conditions. 4. Results and Discussion Thus, for a trafﬁc ﬂow density of around 9 veh/km, the relative reduction in total relative delays without and with lane management was up to 1% in heavy rainfall and up to 4% in light rain and in dry conditions. With the additional increase in trafﬁc ﬂow density, the relative differences according to weather conditions no longer changed with the same trends. With a trafﬁc ﬂow density of about 21 veh/km and with the WB_c measure, the total relative delays were reduced by 8% to 9% in all weather conditions, and by 5% in snow. Again, results of the paired t-test indicated that there is a signiﬁcantly large difference between rD,WB_c (µ = 10.1, σ = 5.4) and rD,NB_ALL (µ = 11.4, σ = 5.8), n = 36, t = 8.2, p < 0.001. The criterion of trafﬁc control on highways when trafﬁc ﬂow is becoming unstable is usually determined by the total equivalent ﬂow rate qe,tot,ALL and the equivalent ﬂow rate in the overtaking lane qe,2,ALL, as well as the standard deviation of speed in the overtaking lane [34,36]. Similarly, the total equivalent ﬂow rate qe,tot,ALL and the proportion of heavy vehicles Phv determine the criterion for trafﬁc management in the case of trafﬁc instability at a large share of heavy vehicles. Considering that free-ﬂow speed varies depending on weather conditions, in the case where the trafﬁc ﬂow rate is a criterion for trafﬁc management, different threshold values of the ﬂow rate threshold should be taken into account, depending on weather and trafﬁc conditions. Therefore, this article demonstrates that it is reasonable to further investigate the effectiveness of the current AHTMS criteria and possibly establish new criteria for trafﬁc management in the case of instability by using other trafﬁc ﬂow characteristics, such as the Coefﬁcient of Variation of Speed CVS [30,46], the trafﬁc ﬂow density kc, or the possibility of changing lanes depending on environmental factors LFRi,c(qe,tot,c), LDRi,c(qe,tot,c) [19]. For comparison, on the basis of the simulation, we calculated the total delays of all vehicles pD,Sc on the simulation model road network, varying the input data and calculating the equivalent trafﬁc ﬂow rate for the case of a highway level terrain according to the HCM methodology [17]. 4. Results and Discussion qe,tot,Sc pD,NB_ALL qe,tot,Sc pD,NB_d qe,tot,Sc pD,NB_lv qe,tot,Sc pD,NB_s qe,tot,Sc pD,NB_w1 qe,tot,Sc pD,NB_w2 qe,tot,Sc pD,NB_w3 (pcu/h) (h) (pcu/h) (h) (pcu/h) (h) (pcu/h) (h) (pcu/h) (h) (pcu/h) (h) (pcu/h) (h) 1575 0.84 1575 0.85 1575 0.73 1575 0.92 1575 0.74 1575 0.79 1575 0.78 2291 1.78 2284 1.81 2291 1.98 2284 2.11 2284 1.83 2284 1.95 2284 1.85 2873 3.03 2873 3.21 2873 3.66 2880 3.56 2873 3.14 2880 3.34 2880 3.45 3448 7.05 3448 6.90 3455 7.13 3441 6.98 3448 7.09 3455 7.18 3455 7.24 4093 11.11 4086 10.75 4051 10.67 4051 11.75 4072 11.30 4058 11.36 4044 11.75 4526 17.91 4540 17.65 4526 18.85 4547 20.84 4547 18.47 4526 19.30 4526 20.58 qe,tot,Sc pD,NB_d/pD,NB_ALL pD,NB_lv/pD,NB_ALL pD,NB_s/pD,NB_ALL pD,NB_w1/pD,NB_ALL pD,NB_w2/pD,NB_ALL pD,NB_w3/pD,NB_ALL (pcu/h) 1575 1.02 0.87 1.10 0.89 0.95 0.94 2291 1.02 1.12 1.19 1.03 1.10 1.04 2873 1.06 1.21 1.17 1.04 1.10 1.14 3448 0.98 1.01 0.99 1.01 1.02 1.03 4093 0.97 0.96 1.06 1.02 1.02 1.06 4526 0.99 1.05 1.16 1.03 1.08 1.15 The line graph in Figure 8b presents pD,Sc as a function of qe,tot,Sc for the scenario with the simulated measure “overtaking ban for HGVs” (WB_c scenarios). In all cases, delays were lower with the trafﬁc control; however, in the qe,tot,Sc interval from 1500 pcu/h to 2900 pcu/h, this difference was not as pronounced as at higher ﬂow rates. In Table 4, in addition to the results on total delays, we present the relative difference, expressed by indices pD, WB_c pD, NB_ALL and pD, WB_c pD, NB_c , where pD,NB_ALL is the total vehicle delay of the reference simulation, pD,NB_c is the total delay without trafﬁc control, and pD,WB_c is the total delay with the trafﬁc control measure in different weather conditions c. Owing to the “overtaking ban for HGVs”, delays were 2% to 3% lower in fair weather conditions and 5% to 7% lower in adverse weather conditions. With higher trafﬁc ﬂow rates, delays could be reduced by up to 21%, regardless of weather conditions, but only up to a certain threshold. As rates exceeded 4500 pcu/h, the relative differences between the delays under different conditions decreased. From the results, similar to the case of relative delays, it appears that trafﬁc management with truck overtaking bans at lower ﬂow rates was not as effective as at ﬂows above 2800 pcu/h. Through trafﬁc simulations, we conﬁrmed that relative trafﬁc delays depend on road weather conditions. 4. Results and Discussion The graph in Figure 8a shows total delays pD,Sc as a function of the total equivalent trafﬁc ﬂow rate qe,tot,Sc for the scenario without special trafﬁc management (NB_c), and that in Figure 8b shows the same scenario with the simulated measure “overtaking ban for HGVs” (WB_c). In both cases, the results of the NB_ALL scenario are also shown as a reference red polyline. Figure 8. Simulated total delays of all vehicles as a function of total equivalent trafﬁc ﬂow rate: (a) for NB_ALL, NB_c scenarios; (b) for NB_ALL, WB_c scenarios. Figure 8. Simulated total delays of all vehicles as a function of total equivalent trafﬁc ﬂow rate: (a) for NB_ALL, NB_c scenarios; (b) for NB_ALL, WB_c scenarios. In addition to the results, Table 3 shows the relative differences, expressed by the index ratio pD, NB_c pD, NB_ALL , where pD,NB_ALL represents the total delays of the vehicles in the reference simulation. In the range of ﬂow rates from 1500 pcu/h to about 2300 pcu/h, delays under bad weather conditions were lower than under reference conditions, except for dry weather In addition to the results, Table 3 shows the relative differences, expressed by the index ratio pD, NB_c pD, NB_ALL , where pD,NB_ALL represents the total delays of the vehicles in the reference simulation. In the range of ﬂow rates from 1500 pcu/h to about 2300 pcu/h, delays under bad weather conditions were lower than under reference conditions, except for dry weather Appl. Sci. 2022, 12, 10169 13 of 17 13 of 17 conditions. This difference may be a result of adverse weather conditions that reduced the speed of vehicles and changed the lane distribution in some way [19]. The ratio value changed with the next step of the trafﬁc ﬂow rate increase. In the qe,tot,Sc interval between 2300 pcu/h and 3500 pcu/h, delays were again shorter. With better weather conditions, the differences over the ﬂow rate value of around 2900 pcu/h decreased. At a ﬂow rate of 3500 pcu/h, the delay differences only increased and no longer ﬂuctuated. At the end of the simulation, total delays increased by 16% in winter conditions, 15% in heavy rain, 8% in moderate rain, and up to 3% in light rain. In dry conditions, a 1% improvement over the reference value was observed. Table 3. Total delays in simulated network depending on qe,tot,Sc for simulation scenarios NB_ALL and NB_c, along with index ratios. 4. Results and Discussion In adverse conditions, these were, on average, 1% to 10% higher than relative delays in dry conditions with the same trafﬁc ﬂow rate, and worst in the case of winter conditions, followed by low visibility and various rain intensities or wetness. For the maximum simulated ﬂow rate of 4000 veh/h with a 10% share of heavy vehicles, i.e., a trafﬁc ﬂow density of slightly more than 20 veh/km, when the characteristics of the trafﬁc ﬂow in the trafﬁc lanes were known, the relative delays were 1% greater in dry weather conditions, 3% to 5% greater in wet conditions, 8% greater in poor visibility, and up to 12% greater in winter conditions than in the case where the trafﬁc ﬂow characteristics were determined independently of weather conditions (_ALL). By simulating the trafﬁc management measure of the truck overtaking ban, we calculated that relative delays were reduced by an average of 15% to 18% compared to a benchmark without trafﬁc management. The largest difference occurred with medium-sized ﬂow rates or a density of about 12 veh/km and was as high as 25% in snowy weather conditions. We found that the Appl. Sci. 2022, 12, 10169 14 of 17 effect of trafﬁc management was greater in the case of adverse weather conditions with the same trafﬁc demand. The relative difference varied depending on the density of trafﬁc ﬂow. Trafﬁc management had the greatest effect in the case of medium density (about 12 veh/km), where the relative differences between weather conditions were small. We also calculated total delays as a function of the equivalent ﬂow rate. In all weather conditions, trafﬁc management could achieve lower delays; however, in the interval of ﬂows from 1500 pcu/h to 2900 pcu/h, this difference was not as large as at higher ﬂow rates. Owing to trafﬁc management, delays were reduced by 2% to 3% in good weather conditions and by 5% to 7% in adverse weather conditions. However, at higher ﬂow rates, delays could be reduced by up to 21%, regardless of weather conditions, but only up to a certain limit. When trafﬁc ﬂow rates exceeded 4500 pcu/h, the relative differences between delays under different conditions decreased. Similar to the case of relative delays, the results show that trafﬁc management with truck overtaking bans at lower trafﬁc demand was not as effective as for ﬂow rates above 2300 pcu/h with a 10% share of heavy vehicles. 4. Results and Discussion Total delays may be equal to or even lower in the case of rain or a wet carriageway. We found that truck overtaking bans had a positive effect when considering the relative difference in total delays but only when the trafﬁc ﬂow rate of all vehicles and heavy vehicles on the carriageway separately exceeded a certain threshold value. As part of our research, we limited the proportion of HGVs in the total ﬂow rate. If we were to vary this, we would achieve more results. With the research, we proved that sensing weather conditions is important in the case of decision-making for lane trafﬁc control. Table 4. Total delays in simulated network depending on qe,tot,Sc for simulated scenarios NB_ALL and WB_c, along with index ratios. qe,tot,Sc pD,NB_ALL qe,tot,Sc pD,WB_d qe,tot,Sc pD,WB_lv qe,tot,Sc pD,WB_s qe,tot,Sc pD,WB_w1 qe,tot,Sc pD,WB_w2 qe,tot,Sc pD,WB_w3 (pcu/h) (h) (pcu/h) (h) (pcu/h) (h) (pcu/h) (h) (pcu/h) (h) (pcu/h) (h) (pcu/h) (h) 1575 0.84 1575 0.85 1575 0.73 1575 0.92 1575 0.74 1575 0.79 1575 0.78 2291 1.78 2284 1.81 2291 1.98 2284 2.11 2284 1.83 2284 1.95 2284 1.85 2873 3.03 2873 3.21 2873 3.66 2880 3.56 2873 3.14 2880 3.34 2880 3.45 3448 7.05 3448 6.90 3455 7.13 3441 6.98 3448 7.09 3455 7.18 3455 7.24 4093 11.11 4086 10.75 4051 10.67 4051 11.75 4072 11.30 4058 11.36 4044 11.75 4526 17.91 4540 17.65 4526 18.85 4547 20.84 4547 18.47 4526 19.30 4526 20.58 qe,tot,Sc pD,WB_d/pD,NB_ALL pD,WB_lv/pD,NB_ ALL pD,WB_s/pD,NB_ALL pD,WB_w1/pD,NB_ALL pD,WB_w2/pD,NB_ALL pD,WB_w3/pD,NB_ALL (pcu/h) 1575 1.02 0.87 1.10 0.89 0.95 0.94 2291 1.02 1.12 1.19 1.03 1.10 1.04 2873 1.06 1.21 1.17 1.04 1.10 1.14 3448 0.98 1.01 0.99 1.01 1.02 1.03 4093 0.97 0.96 1.06 1.02 1.02 1.06 4526 0.99 1.05 1.16 1.03 1.08 1.15 qe,tot,Sc pD,WB_d/pD,NB_d pD,WB_lv/pD,NB_ lv pD,WB_s/pD,NB_s pD,WB_w1/pD,NB_w1 pD,WB_w2/pD,NB_w2 pD,WB_w3/pD,NB_w3 (pcu/h) 1575 0.96 0.94 0.88 1.06 1.01 1.01 2291 0.96 0.84 0.78 0.94 0.89 0.94 2873 0.93 0.78 0.79 0.94 0.89 0.86 3448 0.82 0.81 0.80 0.80 0.80 0.79 4093 0.91 0.94 0.82 0.88 0.88 0.85 4526 0.94 0.90 0.79 0.89 0.86 0.81 5. Conclusions The research presented in this article included an analysis of trafﬁc ﬂow characteristics in different highway trafﬁc lanes with an emphasis on the inﬂuence of weather conditions. With the results of the analyses, we conﬁrmed that weather conditions have different effects on trafﬁc ﬂow and lane distribution. This may be important when monitoring capacity utilisation and other performance criteria of highway trafﬁc control services for the future. In general, we found that, in certain cases, bad weather has a positive effect on the homogeneity of the trafﬁc ﬂow speed and that, under such circumstances, vehicles do not change lanes, which can reduce delays. g y Using a trafﬁc microsimulation on a highway section, we determined vehicle delays as a function of trafﬁc demand in different weather conditions using the example of the trafﬁc Appl. Sci. 2022, 12, 10169 15 of 17 15 of 17 management scenario “overtaking ban for HGVs”. The delays calculated with the trafﬁc model were compared with a reference scenario without trafﬁc management in simulated different weather conditions but with controlled trafﬁc input data. If the effectiveness of the AHTMS measures is measured by the reduction in delays, it would be important to know the result that the same value of delay is achieved for different values of trafﬁc ﬂow density in different weather situations. In practice, the techniques for empirically determining delays can be complex; hence, we can expect the introduction of online trafﬁc models soon. y p p Of course, it would be interesting to investigate whether it is justiﬁed to have a day- time truck overtaking ban independent of trafﬁc ﬂow rates on all highways and whether preference should be given to AHTMS, in which a measure of dynamic control of heavy vehicles would be implemented when needed, depending on trafﬁc demand, in a real case and not hypothetically. We should know that in Slovenia, AHTMSs have already been implemented on highways and that Slovenia has the National Trafﬁc Management Centre, whose functionality also includes the online modelling of trafﬁc ﬂows, including for short-term prediction. An overtaking ban for HGVs was established on the whole dual carriageway highway network in Slovenia in a static way (overtaking is permanent and intermittent on some highways). Through our research, we found that dynamic bans can be more effective when we include the dynamics of trafﬁc demand and environmental conditions. 5. Conclusions At the same time, actual compliance with any strict rules should be investi- gated. The next issue related to the problem of homogenisation of trafﬁc ﬂows is certainly the monitoring and management of recreational vehicles, focusing on motorhomes and vehicles with tourist trailers, which have a maximum authorised speed that is different from other vehicles. Author Contributions: Conceptualization, R.R., R.M. and I.S.; Data curation, R.R., R.M. and I.S.; Formal analysis, R.R., R.M. and I.S.; Funding acquisition, R.R.; Investigation, R.R. and R.M.; Method- ology, R.R., R.M. and I.S.; Project administration, R.R. and I.S.; Resources, R.R. and R.M.; Software, R.R. and R.M.; Supervision, R.R. and I.S.; Validation, R.R., R.M. and I.S.; Visualization, R.R., R.M. and I.S.; Writing—original draft, R.R. and R.M.; Writing—review & editing, R.R., R.M. and I.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding authors. Acknowledgments: The authors would like to thank the Slovenian Infrastructure Agency (DRSI) and the Motorway Company of the Republic of Slovenia (DARS d.d.) for their cooperation and access to trafﬁc and environmental data from their information systems. Acknowledgments: The authors would like to thank the Slovenian Infrastructure Agency (DRSI) and the Motorway Company of the Republic of Slovenia (DARS d.d.) for their cooperation and access to trafﬁc and environmental data from their information systems. Acknowledgments: The authors would like to thank the Slovenian Infrastructure Agency (DRSI) and the Motorway Company of the Republic of Slovenia (DARS d.d.) for their cooperation and access to trafﬁc and environmental data from their information systems. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W4313465157	https://www.intechopen.com/citation-pdf-url/85359	English	null	Flipped Classroom Approach of Teaching Chemistry in Higher Education	IntechOpen eBooks	2,023	cc-by	3,882	Abstract The flipping classroom method has been increasing steadily in acceptance and approval worldwide. In fact, there is a global agreement on the benefits of flipping courses at all levels and different majors. This approach has been largely adopted, specifically at the level of higher education. Our findings revealed an amelioration of the mean student’s success percentage with the use of Edmodo and Moodle during the environmental chem- istry course taught with the flipped approach. This paper reviews the flipped classroom method as an advantageous active learning method and counsels its combination with modern information and communication technology (ICT) for better profit as well. Keywords: chemistry courses, higher education, flipped teaching, ICT, classroom approach Chapter Flipped Classroom Approach of Teaching Chemistry in Higher Education Kaouther Ardhaoui 3. Teacher’s role Create learning conditions based on direct questioning of knowledge, become a facilitator to promote learning, engage in one-on-one interactions with students, correct misunderstandings, personalize learning for each student, use technical equipment suitable for learning conditions, and create interactive discussions and conditions, increase student engagement, share lecture videos as extracurricular activities, provide feedback by applying teaching strategies. 2. Presentation of the flipped course method McNally et al. [8] identify a flipped classroom broadly, if events that have typi- cally and traditionally happened inside the “classroom” (e.g., lectures) occurred outside the session. Crucial essentials of what founds a flipped classroom consist of (a) an opportunity for students to acquire introduction to study content before the class (e.g., recorded lectures), (b) an encouragement for students to prepare for class (e.g., pre-class quizzes), (c) a process to evaluate student understanding (e.g., graded pre-class quizzes), and (d) in-class activities that emphasize on higher-level cognitive activities including active learning, peer learning, and/or problem solving. g g p g p g Additionally, the basic purpose of flipping the classroom is to relocate activities traditionally conducted within the classroom, like lectures, to educational resources that students engage with before attending class. This reallocation is intended to free classroom time to create meaningful learning situations for in-class interaction between students and teachers [9]. Also, Cheng et al. [10] define the flipped classroom instructional strategy as students learning with instructional supporting materials like documents or videos before class and then engaging in interactive and collaborative learning activities that facilitate their understanding, application, analysis, evaluation, and creation during class. Besides, O’Flaherty & Phillips confirm that students who are most profoundly involved will reflect, inquiry, speculate, estimate, and make links between ideas [11]. Otherwise, students who are disconnected seem to take a superficial method to learning by replicating transcripts, converging on disjointed evidences and hopping to deductions. During the flipped course, teachers and students have particu- lar duties. In fact, Ozdamli & Asiksoy resumed and listed these roles as follows [12]: 1. Introduction Essentially, this paper reports the important features of some reviews about flipped courses, especially chemistry courses at the level of higher education, in addition to modern techniques potentially useful to increase the benefits of flipping courses. g g g Essentially, this paper reports the important features of some reviews about flipped courses, especially chemistry courses at the level of higher education, in addition to modern techniques potentially useful to increase the benefits of flipping courses. 1. Introduction According to Bonwell and Eison [1], the term “active learning” has never been spe- cifically defined in educational researches and books. Some general features are usu- ally related to the usage of policies that promote active learning in the class: Students’ duty is not limited to listening. Low importance is given for transmitting information and much more on rising students’ skills. Students are implicated in higher-order reflection (analysis, synthesis, and evaluation), and they are involved in activities (e.g., reading, discussing, and writing). A particular importance and consideration are bestowed on students’ investigation of their own attitudes and standards. Moreover, active learning is defined as any instructional method that engages students in the learning process [2]. The core elements of active learning are student activity and engagement in this learning process. Active learning is often contrasted to the traditional lecture, where students passively receive information from the instruc- tor [2]. This strategy of active learning is generally adopted to improve Students’ Critical Thinking, Performance, Creativity, Motivation, and Communication Skills [3–6]. Furthermore, Bonwell and Eison [1] stated that there is a Serious Problem in Higher Education which is described in eight perceptible discrepancies in the prac- tice of higher education, counting the gap between teaching and learning, the gap between teaching and testing, and the gap between educational research and practice which were also acutely studied by Weinert et al. [7]. A thoughtful discrepancy also 1 Higher Education – Reflections from the Field – Volume 3 occurs between how university educators typically teach (i.e., counting mainly on the lecture method) and how they intend and are supposed to teach (i.e., employ- ing active learning to enable students’ control of subject matter, improve academic capacities, and build personal perceptions and principles). Then there are solutions to abolish this discrepancy by adapting the lecture, performing more inspiring class dis- cussions, and using other tactics related to active learning, such as blended teaching. occurs between how university educators typically teach (i.e., counting mainly on the lecture method) and how they intend and are supposed to teach (i.e., employ- ing active learning to enable students’ control of subject matter, improve academic capacities, and build personal perceptions and principles). Then there are solutions to abolish this discrepancy by adapting the lecture, performing more inspiring class dis- cussions, and using other tactics related to active learning, such as blended teaching. 5. Flipping courses in chemistry Bodner stated that the principal learning theory in chemistry education is con- structivism, which aims to base students’ approach to learning, by absorbing new ideas and information so that it makes sense with what they already know [14]. Teaching underneath the sphere of constructivism would consequently mean that teachers do not just inform students what they are in need to acquire, but deliver structured activities so that they become able to build their knowledge within the strictures of their own prior knowledge [13]. Besides, Bergmann & Sams affirmed that flipping the classroom establishes a framework that ensures students receive a personalized education tailored to their individual needs [15]. Likewise, Bancroft et al. stated that numerous studies represent increasing evidence that flipping chemistry lecture courses have the potential to yield small to moderately significant gains in student academic performance compared to traditional lecture-based courses [16]. While studying the flipped classroom model in higher education, Al-Samarraie et al. revealed that chemistry was the foremost subject that profited from applying such approach. The flipped course was found to enable students’ engagement and self- efficacy in studying by inspiring them to reflect on the topic and work with peers to answer questions and crack issues [17]. q For example, in their evaluation of a flipped-format general chemistry course, Weaver and Sturtevant found that this teaching procedure increased student exam scores and passing rates [18]. In another study on organic chemistry, Fautch showed an improvement in the summative assessment of the students attending the flipped course with a noticeable gain in confidence and passion for the subject [19]. In our previous research [6], we found that flipped courses did not only improve achievement in a notable way, but they also boosted motivation levels. The likeliest explanation for this association between motivation and achievement is that increased motivation, the immediate reaction to a new learning task, is an affective state that involves feelings of arousal, alertness, attention, and concentration and is, therefore, a key initiator of productivity and achievement [20]. Our results were in line with subjective impressions: considering novel learning methods like flipped courses, revealed that these might be not only more motivating in comparison to classic courses, but also added that they might trigger knowledge acquisition. 4. Student’s role Take responsibility for their own learning, watch pre-class lecture videos and use learning materials to prepare for lessons, study at their own pace, interact with 2 Flipped Classroom Approach of Teaching Chemistry in Higher Education DOI: http://dx.doi.org/10.5772/intechopen.109235 teachers and friends as necessary, receive and give feedback, participate in class discussions, and participate in teamwork. As regard to some confusions and misunderstandings of what flipped learning is, the Flipped Learning Network delivered the subsequent definition (Flipped Learning Network, 2014): “Flipped Learning is a pedagogical approach in which direct instruction moves from the group learning space to the individual learning space, and the resulting group space is transformed into a dynamic, interactive learning environment where the educator guides students as they apply concepts and engage creatively in the subject matter” [13]. 6. Combining flipped courses with other technics Observing the results’ evolution of the achievement tests is related to the envi- ronment chemistry course that we have been teaching since 2018. We noticed a continuous improvement in the results of the students attending this course, where the flipped teaching techniques were considered, with the use of applications. In fact, during the university year 2018–2019, a flipped course with paper documents was taught, during the university year 2019–2020, a flipped course with numeric docu- ments displayed on Edmodo was proposed, and during the university year 2019–2020, the same course was taught with Moodle where documents were displayed in addition to interactive activities. The mean student’s success percentage was 74.2% in 2018; 77.3% in 2019; and 81.8% in 2020; such a result is likely to be due to the handling of ICT. This is in accordance with similar studies on the effect of modern technologies of communication on student grades. Actually, chemistry students’ own smartphones, laptops, and tablets and could use appropriate apps to complement traditional forms of learning. There is a positive correlation between the relative grades obtained using mobile applications and the final exam grades [22]. Moreover, according to Guerrero et al., handling mobile applications in the lectures enthused not only collective work but also the use of mobile technologies for studying basic sciences [23]. In the laboratory, this technological skill abridged the average time of practices and led to an important reduction in reagent waste in the experiences as well as improved the number of successes regarding problem samples. In fact, interactive learning is one of the approaches, which is very important to explore in higher education. In addition, the use of modern computer software in educating chemistry makes the basis for rising students’ curiosity in chemistry, delivering knowledge, and combining knowl- edge. Chemical computer software is a program intended to accomplish calculations of complex chemical equations and procedures, the structure of chemicals, their identification, and the presentation of the characteristics of various substances [24]. 5. Flipping courses in chemistry These outcomes are in agreement with those of Weaver and Sturtevant, who, after three years handling ACS (American Chemical Society) standardized exams, executed flipped courses and found that scores in the latter were significantly higher by almost one standard devia- tion when equated with students’ preceding scores in conventional courses [18]. 3 Higher Education – Reflections from the Field – Volume 3 Our results agree, in an additional context, with a similar meta-analysis on the effects of flipped courses on learning results [21]. During this meta-analysis, the author established a strong positive impact of flipped courses and showed the definite potential of face-to-face time and quiz activities, which seem to configure the largest effect size. 7. Discussion Numerous studies have shown the advantages of using the flipped course approach in several disciplines, such as Hew et al.’s second-order meta-analysis of flipped classroom usage across subjects found that the flipped classroom approach improved overall academic performance compared to traditional non-flipped classrooms [25]. In a similar context, according to a systematic review performed by Akçayır and Akçayır, a reverse course leads to positive academic outcomes because it encourages improve- ment in student learning (e.g., enhanced motivation to learn, positive student atti- tudes) [26]. Recently, Jong reported that students from teaching subjects of language education, social and humanities education, and mathematics and science education, appreciated flipped courses as having desirable benefits for attention, relevance, and satisfaction, especially in chemistry teaching at the higher education level [27]. 4 Flipped Classroom Approach of Teaching Chemistry in Higher Education DOI: http://dx.doi.org/10.5772/intechopen.109235 Meanwhile, some challenges and limitations were reported, related essentially to the lack of students’ engagement in the extra class activities [11], the risk of being stubborn at the beginning and may come to class without preparation, and this approach is also seen as increasing rather than relieving teachers’ responsibilities [12]. Consequently, some solutions are proposed in order to counter these issues, such as open teacher–student communication before flipping, showing students how to learn through flipped classrooms, and using gamified learning materials to monitor and motivate students’ studying [28]. y g Additionally, using modern technologies like computers and applications raises the efficiency of teaching and the interest of students in learning. In fact, Ottenbreit- Leftwic et al. enlightened the results of surveys indicating that teachers use tech- nology to address both majors (e.g., creating customized instructional materials, improving classroom management through student engagement) and student needs (e.g., improving student comprehension and equipping students with technology skills) [29]. Similarly, Ertmer & Ottenbreit-Leftwich proposed that teachers’ mind- sets must change to embrace the idea that teaching will not be effective without the appropriate use of information and communication technology (ICT) resources to facilitate student learning [30]. g It is then advisable to combine the flipped course approach with modern technolo- gies. In fact, the unified flipped learning model is wished for including the features of mobile and wireless communication technologies into the flipped classroom model to afford a director for researchers and educators to create operative flipped learning activities and plans for activating students’ learning effortlessly across frameworks [31]. 7. Discussion Furthermore, incorporating game elements into a flipped classroom increases motivation, participation, and learning performance. It is also found that the plat- forms, Moodle and Kahoot, are the most preferred platforms and points, badges, and leaderboards are the most used game elements for gamification [32]. Hence, the flipped course method is recommended in teaching chemistry courses in higher education, especially while combining this method with modern information com- munication technology. 8. Conclusion This paper presents some eminent features of the advantages of adopting a flipped classroom approach, particularly during chemistry courses at the university, with a preference for joining this approach with modern technologies like computer and mobile applications. We found that combining applications like Edmodo and Moodle with the flipped course triggered better success percentages for students than paper- based flipped courses. Such a combination is now considered and generalized to all our courses where Moodle is used to teach chemistry flipped courses related to green chemistry, water treatment, and cosmetic formulation. 5 5 Author details Kaouther Ardhaoui Higher Institute of Applied Biology of Medenine, Arid Region Institute of Medenine, Research Laboratory of Eremology and Combating Desertification, University of Gabes, Gabes, Medenine, Tunisia Address all correspondence to: ardhaouikaouther@gmail.com Higher Education – Reflections from the Field – Volume 3 Author details Kaouther Ardhaoui Higher Institute of Applied Biology of Medenine, Arid Region Institute of Medenine, Research Laboratory of Eremology and Combating Desertification, University of Gabes, Gabes, Medenine, Tunisia Address all correspondence to: ardhaouikaouther@gmail.com © 2022 The Author(s). Licensee IntechOpen. 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https://openalex.org/W2970527307	https://link.springer.com/content/pdf/10.1007/s11469-019-00120-2.pdf	English	null	Perspectives of Frontline Professionals on Palestinian Children Living with Sibling and Parental Drug Use in the West Bank and Gaza Strip	International journal of mental health and addiction	2,019	cc-by	8,984	International Journal of Mental Health and Addiction (2020) 18:1097–1112 https://doi.org/10.1007/s11469-019-00120-2 International Journal of Mental Health and Addiction (2020) 18:1097–1112 https://doi.org/10.1007/s11469-019-00120-2 ORIGINAL ARTICLE Abstract The Occupied Territories of Palestine (OtP) consists of the non-contiguous West Bank including East Jerusalem, and the Gaza Strip. Political and economic tensions and its dense populations compound the impact of drug abuse and addiction in the home. A qualitative study using four focus groups (n = 42) was conducted in West Bank and Gaza Strip explored the experiences of professionals working with Palestinian families and children affected by substance use and addiction in the home. Data were analysed using thematic analysis (TA), and four themes emerged. These were ‘The rising and shifting problem of drug use in Palestine’; ‘Psychosocial causal factors of drug use in Palestine’; ‘The consequences for children and families living with drug use’; and ‘Potential solutions to the problem are complex and multi-faceted.’ The study paints a concerning picture of how drug abuse impacts on Palestinian families subjected to multiple pressures, stigmas, risks and harms relating to their situation. Keywords Children . Parents . Siblings . Drugs . Palestine . Gaza . West Bank . Jerusalem . Palestine . Substance abuse Keywords Children . Parents . Siblings . Drugs . Palestine . Gaza . West Bank . Jerusalem . Palestine . Substance abuse Perspectives of Frontline Professionals on Palestinian Children Living with Sibling and Parental Drug Use in the West Bank and Gaza Strip Mohammed Al-Afifi1 & Leen Abushams2 & Mazen Sakka1 & Maha Shehada1 & Riad Afifi1 & Majed Alloush3 & Afaf Rabee3 & Stephanie Kewley4 & Zara Quigg5 & Mark Whitfield5 & Jim McVeigh5 & Mayyada Wazaify2 & Marie Claire Van Hout5 Published online: 30 August 2019 # The Author(s) 2019 Extended author information available on the last page of the article * Marie Claire Van Hout m.c.vanhout@ljmu.ac.uk Background The Occupied Territories of Palestine (OtP) consists of the non-contiguous West Bank with an area of 3000 km2 and a population of three million, and the Gaza Strip with an area of 262 km2 and a population of two million. According to the United Nations Relief and Works Agency for Palestine Refugees in the Near East (UNRWA), 26.6% are refugees living in 19 camps in the West Bank and Jerusalem, while in the Gaza Strip, 66.2% are refugees living in eight densely populated camps. The crude birth rate in Palestine is 30.5 per thousand, and about * Marie Claire Van Hout m.c.vanhout@ljmu.ac.uk Extended author information available on the last page of the article International Journal of Mental Health and Addiction (2020) 18:1097–1112 1098 40% of refugees are children (Waterston and Nasser 2017). The Al-Aqsa uprising in 2000 resulted in a significant rise in individual and community stress, economic hardship, exposure to political violence, school closures and travel restrictions (Massad et al. 2016). In Gaza Strip, the frequent wars and escalations including the war in 2014 lasted 51 days had resulted in a high number of causalities, fatalities, trauma and fear (Al-Afifi et al. 2015). High unemploy- ment and poverty prevail in both areas, particularly in the Gaza Strip (Palestinian Central Bureau of Statistics (PCBS) 2018). Conditions in the Gaza Strip and the West Bank have facilitated an exponential rise in drug trafficking, drug use and addiction among Palestinians, with increasing prevalence of illicit, new psychoactive substance (NPS), over the counter (OTC) and prescription drug abuse observed among youth, Palestinian women and family members of current drug users (Palestinian National Institute of Public Health 2017a, b; Damiri et al. 2018b; Sweileh et al. 2004). These observed increases have occurred despite religious, legal and cultural constraints. The stigma of addiction across the OtP is significant (Damiri et al. 2018a; Van Hout et al. 2019; Defense for Children International/Palestine Section 2007). Recent situational assess- ments conducted in 2017 have reported that drug trends include marijuana, prescription medications (anti-depressants, Z-hypnotics, benzodiazepines and analgesics) and novel psy- choactive substances (NPS) (‘Sintetique Marijuana’), with reported high dose use of metha- done, morphine, phencyclidine, barbiturates, benzodiazepines and synthetic opioids such as tramadol, and gabapentinoid drugs such as pregabalin (Palestinian National Institute of Public Health 2017a). Damiri et al. Methods A focus group study was conducted in West Bank and Gaza Strip to explore the experiences and knowledge of professionals working with Palestinian families and children affected by substance use and addiction in the home. It was undertaken by an international multi- disciplinary research team. Ethical approval for the study was granted by the University Ethics Committee at Liverpool John Moores University, UK (approval number 19/PHI/005) with further ethical approval granted by the Deanship of Scientific Research at the University of Jordan, Jordan (approval number 413/2019/19) and the ethics committee at the SARC Gaza, Palestine (approval number R-A-01- 2019). The design of the focus group guide was based on research expertise in the field, existing work by team members in both the Gaza Strip and the West Bank and on a systematic review of literature conducted by the team (Van Hout et al. 2019). Participants were recruited purposively through face-to-face, telephone and email strategies (Etikan et al. 2016). Eligibil- ity criteria meant that all participants were over the age of 18 and employed or studying/ researching in professional roles and subjects that would bring them into contact with Palestinian families and children affected by substance use and addiction. Potential participants were sent an information sheet about the study and offered an opportunity to ask further questions about the study before agreeing to participate. Before participating, participants signed a consent form. Participants did not receive any incentive or compensation for participation. Focus groups were conducted by two members of the team, a facilitator and co-facilitator, were audio recorded and supported by note taking. Open-ended questions (Smithson 2000; Kallio et al. 2016) were posited by the focus group facilitator. Participants were asked to describe the following: ‘the current situation with regard to substance abuse and addiction in Gaza or West Bank’; ‘how this situation has changed over time’; ‘how this currently affects Palestinian families and children’. In addition to discussion focusing on the current status of drug use and addiction in the Gaza Strip or the West Bank, the facilitator also explored participants thoughts around possible community and psychosocial interventions needed to reduce the vulnerability of children exposed to substance use in the Gaza Strip or the West Bank, and how non-governmental organisations (NGOs) might better support Palestinian families and children affected by substance use and addiction, was also asked. Background (2018b) reported that in 2018, on the rise since 2013 of trafficking and use of NPS, particularly synthetic cannabinoids, the manufacture of liquid amphetamine and the cultivation of marijuana in the OtP. The 2017 situational assessments estimate that there are now over 80,000 drug users in the OtP, of which 26,500 are high-risk drug users (Palestinian National Institute of Public Health 2017a). Children and young people are particularly at risk of substance abuse and are vulnerable to physical and sexual abuse, exploitation in drug trafficking and at risk of becoming addicts themselves (Defense for Children International/Palestine Section 2006; Palestinian National Institute of Public Health 2017b; Damiri et al. 2018a; Van Hout et al. 2019). Drug-related risk behaviours are higher among males, older youth, in urban areas and refugee camps (Thabet and Dajani 2012; Glick et al. 2018). There has been a sharp increase in familial poverty, children dropping out of school and becoming caregivers, becoming street children by either begging or working with little income, and are at high risk of exploitation (Van Hout et al. 2019). There is potential for drug exposed and traumatised Palestinian children to use drugs themselves, become dependent and their risk of overdose, psychiatric events and HIV/hepatitis C acquisition (Van Hout et al. 2019). The Palestinian National High Committee for the Prevention of Drugs and Psychotropic Substances recognises drug dependence as a multi-factorial health disorder and addressing drug dependence as a disease is highlighted in the past two Palestinian National Health Strategies. The right to health was included in the current Palestinian Drug Law which states the drug addict is a patient and should have the opportunity to access treatment services. Most recently in 2019, the Palestinian Ministry of Health with technical support from the United Nations Office on Drugs and Crime Programme Office in the OtP (UNODC-POPSE) has responded to the growing problem of drug dependence and HIV/hepatitis C, and has established the Palestinian National Rehabilitation Centre (PNRC) based in Bethlehem, West Bank (United Nations Office for Drugs and Crime (UNODC) 2019). This is an encouraging step forward for the West Bank, and yet leaves the Gaza Strip behind, without a treatment International Journal of Mental Health and Addiction (2020) 18:1097–1112 1099 facility and struggling to deal with the societal issue of drug abuse in Palestinian families. Background A greater insight into the experiences of families and children affected by substance use in Palestine is warranted (Van Hout et al. 2019). In order to better understand their needs, the study aimed to explore the perspectives of professionals working with international organisa- tions, non-governmental organisations and government who provide drug treatment to Pales- tinian families in the West Bank and the Gaza Strip. Results Four focus groups were undertaken across the Gaza Strip and the West Bank. A total of 42 participants took part (M = 10.5 SD = 2.5), each focus group lasted on average 89 min (SD = 13.9). Table 1 provides an overview of participants’ demographics and focus group details, and Fig. 1, a thematic map of analysis. Methods All focus groups were audio recorded and fully transcribed into Arabic, and then translated into English by the second author. This was cross-checked for accuracy by the lead author, prior to analysis. Data were analysed using thematic analysis (TA) (Braun et al. 2019). This approach was deemed suited to garner in depth understanding of the impacts of familial drug abuse within the multifaceted socio-political context of Palestine. It underpins phenomenological examina- tion of the experiences of professionals working with Palestinian families and children, from a range of multi-disciplinary perspectives, realities and meanings, due to its mitigation of 1100 International Journal of Mental Health and Addiction (2020) 18:1097–1112 potential cultural and language misinterpretation, and appreciation of the complex social contexts and challenges faced by participants (Nowell et al. 2017; Braun and Clarke 2006; Clarke and Braun 2018). In order to ensure scientific rigour, a quality framework in analysis was used (Braun and Clarke 2006). This involved several key steps: (1) reading and rereading the transcription, individually and in pairs to note early ideas; (2) coding in a systematic and logical manner using a data-driven approach supported by NVivo version 12, and paying attention to interesting concepts and ideas within the data; (3) organisation of codes into corresponding groups using an iterative process in developing themes and subthemes; (4) refining and reviewing of themes by the team as a collective in terms of internal homogeneity and external heterogeneity, examination of coherence of patterns across themes and development of a thematic map; and (5) final clear definition and naming of themes, with data extracts representing and articulating the essence of the theme, and overall analysis. The Rising and Shifting Problem of Drug Use in Palestine Across all focus groups, concerns for the increasing prevalence of drug use and addiction within Palestine communities were reported. The reported increase in drug use and addiction appears to have been a rapid phenomenon. In the Jerusalem focus group, participants reported that since 2015, addiction ‘has risen with [sic] about 200%’ (Male Anti-Drug Authority Officer) particularly with synthetic cannabinoids, and NPS such as ‘Hydro’ for which profes- sionals in Jerusalem asserted that ‘95% of the cases seeking treatment and advice are addicted to it’ (Male Psychologist). Gender differences were also reported, with male Palestinians reported to engage in drug use at a greater rate compared with females although these reports cannot be fully relied upon, as one participant in the Gaza Strip focus group observed that ‘drug use is spreading among females but concealed because of the stigma’ (Male Social worker from Middle Camps). The prevalence of the type of drug consumed in the OtP was also discussed. The Bethlehem focus group noted ‘the Palestinian market is like any market, heroin exists, hashish, crystal, ecstasy, trip, patches/stamps and medical marijuana with 100 NIS/gram, it all exists and [is] very similar to the Israeli market’ (Male Anti-Drug Authority Officer). Consistent across all discussions was the reported use of tramadol, and particularly the Gaza Strip focus group where this opioid was reported to be ‘the most commonly used’. Identified across all discussions was the observation that the choice of drug depends upon availability; once the market has consumed one supply, a new substance surfaces, creating new demand. This was raised in the Ramallah focus group as ‘hydro [synthetic cannabinoids], nice guy [synthetic cannabinoids] and marijuana have been trendy for a long time, but when people became aware [of the harms], they retreated and turned to pills’ (Male Anti-Drug Authority Officer). The Jerusalem focus group also reported a shift toward synthetic cannabinoids stating they recently International Journal of Mental Health and Addiction (2020) 18:1097–1112 1101 Table 1 Demographic detail of four focus groups across the Gaza Strip and the West Bank Participant demographic Focus group Location of focus group Duration minutes Total number Gender (n) Place of residence (n) Profession (n) FG1: Gaza SARC Hall, Gaza City 75 11 Female (3) Male (8) • Gaza and North Governorate (6) • Middle camps, and South (Khanyonis and Rafah) (5) • Teacher/high school (1) • Teacher/primary school (1) • Psychiatry and addiction nurse (2) • Pharmacists drug dispensing (2) • Psychiatric doctor (2) • Lawyer in the Anti-Drug Authority (Gaza) (1) • Social worker (2) FG2: Jerusalem Al-Sadiq Al-Taieb meeting room in Jerusalem 100 10 Female (2) Male (8) • Jerusalem authority (10) • Officers in Anti-Drug Authority (3) • Social worker (3) • Officer in family protection and juvenile police (1) • Psychologists (3) FG3: Bethlehem Palestinian Social and Psychological Professionals’ association - Bethlehem Branch 105 14 Female (6) Male (8) • Bethlehem (12) • Aida Camp (1) • Dora Al-Khalil (1) • Social worker (3) • Officers in Anti-Drug Authority (3) • Social Services volunteer (1) • Law researcher (1) • Officers in the directorate of juvenile and family protection (2) • Psychologists and educational counsellors (2) • Administration members of the Palestinian Social and Psychological Professionals’ Association (2) FG4: Ramallah Al-Sadiq Al-Taieb Hall in Ramallah 75 7 Female (4) Male (3) • Ramallah (5) • Jerusalem suburbs (2) • Officers in Anti-Drug Authority (2) • Officers in the directorate of juvenile and family protection (1) • Officer for the anti-drug section in the Ministry of Social Development (1) • Members and workers of Al-sadiq Al-Taieb association(3) 1102 International Journal of Mental Health and Addiction (2020) 18:1097–1112 Fig. 1 Thematic map ‘Palestinian Children living with sibling and parental drug use’ Fig. 1 Thematic map ‘Palestinian Children living with sibling and parental drug use’ Fig. 1 Thematic map ‘Palestinian Children living with sibling and parental drug use’ ‘witnessed the synthetic cannabinoids to be the most on demand substance in Palestinian streets, but addicts always use it with hashish, they smoke them together, so if an addict wants to quit from Hydro, they seek hashish’ (Male Psychologist). Polysubstance use prevalence was reported across all discussions. Psychosocial Causal Factors of Drug Use in Palestine Most consistent across all four focus groups was the problem of family breakdown, when this was experienced, irrespective of its cause, this resulted in ‘the end of the supervision of the children’ and external peer influences on children became ‘a group of bad friends…drag[ging] him to drugs as a way of releasing the pressure’ (Male Psychologist Jerusalem group). The ease at which Palestinian adults and young people can access drugs was a dominant theme across all focus groups. Several factors were discussed to explain this access, which include ‘today there is a dealer in every village and corner, before; you had to travel to Jerusalem to specific places to get the substance…as well as cheap prices’ (Male Psychologist and Educational Counsellor in Bethlehem); improved manufacturing and distribution channels means ‘it is synthesized locally…in 2018 the West Bank territory became an area of cultivation of drugs through plantations’ (Male Psychologist Jerusalem); vulnerable children are targeted by drug dealers ‘in the promotion of drugs where the penalty is less because they are minors’ (according to a male nurse working in the Psychiatry and Addiction unit in the Gaza Strip); and women and children are given drugs unwittingly, creating dependency and further demand. All participants agreed that the media and improved technologies facilitated the normalisa- tion of drug use, particularly among young people. In the Gaza Strip, participants reported that access to the ‘internet and the proliferation of pornographic and corrupt sites is a major reason, especially after wide spread of smart mobile telephones’ (Male Nurse). Those working in Jerusalem noted the sensationalisation of drugs and its effects through broader film and media outlets such as the ‘internet, TV… Social media…Technology as the main factor’ (Male Anti- Drug Authority Officer) because ‘children imitate what they see’. Two of the four focus groups (Jerusalem and Bethlehem) reported how patriarchal values and beliefs contribute to the drug problem within Palestinian families. Blame, however, was placed on mothers who, in order to protect herself and her family, often hide the problem. One participant in Jerusalem reported how ‘the mother is under pressure whether it was for sexual abuse or to keep the house together for the sake of her daughters’ (Male Juvenile and Family Protection Police Department). Psychosocial Causal Factors of Drug Use in Palestine The lack of social and institutional controls within the OtP was described as predominant factors related to drug use. The political regime and occupation were discussed in the Bethlehem focus group; ‘since 1995 until today, we did not become a Palestinian state and there was no improvement. This general political frustration led to the use of drugs, and recently they brought down the tax on alcoholic beverages … encouraged drinking and stimulated using drugs’ (Male Social Worker). This was echoed by the Jerusalem focus group, where participants alleged that ‘this distribution and revolution is due to the occupation to keep the youth in coma and away from the Palestinian cause, so they help in the drugs revolution’ (Male Social Worker). The occupation and regime appeared to have resulted in an insufficient and ineffective correctional system, whereby laws are not routinely implemented, meaning that services to support those within the correctional system or victims of crime fail. For example, participants from Bethlehem explained how drug dealers capitalise on the demand for drugs by exploiting the law: ‘The Juvenile Law in 2016 considered those under 18 years as a victim and those under 15 considered as a special treatment other than the 15-18 year old. The law was very lenient with this category (under 15), so drug dealers got advantage from this law and exploited the children in trade, distribution, and others. This situation has an impact on the exploitation of children [sic]’ (Male Officer in the Directorate of Juvenile and Family Protection). The issue of poor social controls included local structures (e.g. schools; religious groups). Participants from Bethlehem agreed that the lack of follow-up when children dropped out of school meant that ‘there is no observation and no awareness in schools, which increases the current drug situation’ (Female Volunteer in the Palestinian Social and Psychological International Journal of Mental Health and Addiction (2020) 18:1097–1112 1103 Professionals Association). Those from Ramallah felt the ‘weakness of values and the absence of religious persuasion contributed greatly’ (Female member and worker of the Al-Sadiq Al- Taieb Association) to the drug problem. Psychosocial Causal Factors of Drug Use in Palestine Likewise, participants working in Bethlehem observed how mothers were blamed for failure to fully care for their children particularly when the father is absent: ‘Mothers today doesn’t [sic] take care of her children or follow them because she is busy. The absence of the role of the family and the absence of the father’s authority contributes to the situation’ (Male Anti-Drug Authority Officer). While professionals in the Ramallah focus group suggested drug use as a developmental factor, in that adolescent drug users have a natural curiosity around experimentation; a range of individual factors related to substance abuse in Palestinian communities was described by all groups as interrelated with both situational factors and cultural stigma being factors. The choice to use drugs was primarily reported to be driven by people’s sense of ‘loss of hope… fear, psychiatric distress’ or as a strategy to cope and ‘escape problems’ and obtain ‘temporary relief’ from the fear of violence, poverty and insecurity. Gender was observed by some participants as also determining drug activity and motivation for drug use. For instance, while men use drugs to heighten arousal for ‘sexual reasons’; women use drugs to cope with sexual exploitation. In Jerusalem, women’s addiction was described as ‘linked to prostitution, if the female became addicted to it, then it will begin to interact with sex by using drugs so she will have a higher mood combining drugs and sex’ (Male Anti-Drug Authority Officer). Drug use within women was observed to be strongly related to sexual abuse within the home, women International Journal of Mental Health and Addiction (2020) 18:1097–1112 1104 and girls tended to be introduced to drugs ‘usually by her husband if he was a user or her brother or even her father’ (Male Anti-Drug Authority Officer). Indeed, participants from Ramallah noted how women and girls ‘are mostly victims, whether friends or husband or employment or exploitation or others, I haven’t seen a girl who started drugs on her own’ (Male Anti-Drug Authority Officer) not only are they sexually abused but are at risk of being ‘killed for her [drug] abuse’ (Male Anti-Drug Authority Officer). Stigma of the individual user and their family was observed as underpinning the challenges of help-seeking behaviours for addiction, and resulted in hidden use, and attempts to deal with the issue themselves. Psychosocial Causal Factors of Drug Use in Palestine In Bethlehem, participants reported that ‘parents know that their son is addicted, but they won’t turn [sic] him to a rehabilitation centre’ (Female Social Worker). In the Gaza Strip, they too recognised people not seeking treatment because of the ‘stigma and its social dangers’. In Jerusalem, participants described how families ‘usually kick the addict out’ leading to ‘family disintegration and stigma’. The issue of stigma is also highly gendered; whereby, a female addict ‘is more rejected from society than male’ (Male Social Worker in Bethlehem) and most treatment centres will not ‘accept females unless it is a critical case’ (Female Psychologist and Educational Counsellor). One of the reasons for this gender inequality discussed in all focus groups was that ‘society blames women more and stigmatise her’. When a female is addicted to drugs, a Ramallah focus group participant stated, the family response was to hide the situation: ‘They make sure to keep it confidential and no one knows because the topic of drugs is always related to ethics and honour. She may never get married and even her married sisters may become divorced. Their view is to protect the house and the family from the great shame she brought’ (Female staff Ministry of Social Development). While the stigma of addiction persists, according to two focus groups (Bethlehem and Ramallah), there appeared a shift in perspective on addiction to that of a medical issue in younger generations. Participants in the Bethlehem focus group reported a shift toward one of care and compassion, it was noted: ‘if people knew this information about a person they will keep dealing with him, accept him and see him...the addict is a patient not a criminal… previously the addict hiding in shame and now he is declaring his using of drugs’ (Female Social Worker). The Consequences for Children and Families Living with Drug Use All focus groups recognised the significant vulnerability of children living in a household with either a parent or sibling using drugs. One particular issue observed is that children were at risk of physical and sexual violence; ‘inside the house violence may be implanted [sic] on them because the addicted father or adult is usually violent especially in the case of a missing dose, and resorts to beating his children’ (Male officer in Juvenile and Family Protection in Ramallah). Those working in Bethlehem reported how ‘kids are sexually assaulted…the child stops feeling safe in his family and the family becomes a source of fear. So, the child starts resorting to other people who may also exploit him’ (Male Social Worker). In the Gaza Strip, one participant said; ‘there have been several cases of sexual exploitation of children who have left school’ (Male Primary school teacher). Children dropping out of school to help support the economic needs of the family were described as another consequence of drug use within the family. Likewise, ‘a lot of bullying is prevalent’ in schools ‘especially where the mother or father are addicted, is a targeted group and is exploited’ (Female Psychologist in Jerusalem). Children were also observed to become addicts or drug dealers themselves when drug use is in the family. One participant in International Journal of Mental Health and Addiction (2020) 18:1097–1112 1105 Bethlehem reported ‘cases where the father exploited his children to bring drugs, promote and distribute, and because the influence of the father on children is great, they accidentally use drugs and become addicted’ (Male Anti-Drug Authority Officer). Bethlehem reported ‘cases where the father exploited his children to bring drugs, promote and distribute, and because the influence of the father on children is great, they accidentally use drugs and become addicted’ (Male Anti-Drug Authority Officer). Children’s social, emotional and psychological development was observed to become impaired when drug use is in the family. In Bethlehem, participants felt that through learnt behaviour and poor role modelling the ‘disintegration of the personal structure of the child’ leads ‘to many problems in the future’ (Male Psychologist and Educational Counsellor). In Jerusalem, a participant observed, particularly ‘from the early years of age when he witnesses these behaviours, he will copy them’ (Male Social Worker). The Consequences for Children and Families Living with Drug Use The second consequence experi- enced by children and families was observed to be increased criminality, both in terms of becoming victims of crime as well as becoming criminalised themselves. Women and children are vulnerable to being victims of sexual and violent assaults, including murder. Examples were reported across all focus groups, for example, a woman was killed in Bethlehem by her husband ‘with the knowledge of his family who didn’t try to help’ (Female Psychologist and Educational Counsellor). Violence was also viewed to be used to help fund drug use in the Gaza Strip, where there are ‘criminal cases filed for thefts and armed robberies and in some cases, killing for the money to buy the drug’ (Male Primary School Teacher) and Palestinian women are sexually exploited to secure finances or the safety of their family. Young people were viewed to be at risk of becoming criminalised. In Jerusalem, participants reported if the father breaks ‘the law, he will be put away in jail’; therefore, children ‘steal for the sake of getting money for house expenditure because there is no provider to depend on’ (Male Juvenile and Family Protection Police Department). As children drop out of school, they turn to the streets to ‘beg and steal’ (Gaza Strip) or children themselves become part of the distribution chain and begin to deal drugs for others, they ‘smuggle drugs because they are the most innocent category in society so a dealer can get use of this point’ (Female Psychologist in Jerusalem) or, as reported in Bethlehem, this becomes part of the family norm as the ‘father exploit(s) his children to bring drugs, promote and distribute’ (Male Anti-Drug Authority Officer). Drug use within Palestinian families appeared deeply connected with family disintegration; all focus groups described how families become unsafe and broken: ‘The family of the addict is a sick family’ (Male Psychologist and Educational Counsellor in Bethlehem). This destruc- tion is caused by an ‘absence of social security. The Consequences for Children and Families Living with Drug Use This issue has affected all communities and families even good families because it has instilled fear for their children and others, the absence of laws’ (Female Administrator in the Palestinian Social and Psychological Profes- sionals Association) in which ‘the impact of drugs on the economic situation is negative, in terms of their deviation due to unemployment and also affects security and safety and increase crime, it is all linked together’ (Female Law Researcher Department of Social Development in Bethlehem). Those working in the Gaza Strip highlighted how ‘many divorces are registered because of the husband’s addiction and the resulting problems’ (Male Pharmacist in the Psychiatric Department) which doubly stigmatises the family as ‘the stigma of addiction affects the entire family and affects their social relationship, especially in marriage’ (Female psycho social support in the Mental Hospital). As a result of this breakdown, children’s futures are compromised which places jeopardy on the overall Palestinian family’s financial security. Potential Solutions to the Problem Are Complex and Multi-Faceted Potential solutions to deal with the myriad of factors and challenges faced by drug-using families in Palestine were discussed. Awareness raising and education were a persistent theme. International Journal of Mental Health and Addiction (2020) 18:1097–1112 1106 While awareness levels appear to be improving across the Gaza Strip and the West Bank, there remain challenges, requiring greater investment of effort across a range of domains, including education, health, family, religious communities and social media. In Jerusalem, one partici- pant noted: ‘the child in his early stages spends most of his time being at school, there comes the role of the Ministry of Education in strengthening the social workers and psychologists in staying in constant contact with children who suffer from this problem at home’ (Male Anti- Drug Authority Officer). Schools and educational institutions were viewed to be the most logical place to start early education around the harms of drug use, and other health concerns, such as smoking. Those working in the Gaza Strip argued that there is a real need because ‘fighting child smoking…is widespread among them and is a major entry point for addiction’ (Male Psychiatric Physician in the Mental Hospital). In addition to raising children’s’ aware- ness, the need for enhanced parenting support and family education was warranted. Those in Bethlehem suggested there ought to be ‘awareness for their parents, they need to have training courses and awareness of how to deal with their children, how to become leaders and how to deal with the problem’ (Female Administrator in the Palestinian Social and Psychological Professionals Association). Interventions were advised to be delivered in the family home, through ‘visits to families in their homes in order to raise awareness to avoid the problem’ (Female Psychologist and Educational Counsellor). One participant in Jerusalem stated that they should ‘not raise awareness about the child alone but to the mother and father as well, and care about the psychological emptying of them. I want to work on the whole house with all its members not only the children’ (Male Anti-Drug Authority Officer). Awareness regarding access to treatment and how treatment might help people is also required across the OtP, ‘awareness shouldn’t create intimidation, but…keep the message positive with a positive impact on the community’ (female Ministry of Social Development in Ramallah). Potential Solutions to the Problem Are Complex and Multi-Faceted Participants across all discussion in the West Bank and the Gaza Strip observed the need to improve security and develop greater social controls across the OtP, for example, through greater supervision in schools or in families. It was not clear how these controls might be operationalised; however, one participant in Jerusalem argued legal restraints are needed: ‘Follow-up of parents to their children, when my son returns at 1am at night and I do not know where he was at that time, this is absolutely wrong, there must be a follow-up and strict laws in the house, so things are not left alone’ (Male Juvenile and Family Protection Police Department). In Ramallah and Bethlehem, participants felt that children should be removed from the family and sent to ‘institutions that foster children from families who are exposed and affected by the abuse of one parent’ (Female Ministry of Social Development in Ramallah). Improving access to treatment was highlighted, alongside the need for enhanced resourcing of drug rehabilitation centres. While primary health care and mental health clinics have begun to emerge in the Gaza Strip, drug rehabilitation centres do not appear to be a priority. Access and uptake to treatment were also observed to be dependent on gender and cost. Those in Bethlehem highlighted that one centre does not accept women causing ‘injustice and stigma greater for females than males’ (Female Psychologist and Educational Counsellor). While ‘in Huda society, addicts were treated free of charge at the expense of the association’ (Female Psychologist in Jerusalem), this is not the case for all provinces, focus groups suggested access to treatment should be free. Treatment for younger addicts was also advised to be targeted toward their needs in which professionals could ‘work with families and children to receive services and subsequent care’ (Female Law Researcher in Bethlehem). All focus groups spoke about the need for greater investment in strength-based approaches to tackle the issue of drug use, particularly when attempting to help prevent children becoming International Journal of Mental Health and Addiction (2020) 18:1097–1112 1107 addicts. Suggestions included ‘youth-to-youth programs’ (Gaza Strip) or peer mentoring schemes, which would provide ‘good, trained friends within specific programs that pass certain behaviours to their peers. Passing a specific message from a peer to peer’ (Male Social Worker in Jerusalem). Potential Solutions to the Problem Are Complex and Multi-Faceted Activities are required that would draw on the strengths and skills of young people helping them ‘improve their skills and with other things that occupy them from the streets’ (Female Psychologist and Educational Counsellor in Bethlehem); ‘open sports clubs and amusement parks under the supervision of the officials’ (Female Law Researcher in Bethlehem); and to ‘take advantage of his energy in a positive way and expand his hobbies’ (Male Anti-Drug Authority Officer in Jerusalem). Not only would these approaches help children’s development, but these may help them deal with some of the problems and challenges they face within their communities ‘finding clubs or places reserved for children for extracurricular activities where they are trained on how they face the problem’ (Male Social Worker in Jerusalem). Participants wanted to capitalise on the free time children have by occupying their time productively exploiting all opportunities by ‘teaching them professions, activities, ideas and organise their time toward the positive side to avoid their falling addiction’ (Female Administrator in the Palestinian Social and Psychological Professionals Association); ‘kill leisure time, and register them in clubs and activities’ (Male Officer in the Directorate of Juvenile and Family Protection in Bethlehem); ‘intensify youth camps especially in the summer’ (Male Pharmacist in the Psychiatric Department in the Gaza Strip); and ‘after school there is a lot of free time and in the summer vacation as well, so we have to support the idea of summer camps’ (Male Social Worker in Jerusalem). One of the key messages was best summed up by one Jerusalem participant who noted that young people ‘are like a tree, if we water it right it bears fruit, but if we neglect it, it will be dry’ (Male Anti-Drug Officer). Discussion There has been little research or response activity to Palestinian children’s unique needs relating to trauma and vulnerabilities caused by drug abuse in the home United Nations Office for Drugs and Crime (UNODC) 2019; (Van Hout et al. 2019). This unique study represents a first attempt to understand the issue from the perspectives of those working in the field and highlights the urgent need to respond to the needs of Palestinian children exposed to drug abuse in the home. The impact of drug abuse on these children, their siblings and their parents is significant, notwithstanding the existing traumas, instability and conflict in the OtP. The situation of children and their mothers is exacerbated by the hidden nature of drug abuse within their families. This focus group study underpins and expands the evidence base highlighted in the team’s review of literature (Van Hout et al. 2019). They continue to paint a concerning picture of families being subjected to multiple pressures and exposed to a multitude of risks including use of drugs as a coping mechanism for trauma, stigmatisation for that drug use, restricted access to services, family breakdown, sexual abuse, criminality and lack of agency over day to day existence. Of particular concern is the stigma experienced by women (and mothers) using drugs, many of whom have experienced multiple trauma including sexual assault, which adds to an already long list of additional pressures for them which may add to their rationale for using drugs. When these women feel unable to access support, the fall out includes inevitable suffering and increased risk for their children who will have already experienced trauma themselves through the occupation. 1108 International Journal of Mental Health and Addiction (2020) 18:1097–1112 Some of the issues are not wholly different to the issues currently facing communities across the world. For instance, focus groups noted the increased presence of NPS, a relatively recent phenomenon even in affluent Western countries, which demonstrates that the market for illicit substances is evolving as rapidly in the OtP as elsewhere, and this in itself creates new issues for authorities, not least because of the lack of evidence for long-term use of such substances (Mounteney et al. 2016). Added to stigma (Defence for Children International/ Palestine Section 2007; Damiri et al. 2018a; Van Hout et al. Discussion 2019;, the poor social services, social and political tensions and inability of the Palestinian law enforcement to police the influx of drugs into the West Bank, East Jerusalem and Gaza compound the issue (Massad et al. 2016). The perceived similarity between the Palestinian and Israeli drug markets highlights the ease with which substances are entering the Palestinian areas and the probability that, because of the blockade, drugs are entering the OtP through Israeli routes whether official (a concern raised by some participants) or not. Some discussion in focus groups highlighted the negative consequence of reduced punishments for children who were caught selling illicit substances, although the evidence is that by decriminalising or lessening penalties for drug use at least, this has benefits to societies as they treat drug use as a health issue rather than a criminal issue (Volkow et al. 2017). This is evidenced in the current Palestinian Drug Law referring to the drug addict as a patient not a criminal, and makes it more likely that the individual concerned would seek help for their drug use if they viewed it as becoming problematic, and for parents, this would have the knock-on effect of limiting the potential for damage to their wider families. In Bethlehem, this was becoming the case increasingly, and the benefits of this approach might encourage other regions to facilitate non-punitive programmes. This is particularly of interest given the recent 2019 opening of the PNRC in Bethlehem which provides free drug dependence treatment to women and men (United Nations Office for Drugs and Crime (UNODC) 2019). Improving access to treatment would also be beneficial to the population, but the financial constraints placed on operation of support systems to Palestinians by the Israeli authorities make expanding this kind of activity more difficult. Of greater importance is the general lack of social structures noted such as schools or religious groups, which provide less opportunity for engagement and greater opportunity for individuals to seek other forms of past times in the absence of contact with other people in these settings. This is again not unique to the OtP, as cuts to education or social support structures due to austerity have proven in other countries (Abramsky 2019), but the pressures on Palestinian infrastructure and its social structures are clearly impacting on more than just education or religious practice. Discussion The identified family breakdown, which has been a consistent theme of Palestinian life for many years, provides one less fall-back support structure for individuals already living without a safety net. However, because of the importance placed on social structures, they can also cause damage in terms of stigma for individuals who fall outside of those defined boundaries, and this is clearly a barrier to families accessing help when needed. This stigma may be more prominent in predominantly Muslim societies (Arfken and Ahmed 2016). Problematic substance use can often be a coping mechanism for pressures on an individual experienced through the conditions in which they live. While suggestions around potential solutions focussed on control strategies, it is difficult to believe that reducing the use of drugs would be possible to any great extent in a country which has experienced long-term occupation with regular conflict, and the trauma that this engenders in children from an early age. Substances including heroin have been used for instance by personnel in areas of conflict globally over a long period of time as a way of coping with the horror of violence, and this is International Journal of Mental Health and Addiction (2020) 18:1097–1112 1109 recognised by one of the participants who explained drug use by ‘loss of hope…fear [and] psychiatric distress’, but most people using drugs in areas of conflict cease when removed from that combat zone (Robins et al. 2010). While Palestinian children experience trauma on a daily basis through their lived experience of the occupation which leads to substance use, it may still be possible to reduce the harms associated with drug use by emphasising education around types of substances, routes of administration and the effect on mental health. This was recognised by discussion here of the various mediums in which education and awareness could take place, and particularly using social media channels can be a way of cost effectively disseminating health messages to younger people through peer-to-peer sharing (Evans et al. 2017). Visits to households by health visitors would also be beneficial but could be labour intensive if not focussed on particular sections of the population; however, training for existing staff around substance use would potentially be a feasible action. Discussion Removing children from Palestinian families with problematic substance use on the other hand could adversely affect the child if they experience detachment from their families from this process and control strategies for substance use have regularly had poor outcomes associated with them (Maciel and de Vargas 2015). While some of these interventions may be helpful, the context is an occupation and blockade which is causing substantial suffering to the population and some focus group members indeed believed that the high levels of drug use were part of a strategy of population control. Children regularly experience multiple adverse childhood experiences (ACEs) includ- ing depression, anxiety and associated post-traumatic stress disorder (PTSD) which are all well-known risk factors for heightened levels of drug use, and this was acknowledged too by the focus groups (Volpe et al. 2017). The role of ACEs within an environment of occupation and political violence remains unstudied and may be key to developing effective intervention that promotes resilience within this young vulnerable population in the OtP. Attempting to reduce harm while not tackling the underlying causes of why individuals might use a substance will at best provide small benefits to the person concerned and the wider family. Because this area is under-researched, it is a hidden and very stigmatised social problem in Palestinian society, as this is only a small qualitative study of frontline professional perspectives, it is difficult to make generalisations but the speed at which participants felt the situation had escalated over the last few years in the OtP. We recognise this means that action is needed now to prevent further escalation of the issues concerned. At a time when civil society is considered by some to be at breaking point and the recent ‘right to return’ protests were described by the United Nations as ‘a call for help from a population in despair’ (United Nations/The question of Palestine 2019), it is clear that inaction on this issue may contribute to further social unrest for an already besieged population. References Abramsky, S. (2019). The price of austerity in England. Nation, 308(8), 22–25 Al-Afifi, M. F., Sakka, M., Shehada, M., & Afifi, R. (2015). 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Conclusion The continued political and economic tensions and dense populations living in the West Bank and Gaza Strip have compounded the impact of drug abuse and addiction in the home. This unique study has illustrated the experiences of professionals working with Palestinian families and children affected by substance use and addiction in the home. It paints a concerning picture of how drug abuse impacts on Palestinian children, mothers and parents subjected to multiple pressures, stigmas, risks and harms relating to their situation, and underscores the urgent need for a united and strategic response. 1110 International Journal of Mental Health and Addiction (2020) 18:1097–1112 Funding Information The study was funded by the Global Challenges Research Fund (GCRF) Small Grants Scheme 2019, Liverpool John Liverpool John Moore’s University, 2019 to Professor Marie Claire Van Hout. Conflict of Interest The authors declare that they have no conflict of interest. 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Harm reduction: an alternative to the failure of the war on drugs. Cogitare Enferm, 20(1), 205–208. Massad, S. G., Shaheen, M., Karam, R., Brown, R., Glick, P., Linnemay, S., & Khammash, U. (2016). Substance use among Palestinian youth in the West Bank, Palestine: a qualitative investigation. BMC Public Health, 16(1), 800. Mounteney, J., Griffiths, P., Sedefov, R., Noor, A., Vicente, J., & Simon, R. (2016). The drug situation in Europe: an overview of data available on illicit drugs and new psychoactive substances from European monitoring in 2015. Addiction, 111(1), 34–48. ( ) Nowell, L. S., Norris, J. M., White, D. E., & Moules, N. J. (2017). Thematic analysis: striving to meet the trustworthiness criteria. International Journal of Qualitative Methods, 16(1), 1609406917733847. 1111 International Journal of Mental Health and Addiction (2020) 18:1097–1112 Palestinian Central Bureau of Statistics (PCBS). (2018). Press report on the Labour Force Survey results. Ramallah - Palestine. Retrieved from https://www.pcbs.gov.ps/portals/_pcbs/PressRelease/Press_En_13-2- 2019-LF-e.pdf. Last accessed on 2 July 2019. p y Palestinian National Institute of Public Health. (2017a). Estimating the extent of illicit drug use in Palestine. Retrieved from https://www.unodc.org/documents/middleeastandnorthafrica/Publications/Estimating_the_ Extent of Illicit Drug Use in Palestine.pdf. Last accessed on 2 July 2019. _ _ _ g_ _ _ p y Palestinian National Institute of Public Health. (2017b). Illicit drug use in Palestine. Retrieved from https://www. unodc.org/documents/publications/Illicit_Drug_Use_in_Palestine.pdf. Last accessed on 2 July 2019. Robins, L., Helzer, J., Hesselbrock, M., & Wish, E. (2010). Vietnam veterans three years after Vietnam: how our study changed our view of heroin. The American Journal on Addictions, 19(3), 203–211. Smithson, J. (2000). Using and analysing focus groups: limitations and possibilities. International Journa Social Research Methodology, 3(2), 103–119. Sweileh, W. M., Arafat, R. T., Al-Khyat, L. S., Al-Masri, D. M., & Jaradat, N. A. (2004). A pilot study to investigate over-the-counter drug abuse and misuse in Palestine. Saudi Medical Journal, 25(12), 2029–2032. Thabet, A. A. M., & Dajani, J. K. (2012). Substance abuse among Palestinians in the West Bank and Gaza Strip. References Arab Journal of Psychological Science, 36, 76–78. Sweileh, W. M., Arafat, R. T., Al-Khyat, L. S., Al-Masri, D. M., & Jaradat, N. A. (2004). A pilot study investigate over-the-counter drug abuse and misuse in Palestine. Saudi Medical Journal, 25(12), 2029–20 investigate over-the-counter drug abuse and misuse in Palestine. Saudi Medical Journal, 25(12), 2029–2032. Thabet, A. A. M., & Dajani, J. K. (2012). Substance abuse among Palestinians in the West Bank and Gaza Strip. Arab Journal of Psychological Science, 36, 76–78. Thabet, A. A. M., & Dajani, J. K. (2012). Substance abuse amo Arab Journal of Psychological Science, 36, 76–78. United Nations Office on Drugs and Crime (UNODC). (2019). Independent Evaluation of PSEY13. Supporting the establishment of evidence-based drug dependence treatment and rehabilitation system for the Palestine National Rehabilitation Centre. Retrievd from https://www.unodc.org/documents/evaluation/Independent_ Project_Evaluations/2019/PSEY13_Final_Independent_Project_Evaluation_Jan_2019.pdf. Last accessed on 2 July 2019. United Nations/The question of Palestine. (2019). The UN independent commission of inquiry on the 2018 Gaza protests. Retrieved from https://www.un.org/unispal/document/un-independent-commission-of-inquiry-on- protests-in-gaza-presents-its-findings-press-release/. Last accessed on 2 July 2019. Van Hout, M. C., Al-Afifi, M. F., Abushams, L., Kewley, S., Quigg, Z., Whitfield, M., et al. (2019). Palestinian children’s experiences of drug abuse in the home in the occupied territories of Palestine: a scoping review of extant literature. International Journal of Mental Health and Addiction, 1–14. https://doi.org/10.1007 /s11469-019-00085-2. Volkow, N. D., Poznyak, V., Saxena, S., Gerra, G., & Network, U. I. I. S. (2017). Drug use disorders: impact of a public health rather than a criminal justice approach. World Psychiatry, 16(2), 213–214. Volpe, E. M., Rodriguez, L., & Read, J. P. (2017). The relationship between trauma and PTSD and the influence of alcohol and drug use. In Alcoholism-clinical and experimental research (Vol. 41, p. 148A). Hoboken: Wiley. Waterston, T., & Nasser, D. (2017). Access to healthcare for children in Palestine. BMJ Paediatrics Open, 1(1). Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Affiliations Mohammed Al-Afifi1 & Leen Abushams2 & Mazen Sakka1 & Maha Shehada1 & Riad Afifi1 & Majed Alloush3 & Afaf Rabee3 & Stephanie Kewley4 & Zara Quigg5 & Mark Whitfield5 & Jim McVeigh5 & Mayyada Wazaify2 & Marie Claire Van Hout5 Mohammed Al-Afifi m.afifi47@gmail.com Leen Abushams leen_abushams@hotmail.com Mazen Sakka sakkamazen@gmail.com Maha Shehada maha702000@gmail.com Riad Afifi afifiriad@gmail.com Mohammed Al-Afifi m.afifi47@gmail.com International Journal of Mental Health and Addiction (2020) 18:1097–1112 1112 Majed Alloush al_sadiq22000@yahoo.com Afaf Rabee afafrabee@yahoo.com Stephanie Kewley s.kewley@ljmu.ac.uk Zara Quigg z.a.quigg@ljmu.ac.uk Mark Whitfield m.whitfield@ljmu.ac.uk Jim McVeigh j.mcveigh@ljmu.ac.uk Mayyada Wazaify m.wazaify@ju.edu.jo 1 Substance Abuse Research Centre (SARC), Gaza, Palestine 2 School of Pharmacy, University of Jordan, Amman, Jordan 3 Al-Sadiq Al-Taieb Association, Ramallah, Palestine 4 School of Science, Liverpool John Moore’s University, Liverpool, UK 5 Public Health Institute (PHI), Liverpool John Moore’s University, Liverpool, UK Majed Alloush al_sadiq22000@yahoo.com Afaf Rabee afafrabee@yahoo.com Stephanie Kewley s.kewley@ljmu.ac.uk Zara Quigg z.a.quigg@ljmu.ac.uk Mark Whitfield m.whitfield@ljmu.ac.uk Jim McVeigh j.mcveigh@ljmu.ac.uk Mayyada Wazaify m.wazaify@ju.edu.jo 1 Substance Abuse Research Centre (SARC), Gaza, Palestine 2 School of Pharmacy, University of Jordan, Amman, Jordan 3 Al-Sadiq Al-Taieb Association, Ramallah, Palestine 4 School of Science, Liverpool John Moore’s University, Liverpool, UK 5 Public Health Institute (PHI), Liverpool John Moore’s University, Liverpool, UK 1 Substance Abuse Research Centre (SARC), Gaza, Palestine 2 School of Pharmacy, University of Jordan, Amman, Jordan 3 Al-Sadiq Al-Taieb Association, Ramallah, Palestine 4 School of Science, Liverpool John Moore’s University, Liverpool, UK 5 Public Health Institute (PHI), Liverpool John Moore’s University, Liverpool, UK
https://openalex.org/W2560517286	https://escholarship.org/content/qt1qb6c91j/qt1qb6c91j.pdf?t=oz1jrq	English	null	4D flow streamline characteristics of the great arteries twenty years after Lecompte and direct spiral arterial switch operation (DSASO) in simple TGA	Global cardiology science & practice	2,016	cc-by	1,655	UC Irvine UC Irvine Previously Published Works Title 4D flow streamline characteristics of the great arteries twenty years after Lecompte and direct spiral arterial switch operation (DSASO) in simple TGA https://escholarship.org/uc/item/1qb6c91j Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org Images in cardiology O P E N A C C E S S O P E N A C C E S S g gy 4D flow streamline characteristics of the great arteries twenty years after Lecompte and direct spiral arterial switch operation (DSASO) in simple TGA Hans-Hinrich Sievers1, Léon M. Putman1, Arash Kheradvar2, Dominik Gabbert3, Philip Wegner3, Jens Scheewe3, Mona Salehi-Ravesh3, Hans-Heiner Kramer3, Carsten Rickers3 http://dx.doi.org/ 10.21542/gcsp.2016.29 Submitted: 18 August 2016 Accepted: 12 September 2016 c⃝2016 The Author(s), licensee Magdi Yacoub Institute. This is an open access article distributed un- der the terms of the Creative Com- mons Attribution license CC BY-4.0, which permits unrestricted use, dis- tribution and reproduction in any medium, provided the original work is properly cited. 1 Department of Cardiac and Thoracic Vascular Surgery, University Hospital of Schleswig-Holstein, Campus Lue- beck, Ratzeburger Allee 160, 23538 Luebeck, Germany 2 University of California Irvine, Edwards Lifesciences Center of Advanced Car- diovascular Technology, Irvine, CA 92697 3 Department of Congenital Heart Dis- ease and Pediatric Cardiology, Uni- versity Hospital of Schleswig-Holstein, Campus Kiel, Arnold-Heller-Str. 3, 24105 Kiel, Germany Email: Hans-Hinrich.Sievers@uksh.de 1 Department of Cardiac and Thoracic Vascular Surgery, University Hospital of Schleswig-Holstein, Campus Lue- beck, Ratzeburger Allee 160, 23538 Luebeck, Germany 2 University of California Irvine, Edwards Lifesciences Center of Advanced Car- diovascular Technology, Irvine, CA 92697 BACKGROUND Simple TGA accounts for 5–7% of patients with congenital heart malformations and, untreated, has a bad prognosis with nearly 90% cases leading to death within one year. After a breakthrough of surgery for simple TGA inaugurating the two-stage arterial switch operation (ASO) by Yacoub in 19773, the door was opened for successful anatomical correction. In the early 1980s, the heart-lung machine technology was refined to allow for new- borns to benefit from primary ASO. In 1981 the Lecompte technique4 was developed and used routinely to the present day. This technique transposes the pulmonary bifurcation in front of the aorta, which does not warrant spiral physiological blood flow in the great arteries (Figure 1). In outgrown patients some shortcomings of this technique have surfaced1, calling for new techniques2. In the early 1990s we performed a consecutive series of six patients performing a DSASO (for details see reference5) and re-evaluated these patients twenty years after the operation by MRI technique (for technical details see reference5) showing promising results5. One example is presented here. The blood flow streamlines in the great arteries (Figure 2) show the spiral configuration comparable to a normal person (male; 33.4 years, no cardiac anomaly by echocardiography) (Figure 3) and more physiological than the streamlines in the Lecompte technique (Figure 1) with the blood flow vectors not in a spiral arrangement. There was no semilunar valve dysfunction in the DSASO patient. At the operation the TGA patient was 4 days old and had simple TGA with the aortic root 31◦to the right of the pulmonary root. Figure 2 shows also that twenty years after DSASO the aortic or neo-pulmonary root has rotated somewhat to left indicating a potential morphogenetic adaptation to alter flow conditions during early age. ABSTRACT Transposition of the great arteries (TGA) is caused by discordance between the great arteries and the ventricles. If left untreated, this anomaly has a disastrous perspective. More recent surgical approach for correction includes the Lecompte technique in which the pulmonary bifurcation is transposed anterior to the aorta, which may be less physiologic. Although the early results are excellent, there is potential for future problems involving the great arteries and semilunar valves1. These potential problems necessitate the development of other improved surgical techniques2. Here we report an MRI 4D flow study related to a case of simple TGA whose primary surgical correction – direct spiral arterial switch operation (DSASO) – was performed twenty years ago in an attempt to restore physiologic arrangement among the great arteries and semilunar valves. *Email: Hans-Hinrich.Sievers@uksh.de Submitted: 18 August 2016 Accepted: 12 September 2016 c⃝2016 The Author(s), licensee Magdi Yacoub Institute. This is an open access article distributed un- der the terms of the Creative Com- mons Attribution license CC BY-4.0, which permits unrestricted use, dis- tribution and reproduction in any medium, provided the original work is properly cited. Cite this article as: Sievers HH, Putman LM, Kheradvar A, Gabbert D, Wegner P, Scheewe J, Salehi- Ravesh M, Kramer HH, Rickers C. 4D flow streamline characteristics of the great arteries twenty years after Lecompte and direct spiral arterial switch operation (DSASO) in simple TGA, Global Cardiology Science and Practice 2016:29 http://dx.doi.org/10.21542/gcsp.2016.29 Page 2 of 4 Sievers et al. GCSP 2016:29 Page 2 of 4 Sievers et al. GCSP 2016:29 DISCUSSION Normal anatomy is always the optimal solution in nature warranting normal physiology. This holds also true for surgery in congenital cardiac diseases. TGA is characterized by aorto-ventricular discordance. Theoretically re-transposing the great arteries would be Figure 1. Blood streamlines twenty years after the Lecompte technique showing the flow acceleration particularly at the pulmonary bifurcation. Figure 1. Blood streamlines twenty years after the Lecompte technique showing the flow acceleration particularly at the pulmonary bifurcation. Page 3 of 4 Sievers et al. GCSP 2016:29 g 4 Sievers et al. GCSP 2016:29 Figure 2. Blood streamlines twenty years after direct spiral arterial switch operation (DSASO). Figure 2. Blood streamlines twenty years after direct spiral arterial switch operation (DSASO). Figure 3. Blood streamlines in a healthy volunteer. Figure 3. Blood streamlines in a healthy volunteer. the optimal solution. The near normal blood streamlines of the great arteries twenty years after DSASO provide some evidence that this re-transposition of the great arteries might be possible with excellent results. These data may stimulate a re-thinking on the optimal surgical technique of simple TGA preferably in cases with less rotation of the aortic root to the right of the pulmonary root. Refined operative techniques such as deliberately dissecting the arch and pulmonary arteries till the hilum to get more length of the great arteries as well as the transfer of the left coronary orifice as deep and posterior as possible in the related sinus of the pulmonary root including special anastomotic techniques like the trap door may be of advantage to prevent potential left coronary artery distortion by the rotation process of the neo-pulmonary root. In some cases an elongation of the pulmonary artery with a strip of autologous pericardium may allow for tension and torsion free anastomosis6,7. the optimal solution. The near normal blood streamlines of the great arteries twenty years after DSASO provide some evidence that this re-transposition of the great arteries might be possible with excellent results. These data may stimulate a re-thinking on the optimal surgical technique of simple TGA preferably in cases with less rotation of the aortic root to the right of the pulmonary root. DISCUSSION Refined operative techniques such as deliberately dissecting the arch and pulmonary arteries till the hilum to get more length of the great arteries as well as the transfer of the left coronary orifice as deep and posterior as possible in the related sinus of the pulmonary root including special anastomotic techniques like the trap door may be of advantage to prevent potential left coronary artery distortion by the rotation process of the neo-pulmonary root. In some cases an elongation of the pulmonary artery with a strip of autologous pericardium may allow for tension and torsion free anastomosis6,7. Page 4 of 4 Sievers et al. GCSP 2016:29 Page 4 of 4 Sievers et al. GCSP 2016:29 LESSONS LEARNED This report suggests that recreating ‘normal’ anatomical relationship during surgical treatment of complex congenital heart disease could have important functional implications. Furthermore, post-operative studies of 4D blood flow could provide important insights in planning future operations. REFERENCES [1] Hazekamp MG. Long-term follow-up after the arterial switch operation: Not as perfect as we would have hoped? J Thorac Cardiovasc Surg. 2015;149:968. p J g 5 49 9 [2] Agnoletti G, Ou P, Celermajer DS, Boudjemline Y, Marini D, Bonnet D, et al. Acute angulation of the aortic arch predisposes a patient to ascending aortic dilatation and aortic regurgitation late after the arterial switch operation for transposition of the great arteries. J Thorac Cardiovasc Surg. 2008;135:568–72. [2] Agnoletti G, Ou P, Celermajer DS, Boudjemline Y, Marini D, Bonnet D, et al. Acute angulation of the aortic arch predisposes a patient to ascending aortic dilatation and aortic regurgitation late after the arterial switch operation for transposition of the great arteries. J Thorac Cardiovasc Surg. 2008;135:568–72. ; 35 5 7 [3] Yacoub MH, Radley-Smith R, Maclaurin R. Two-stage operation for anatomical correction of transpositio of the great arteries with intact interventricular septum. Lancet. 1977;1:1275–8. g p 977; 75 [4] Lecompte Y, Zannini L, Hazan E, Jarreau MM, Bex JP, Tu TV, et al. Anatomic correction of transposi the great arteries. J Thorac Cardiovasc Surg. 1981;82:629–31. g p 97 [4] Lecompte Y, Zannini L, Hazan E, Jarreau MM, Bex JP, Tu TV, et al. An the great arteries. J Thorac Cardiovasc Surg. 1981;82:629–31. [5] Rickers C, Kheradvar A, Sievers HH, Falahatpisheh A, Wegner P, Gabbert D, et al. Is the Lecompte Technique the last word on transposition of the great arteries repair for all patients? A magnetic resonance imaging study including a spiral technique two decades postoperatively. Interactive CardioVasc Thoracic Surgery. 2016; in press. g y ; p [6] Sievers HH, Scharfschwerdt M, Putman LM. In vitro evaluation of physiological spiral anastomoses for the arterial switch operation in simple transposition of the great arteries: a first step towards a surgical alternative? Interact Cardiovasc Thorac Surg. 2015;21:157–62. g [7] Sievers H-H, Kheradvar A, Kramer H-H, Rickers C. 3D Heart Model and 4D Flow MRI 20 Years after Spiral Arterial Switch Operation. Thorac Cardiovasc Surg Rep. 2016.
https://openalex.org/W2339877260	https://www.matec-conferences.org/articles/matecconf/pdf/2016/16/matecconf_spbwosce2016_01002.pdf	English	null	Steel and composite structural shells in the construction of high-rise building	MATEC web of conferences	2,016	cc-by	3,797	a Corresponding author : redish132132@yandex.ru Steel and composite structural shells in the construction of high-rise building Andrey Redkin 1,a, Mikhail Baranovskii 1 and Vladimir Tarasov 1 1 St. Petersburg State Polytechnical University, Polytechnicheskaya st. 29, 195251, St. Petersburg, Russia Andrey Redkin 1,a, Mikhail Baranovskii 1 and Vladimir Tarasov 1 Andrey Redkin 1,a, Mikhail Baranovskii 1 and Vladimir Tarasov 1 1 St. Petersburg State Polytechnical University, Polytechnicheskaya st. 29, 195251, St. Petersburg, Russia Abstract. In modern construction thin-shell structures are very popular because they can be easily used to make different constructive and architectural shapes. The main value of such constructions is the ability to use them as load-bearing structures and as the element of architectural image of building at the same time. Thin-shell structures allow us to solve the problems of construction rigidness and stableness, therefore it is rational to use shells in high- rise building structures. At the same time construction of high-rise buildings is connected with a number of special requirements. Disregard of those requirements can cause destruction of the building. In this research there were considered some challenges of high-rise building design on the business complex example. The comparative analysis of two various bearing shells was made. The first case was a metal grid shell, and the second was the combination of metal elements and reinforced concrete ribs. The reinforced concrete ribs (stiffeners) were used to improve the rigidness of the whole structure and to increase the bearing capacity. DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences / 0 0 ( 201 ) conf Web of Conferences MATEC atec m , 6 6 0 , 201 5 1 201 0 0 6 0 1 5 3 3 2 2 DOI: 10.1051/ C ⃝Owned by the authors, published by EDP Sciences / 0 0 ( 201 ) conf Web of Conferences MATEC atec m , 6 6 0 , 201 5 1 201 0 0 6 0 1 5 3 3 2 2 Steel and composite structural shells in the construction high-rise building Andrey Redkin 1,a, Mikhail Baranovskii 1 and Vladimir Tarasov 1 1 St. Petersburg State Polytechnical University, Polytechnicheskaya st. 29, 195251, St. Petersburg, Russia 4 1 Introduction High-rise buildings are different from ordinary buildings and have a number of features in design process and also during construction. The distinctive features are: High-rise buildings are different from ordinary buildings and have a number of features in design process and also during construction. The distinctive features are: A significant influence of horizontal dynamic loads (wind loads); A significant influence of horizontal dynamic loads (wind loads); Heavy load on bearing structures and foundations, in comparison with ordinary buildings; Increased requirements to account the anthropogenic and environmental factor influence. The most important factors are: earthquakes, floods, fires, terrorist acts and solar radiation; Difficult connections between steel and concrete bearing elements; All these features must be considered in the process of high-rise building structural scheme selection. All these features must be considered in the process of high-rise building structural scheme selection. 4 MATEC Web of Conferences 2 Literature review For the first time bearing shells were used in the construction by Russian engineer C.G. Shukhov in 1896. He developed and patented three types of load-bearing grid shells: hanging, convex and tower- shell (patents of the Russian Empire No. 1894, No. 1895, No. 1896; March 12, 1899). Nowadays the theoretical basics of bearing shell mechanics are well studied. The research results are stated in monographs: V.Z. Vlasov - “Stroitelnaya mehanika obolochek”, A.P. Filin -“Elementyi teorii obolochek”, K.Z. Galimova - “Matematicheskaya teoriya obolochek”, H.M. Mushtari - “Nelineynaya teoriya obolochek”, V.V. Novozhilov - "Teoriya tonkih obolochek”, A.L. Goldenveizer - “Teoriya tonkih uprugih obolochek” and others. Researches of many scientists are devoted for the creation and development of methods: E.L. Axelrad - “Gibkie obolochki”, Yu.P. Artyuhin and P.G. Velikanov - “Raschyot anizotropnyih obolochek metodom granichnyih elementov”, D.V. Vaynberg - “Matrichnyie algoritmyi v teorii obolochek vrascheniya” and others. The plasticity and creep theories are very important in bearing shell calculations. Researches in this area were made by scientists: R.A. Arutyunyan, G.I. Byikovtsev, R.A. Vasin, A.A. Vakulenko, M.A. Grekov, A.A. Gvozdev, G. Grinberg, O.Yu. Dinariev, A.S. Dehtyar and others. The plasticity and creep theories are very important in bearing shell calculations. Researches in this area were made by scientists: R.A. Arutyunyan, G.I. Byikovtsev, R.A. Vasin, A.A. Vakulenko, M.A. Grekov, A.A. Gvozdev, G. Grinberg, O.Yu. Dinariev, A.S. Dehtyar and others. In the twentieth century bearing shells were widely adopted in construction, and the number of projects with this scheme was increasing each decade. Therefore, new problems and difficulties in the construction appear in process of bearing shell technology development [1-29], but many of them are already well studied [29-37]. 3 Issue definition The aim of the research was to determine the rules of high-rise building structural scheme and materials selection. And also optimal structural scheme determination from two different variants. 4 Materials and Methods The research object was 41-storey business complex with two various types of bearing shell. General view of the research object is shown on the Fig. 1. Figure 1. General view of the research object. Figure 1. General view of the research object. 01002-p.2 SPbWOSCE-2015 The analytical model in case with steel shell elements in SCAD MATEC Web of Conferences Figure 3. The analytical model in case with steel shell elements in SCAD MATEC Web of Conferences MATEC Web of Conferences MATEC Web of Conferences Figure 3. The analytical model in case with steel shell elements in SCAD Figure 3. The analytical model in case with steel shell elements in SCAD Reinforced concrete shell elements and the central core walls are made of B30class concrete, load- bearing columns – of B60 class concrete. The concrete ribs thickness changes along height from 400 to 200 mm. The columns diameter changes along height from 1000 mm in the lower floors to 300 mm at the top. SPbWOSCE-2015 The building has a "shamrock" shape on the plan, also, the horizontal cross-section changes along the height of the building. Due to the architectural features and the height of the building it was decided to apply the "load-bearing outer shell in combination with a central core" [38]. Central load- bearing core (stiff trunk) are usually made of reinforced concrete, steel and its combinations. Stiff trunk perform the role of rigid vertical consoles fixed in the soils and perceives predominantly horizontal loads. The rigidity of the trunk depends on the shape and cross-sectional area of the stair- elevator site, which sizes are determined according to the number of elevators and stairs requirements. The core is rigidly connected with the metal bearing shell by monolithic reinforced concrete slabs. Bearing structures can be prefabricated or monolithic. In the initial period of high-rise building development bearing structures were normally made from steel. Currently, bearing structures are normally made from reinforced concrete, because this material has better fire resistance and lower cost, and its strength characteristics under compression is quite the same. Nowadays B80 and B100 class concretes are created, although in construction practice are mainly used lower classes of high- strength concretes (B60 and B70), because with the concrete class growth increases its cost, fragility and decreased fire resistance. The grid shell bearing system was selected because of insolation requirements for the building facades. The elements of the grid shell were tubes of square section with 200 mm side and 14 mm thickness. The steel was selected as a material for shell rods because of tensile stresses, which concrete cannot resist. The difference between the two cases of structural schemes was: in the first case, each wing was locked by reinforced concrete elements to improve the stiffness and to make building heavier in these parts. In the second case, instead of the stiffeners on the building edges metal shell with the same configuration as in other parts of the building was used. The analytical models of both cases were made in the SCAD Office software complex. Loads were applied according to the code “Nagruzki i vozdeystviya” [39], including equivalent wind load that was applied statically. Analytical models are shown on Fig. 2,3. Figure 2. The analytical model in case with reinforced concrete stiffeners in SCAD Figure 2. The analytical model in case with reinforced concrete stiffeners in SCAD 01002-p.3 Figure 3. Results d D iscussion an 5 In the SCAD Office finite element environment two selected structural cases were calculated on the rigid foundation. 1-st and 41-st floors were selected for the calculation results comparison .They show the values that characterize the construction work: stresses in the elements, deformations. On the first floor compressive stresses in bearing structures from the vertical loads and bending from the wind are maximum. On the 41-st floor stresses in the elements from the force effects are very small, but there are large values of the total vertical deformations. Deformations of the slab of the first floor on Z-axis and stresses in the shell elements are shown on Fig 4,5,6,7. g , , , Figure 4. First floor slab deformations on axis Z (mm) in case with reinforced concrete stiffeners -18.35 -17.10 -15.84 -14.58 -13.33 -12.07 -10.81 -9.56 -8.30 -7.05 -5.79 -4.53 -3.28 -2.02 Figure 4. First floor slab deformations on axis Z (mm) in case with reinforced concrete stiffeners 01002-p.4 01002-p.4 01002-p.4 Figure 5. First floor slab deformations on axis Z (mm) in case with steel shell elements -9.02 -7.82 -6.62 -5.41 -4.21 -3.01 -1.80 -17.45 -16.24 -15.04 -13.84 -12.63 -11.43 -10.23 SPbWOSCE-2015 Figure 5. First floor slab deformations on axis Z (mm) in case with steel shell elements Figure 6. Stresses (N, [T]) in the shell elements in case with reinforced concrete stiffeners -414.18 -369.73 -325.29 -280.84 -236.39 -191.94 -147.49 -103.05 -58.60 -14.15 30.30 74.74 119.19 163.64 Figure 6. Stresses (N, [T]) in the shell elements in case with reinforced concrete stiffeners Figure 6. Stresses (N, [T]) in the shell elements in case with reinforced concrete stiffeners 01002-p.5 MATEC Web of Conferences Figure 7. Stresses (N, [T]) in the shell elements in case with steel shell elements -498.59 -448.12 -397.65 -347.17 -296.70 -246.23 -195.75 -145.28 -94.80 -44.33 6.14 56.62 107.09 157.56 MATEC Web of Conferences Figure 7. Stresses (N, [T]) in the shell elements in case with steel shell elements First floor slab deformations in two different cases (Fig.4,5) are almost identical. In case with reinforced concrete stiffeners tension is less, the maximum compressive stress is 414.18 t, while in case with steel shell elements - 498.59 t. The difference is 20%. This shows that there is a stresses redistribution: in case with steel shell elements more weight falls on the shell rods along the glazed facades of the building. Results d D iscussion an 5 Stresses in the first floor columns in case with reinforced concrete stiffeners (T) -1676.97 -1648.47 -2213.86 -2186.45 -2761.40 -2719.79 -1647.18 -2173.84 -2743.39 -2745.01 -2175.16 -1649.55 -2760.15 -2210.74 -1676.20 -1645.48 -2183.16 -2719.59 -2209.65 -2361.19 -2768.62 -2771.21 -2362.61 -2211.18 -2773.79 -2716.72 -2347.43 -2165.86 -2381.90 -2190.80 -2772.25 -2382.85 -2197.58 -2164.00 -2348.54 -2715.01 MATEC Web of Conferences -1647.18 -2173.84 -2743.39 -2745.01 -2175.16 -1649.55 Figure 10. Stresses in the first floor columns in case with reinforced concrete stiffeners (T) Figure 11. Stresses in the first floor columns in case with steel shell elements (T) In columns along the edges of the wings difference is much more: in case with steel shell elements compressive stress in the column is 2211.18 t, In case with reinforced concrete stiffeners – 1676.97 t. -1676.97 -1648.47 -2213.86 -2186.45 -2761.40 -2719.79 -2173.84 -2743.39 -2745.01 -2175.16 -2760.15 -2210.74 -1676.20 -1645.48 -2183.16 -2719.59 -2209.65 -2361.19 -2768.62 -2771.21 -2362.61 -2211.18 -2773.79 -2716.72 -2347.43 -2165.86 -2381.90 -2190.80 -2772.25 -2382.85 -2197.58 -2164.00 -2348.54 -2715.01 Figure 10. Stresses in the first floor columns in case with reinforced concrete stiffeners (T) -1676.97 -1648.47 -2213.86 -2186.45 -2761.40 -2719.79 -2760.15 -2210.74 -1676.20 -1645.48 -2183.16 -2719.59 -1676.97 -1648.47 -2213.86 -2186.45 -2761.40 -2719.79 -2760.15 -2210.74 -1676.20 -1645.48 -2183.16 -2719.59 Figure 10. Stresses in the first floor columns in case with reinforced concrete stiffeners (T) -2209.65 -2361.19 -2768.62 -2771.21 -2362.61 -2211.18 -2773.79 -2716.72 -2347.43 -2165.86 -2381.90 -2190.80 -2772.25 -2382.85 -2197.58 -2164.00 -2348.54 -2715.01 -2773.79 -2716.72 -2347.43 -2165.86 -2381.90 -2190.80 -2772.25 -2382.85 -2197.58 -2164.00 -2348.54 -2715.01 -2773.79 -2716.72 -2347.43 -2165.86 -2381.90 -2190.80 -2772.25 -2382.85 -2197.58 -2164.00 -2348.54 -2715.01 Figure 11. Stresses in the first floor columns in case with steel shell elements (T) Figure 11. Stresses in the first floor columns in case with steel shell elements (T) Figure 11. Stresses in the first floor columns in case with steel shell elements (T) In columns along the edges of the wings difference is much more: in case with steel shell elements compressive stress in the column is 2211.18 t, In case with reinforced concrete stiffeners – 1676.97 t. Results d D iscussion an 5 g 41-st floor on Z-axis slab deformations in two different cases are presented on Fig. 8, Figure 8. 41-st floor slab deformations on axis Z (mm) in case with reinforced concrete stiffeners -94.54 -89.72 -84.9 -80.08 -75.25 -70.43 -65.61 -60.79 -55.97 -51.15 -46.32 -41.50 -36.68 -31.86 Figure 8. 41-st floor slab deformations on axis Z (mm) in case with reinforced concrete stiffeners 01002-p.6 Figure 9. 41-st floor slab deformations on axis Z (mm) in case with steel shell elements -69.52 -63.21 -56.91 -50.60 -44.30 -37.99 -31.69 -113.65 -107.34 -101.04 -94.74 -88.43 -82.13 -75.82 SPbWOSCE-2015 SPbWOSCE-2015 Figure 9. 41-st floor slab deformations on axis Z (mm) in case with steel shell elements Deformations of the 41-st floor slabs in different cases are considerably different. Deformations in case with reinforced concrete stiffeners is less approximately on 60-75 mm (the difference is about 73%). Furthermore, it is obvious that in case with steel shell elements, all slab in the wing parts fall on 70-80 mm comparative the central core. With increasing height of the building slabs have increasing slope from the central core to the wing edges. As a consequence, loads on the intermediate supporting columns increases. In case with reinforced concrete stiffeners stresses are redistributed due to changes in the stiffness of the whole building. Massive reinforced concrete ribs better than the metal grid shell perceive compressive loads. Because of their greater rigidity, despite the higher stresses, that they perceive, there are also smaller deformations. In this case slab doesn't fall from the central core to the edges of the building. Slab works as a plate on two relatively rigid supports and the maximum 55 mm deflection is in the span. In case with steel shell elements the slab at the junction to the trunk, rests on a rigid support, and on the other side (on the edge of the wing) relies on the support with a certain deformability, that leads to the whole slab tilt. In case with steel shell elements compressive stress in the most loaded central columns is 2773.79 t, in case with reinforced concrete stiffeners – 2761.40 t. The values are almost identical (difference less than 0.5%), the difference is negligible. First floor column stresses in both cases are shown on Fig. 10,11. 01002-p.7 Figure 10. 01002-p.8 01002-p.8 SPbWOSCE-2015 The difference is about 32 %. The first floor columns reinforcement calculation was made in program “Arbat” and it is presented in the Tab. 1. Table 1. Layouts and diameters of the rods of reinforcement in both cases Construction case Longitudinal reinforcement Cross reinforcement Layout of a longitudinal reinforcement In case with steel shell elements (compressive stress in the columns is 2773.79 [T]) Longitudinal reinforcement – 20 rods, diameter 32 [mm] Cross reinforcement along the X-axis has 10 mm diameter, rod spacing - 200 [mm] Cross reinforcement along the Y-axis has 10 mm diameter, rod spacing - 200 [mm] In case with reinforced concrete stiffeners (compressive stress in the columns is 2211.18 [T]) Longitudinal reinforcement – 15 rods, diameter 20 [mm] Cross reinforcement along the X-axis has 10 mm diameter, rod spacing - 200 [mm] Cross reinforcement along the Y-axis has 10 mm diameter, rod spacing - 200 [mm] From the results it is obvious that the columns in the variation with the metal shell must be reinforced considerably stronger - required 20 rods, diameter - 32 mm, while in the construction with The difference is about 32 %. The first floor columns reinforcement calculation was made in program “Arbat” and it is presented in the Tab. 1. Layout of a longitudinal reinforcement In case with reinforced concrete stiffeners (compressive stress in the columns is 2211.18 [T]) From the results it is obvious that the columns in the variation with the metal shell must be reinforced considerably stronger - required 20 rods, diameter - 32 mm, while in the construction with stiffeners – 15, diameter - 20 mm. From the results it is obvious that the columns in the variation with the metal shell must be reinforced considerably stronger - required 20 rods, diameter - 32 mm, while in the construction with stiffeners – 15, diameter - 20 mm. 041 (2014) ( ) 5. S. L. Chan, J. of Const. St. Res. 57, 1217–1231 (2001) 6. M. Baranovskiy, V. Tarasov, S. Zimin, Appl. Mech. and Mater. 725-726, 774-78 7. Y. Hea, X. Zhoua, X. Zhanga, Thin-Wall. Str. 60, 1–11 (2012) 8. V. S. Karpilovskiy, E. Z. Kriksunov, A. A. Malyarenko, SCAD Office. Formirovaniye secheniy i raschet ikh geometricheskikh kharakteristik (Izd. ASV, 2011) 9. V. S. Karpilovskiy, E. Z. Kriksunov, A. A. Malyarenko, SCAD Office. Vychislitelnyy kompleks SCAD (Izd. SKAD SOFT, 2011) 10. N. Vatin, J. Havula, L. Martikainen, A. Sinelnikov, A. Orlova, S. Salamakhin, Adv. Mater. Res. 945-949, 1211-1215 (2014) 11. Kompleks programm SCAD office - instrumentariy inzhenera-proyektirovshchika, (PGS, 3, 56, 2007) , 60, 12. Kompleks programm SCAD Office - instrumentariy inzhenera – proyektirovshchika, (PGS, 4 2007) 13. Z. Shubber, The development of the typical roof structure project based on trusses made of roll- welded rhs pipes: Bachelor’s Thesis (Civ. and Const. Eng., Lappeenranta: Saimaa University of Applied Sciences, 80, 2013) pp ) 14. R. Kamnik, B. Kovačič, B. Pribicević, A. Ðapo, Geodetski List, 69 (3), 171-188 (2015) 15. N. Vatin, J. Havula, L. Martikainen, A. Sinelnikov, A. Orlova, S. Salamakhin, Proceedings of the International Conference “Innovative Materials, Structures and Technologies”, 187 (2014) 16. D. Trubina, D. Abdulaev, E. Pichugin, V. Rybakov, M. Garifullin, O. Sokolova, Appl. Mechanics and Mater. 725–726, 752–757 (2015) 17. D. Trubina, D. Abdulaev, E. Pichugin, V. Rybakov, Appl. Mech. and Mater. 633–634, 982–990 (2014) 18. D. Trubina, D. Abdulaev, E. Pichugin, V. Rybakov, Appl. Mech. and Mater. 633–634, 1133– 1139 (2014) 19. M. V. Ananina, N. A. Beresneva, L. L. Shurovkina, Const. of Uniq. Build.s and Str. 7 (22), 54–70 (2014) 20. M. Garifullin, D. Trubina, N. Vatin, Appl. Mech. and Mater. 725–726, 697–702, (20 21. V. Rybakov, A. Sergey, Appl. Mech. and Mater. 725–726, 746–751 (2015) T. Nazmeeva, R. Guslinscky, Adv. Mater. Res. 941–944, 1871–1875 (2014) 22. N. Vatin, T. Nazmeeva, R. Guslinscky, Adv. Mater. Res. 941–944, 1871–187 23. B. Kovačič, R. Kamnik, International Journal for Engineering Modelling, 20 (1-4), 77-84 (2007) 24 N Vatin A Sinelnikov M Garifullin D Trubina Appl Mech and Mater 633 634 1037 1041 23. B. Kovačič, R. Kamnik, International Journal for Engineering Modelling, 20 (1-4), 7 23. B. Kovačič, R. Kamnik, International Journal for Engineering Modelling, 20 (1-4), 77-84 (2007) 24. N. Vatin, A. Sinelnikov, M. Garifullin, D. Trubina, Appl. Mech. and Mater. 633–634, 1037–1041 (2014) 24. N. Conclusions 6 According to the calculation results and two variations of building structures comparison, it is possible to say that it is advisable to the use the combined shell of reinforced concrete and steel elements. Grid metal bearing shell, at first, reduces the weight of building bearing structures, and secondly, allows to satisfy the insolation requirements. Stiffeners play the role of additional rigid trunks. They unload metal shell and the intermediate columns inside the building. 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https://openalex.org/W4210949976	https://publikationen.bibliothek.kit.edu/1000143265/148445924	English	null	Oil Droplet Coalescence in W/O/W Double Emulsions Examined in Models from Micrometer- to Millimeter-Sized Droplets	Colloids and interfaces	2,022	cc-by	14,716	Keywords: DCTA; microﬂuidics; surfactant interaction; interfacial properties; PGPR; Tween 40 Citation: Leister, N.; Yan, C.; Karbstein, H.P. Oil Droplet Coalescence in W/O/W Double Emulsions Examined in Models from Micrometer- to Millimeter-Sized Droplets. Colloids Interfaces 2022, 6, 12. https://doi.org/10.3390/ colloids6010012 Citation: Leister, N.; Yan, C.; Karbstein, H.P. Oil Droplet Coalescence in W/O/W Double Emulsions Examined in Models from Micrometer- to Millimeter-Sized Droplets. Colloids Interfaces 2022, 6, 12. https://doi.org/10.3390/ colloids6010012 Nico Leister * , Chenhui Yan and Heike Petra Karbstein Nico Leister * , Chenhui Yan and Heike Petra Karbstein Institute of Process Engineering in Life Sciences, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany; ufefa@student.kit.edu (C.Y.); heike.karbstein@kit.edu (H.P.K.) * Correspondence: nico.leister@kit.edu Abstract: Water-in-oil-in-water (W1/O/W2) double emulsions must resist W1–W1, O–O and W1–W2 coalescence to be suitable for applications. This work isolates the stability of the oil droplets in a double emulsion, focusing on the impact of the concentration of the hydrophilic surfactant. The sta- bility against coalescence was measured on droplets ranging in size from millimeters to micrometers, evaluating three different measurement methods. The time between the contact and coalescence of millimeter-sized droplets at a planar interface was compared to the number of coalescence events in a microﬂuidic emulsion and to the change in the droplet size distributions of micrometer-sized single and double emulsions. For the examined formulations, the same stability trends were found in all three droplet sizes. When the concentration of the hydrophilic surfactant is reduced drasti- cally, lipophilic surfactants can help to increase the oil droplets’ stability against coalescence. This article also provides recommendations as to which purpose each of the model experiments is suited and discusses advantages and limitations compared to previous research carried out directly on double emulsions. Citation: Leister, N.; Yan, C.; Karbstein, H.P. Oil Droplet Coalescence in W/O/W Double Emulsions Examined in Models from Micrometer- to Millimeter-Sized Droplets. Colloids Interfaces 2022, 6, 12. https://doi.org/10.3390/ colloids6010012 Academic Editors: Eduardo Guzmán and Armando Maestro Received: 13 December 2021 Accepted: 26 January 2022 Published: 8 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. 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/). colloids and interfaces colloids and interfaces colloids and interfaces 1. Introduction The oil phase (yellow) has a lipophilic surfac- tant dissolved within to stabilize the water droplets. The outer water phase (dark blue) has a hydro- philic surfactant to stabilize the oil droplets. This work focuses on the coalescence of oil droplets. Figure adapted from Leister and Karbstein [4]. Figure 1. Possible coalescence paths in a monodispersed double emulsion. In the inner water phase (light blue), an active ingredient can be encapsulated. The oil phase (yellow) has a lipophilic surfactant dissolved within to stabilize the water droplets. The outer water phase (dark blue) has a hydrophilic surfactant to stabilize the oil droplets. This work focuses on the coalescence of oil droplets. Figure adapted from Leister and Karbstein [4]. Figure 1. Possible coalescence paths in a monodispersed double emulsion. In the inner water phase (light blue), an active ingredient can be encapsulated. The oil phase (yellow) has a lipophilic surfac- tant dissolved within to stabilize the water droplets. The outer water phase (dark blue) has a hydro- philic surfactant to stabilize the oil droplets. This work focuses on the coalescence of oil droplets. Figure adapted from Leister and Karbstein [4]. Figure 1. Possible coalescence paths in a monodispersed double emulsion. In the inner water phase (light blue), an active ingredient can be encapsulated. The oil phase (yellow) has a lipophilic surfactant dissolved within to stabilize the water droplets. The outer water phase (dark blue) has a hydrophilic surfactant to stabilize the oil droplets. This work focuses on the coalescence of oil droplets. Figure adapted from Leister and Karbstein [4]. Concerning the surfactant interactions, an often-discussed topic is whether the hy- drophilic surfactant has a positive or a negative influence on the encapsulation efficiency. The general observation for PGPR-stabilized water droplets is that smaller hydrophilic surfactants are more likely to increase release than high-molecular-weight polymers [7,13,14]. Surprisingly, the outer interface, and thus O–O coalescence, is less commonly discussed in terms of the interactions between hydrophilic and lipophilic surfactants. Neumann et al. [15] published a study in which the influence of PGPR on the stability of oil droplets with different types of polyvinyl alcohol as hydrophilic surfactants is pre- sented. Gülseren and Corredig [16] examined the properties of interfaces covered with PGPR and proteins in general, but focused more on the resulting stability of inner water droplets. 1. Introduction Since oil coalescence is the main reason behind the macroscopic instabilities in double emulsions, such as changes in viscosity and creaming, it is important to under- stand the composition of the outer interface and the resulting stability against O–O coa- Concerning the surfactant interactions, an often-discussed topic is whether the hy- drophilic surfactant has a positive or a negative inﬂuence on the encapsulation efﬁ- ciency. The general observation for PGPR-stabilized water droplets is that smaller hy- drophilic surfactants are more likely to increase release than high-molecular-weight poly- mers [7,13,14]. Surprisingly, the outer interface, and thus O–O coalescence, is less com- monly discussed in terms of the interactions between hydrophilic and lipophilic surfactants. Neumann et al. [15] published a study in which the inﬂuence of PGPR on the stability of oil droplets with different types of polyvinyl alcohol as hydrophilic surfactants is presented. Gülseren and Corredig [16] examined the properties of interfaces covered with PGPR and proteins in general, but focused more on the resulting stability of inner water droplets. Since oil coalescence is the main reason behind the macroscopic instabilities in double emulsions, such as changes in viscosity and creaming, it is important to understand the composition of the outer interface and the resulting stability against O–O coalescence as well. lescence as well. The stability of the oil droplets is especially in focus when lower concentrations of hydrophilic surfactants are suggested to enhance the encapsulation efficiency [8,13,17]. Since some hydrophilic surfactants are known to decrease the inner water droplets’ stability with higher concentrations [7,18], one way to produce stable double emulsion formulations could lie in the drastic decrease in the hydrophilic surfactants’ concentration. According to previous studies, this could help to keep the inner water droplets stable, but the oil droplets might then be more prone to coalescence. Therefore, this study focuses on the question of how drastically the concentration of hydrophilic surfactants can be g y g The stability of the oil droplets is especially in focus when lower concentrations of hydrophilic surfactants are suggested to enhance the encapsulation efﬁciency [8,13,17]. Since some hydrophilic surfactants are known to decrease the inner water droplets’ stability with higher concentrations [7,18], one way to produce stable double emulsion formulations could lie in the drastic decrease in the hydrophilic surfactants’ concentration. 1. Introduction Water-in-oil-in-water (W1/O/W2) double emulsions are disperse systems, in which oil droplets within a continuous water phase are ﬁlled with smaller water droplets. This structure allows the encapsulation and targeted release of water-soluble substances e.g., vitamins or iron in foods [1] or proteins and peptides in pharmaceutics and cosmetics [2]. To distinguish the continuous water phase from the water phase with the encapsulated substance, the inner water phase is commonly called W1 and the outer water phase W2. Despite the large quantity of research that can be found on a variety of formulations for double emulsion applications, products with this structure are still seldomly found in the market because of stability problems. Academic Editors: Eduardo Guzmán and Armando Maestro Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. y p Double emulsions tend to be more unstable than single emulsions due to the two differently curved interfaces [3]. These interfaces must be stabilized against three different coalescence paths, as shown in Figure 1 [4]. From the idealized starting point on the left side, the following coalescence mechanisms can occur: W1–W1, the coalescence of inner water droplets; W1–W2, the release of inner water droplets into the continuous phase; and O–O, the coalescence of (ﬁlled) oil droplets. To prevent or slow down coalescence, at least two surfactants are added. To keep the inner water droplets emulsiﬁed within the oil, most research is performed on the oil-soluble surfactants polyglycerol polyricinoleate (PGPR) and Span 80 [5]. For stabilizing the oil droplets, a variety of different water-soluble surfactants were applied and compared in different studies [6,7]. These ranged from short- chained ionic [8] and non-ionic [9] surfactants over synthetic polymers [10] to biopolymers, such as pectins and proteins [11]. Since the two different surfactants distribute at both interfaces and build mixed ﬁlms, the resulting stability of a double emulsion is dependent on how well the two surfactants interact at the interfaces [12]. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/colloids Colloids Interfaces 2022, 6, 12. https://doi.org/10.3390/colloids6010012 2 of 17 stribute lsion is Colloids Interfaces 2022, 6, 12 Figure 1. Possible coalescence paths in a monodispersed double emulsion. In the inner water phase (light blue), an active ingredient can be encapsulated. 1. Introduction According to previous studies, this could help to keep the inner water droplets stable, but the oil droplets might then be more prone to coalescence. Therefore, this study focuses on the question of how drastically the concentration of hydrophilic surfactants can be decreased without destabilizing the oil droplets completely. decreased without destabilizing the oil droplets completely. One challenge that must be overcome to evaluate the stability of isolated oil droplets is to eliminate some of the parameters that affect droplet size. In emulsions in general, the One challenge that must be overcome to evaluate the stability of isolated oil droplets is to eliminate some of the parameters that affect droplet size. In emulsions in general, the droplet size directly after break-up is, apart from the stability against coalescence itself, dependent on the interfacial tension and viscosity ratio between the phases [19]. When comparing different formulations for double emulsions, the interfacial tension is dependent on the surfactant concentrations and their combination. The viscosity of the disperse W1/O phase is a function of the ﬁlling degree of the oil droplets with inner water and can change when the encapsulated water phase is (partly) released during the process. Colloids Interfaces 2022, 6, 12 3 of 17 3 of 17 The change in the oil droplet size during storage is affected by two additional mecha- nisms apart from O–O coalescence: the release of the inner phase by W1–W2 coalescence leads to shrinking droplets and the diffusion of water between the phases can lead to both, increasing and decreasing the droplet sizes, depending on the osmotic pressure in the W1 phase [4]. For these reasons, the direct estimation of coalescence stability from measured oil droplet sizes in double emulsions is always associated with additional assumptions. To overcome these limitations and to be able to perform experiments more quickly, several models of double emulsion have been developed in other studies. One the one hand, the general focus of these models of double emulsion is the decoupling of the production process from the instabilities. In all top-down emulsiﬁcation processes (e.g., high-pressure homogenization, rotor–stator systems), the ﬁnal droplet size is a result of break-up, collision and coalescence at the same time [20]. This results in a process-formulation combination- speciﬁc droplet size distribution. 1. Introduction When the emulsion droplets are produced in a bottom-up approach (e.g., membrane emulsiﬁcation or microﬂuidics), the droplet size can be more easily set to a ﬁxed size [21]. On the other hand, the models can simplify double emulsions to single emulsions, where only one coalescence mechanism is possible, which allows the mechanism to be examined more easily [22]. Additionally, the time and material needed for the examination of each surfactant combination can be reduced. When only examining the coalescence of oil droplets in double emulsions, the inner water droplets can be neglected [15]. When the lipophilic surfactant is added to the oil phase nonetheless, the molecular arrangement of the surfactant molecules at the O/W interface is likely to be the same, as should be the observed stability. The most simpliﬁed version of a coalescence process is a single droplet coalescing at a planar interface with an experimental setup, often called “Diffusion and Coalescence Time Analyzer” (DCTA) [23]. This experiment was used previously to describe the coalescence of oil droplets [24] and water droplets [25] for different surfactant combinations. The time between the contact of the droplet with the interface is called coalescence time and can be used as a value to describe the stability of the ﬁlm between two interfaces [4,26]. This experiment can be performed quickly and with little equipment, but the scatter of data points limits the conclusions as to signiﬁcant differences in stability between two formulations. With microﬂuidic chips of coaxial glass capillaries, monodispersed droplets with a variation in diameter of less than 5% can be produced [21]. The droplet ﬂuid ﬂows through a small tip, allowing droplet break-up in co-ﬂow in the dripping regime [27,28]. To determine the stability of a monodisperse emulsion, the number of coalescence events can be calculated [29]. When two droplets of the same size and volume coalesce, their diameters increase by 3√ 2 ≈1.26; when three droplets coalesce, their diameters increase by 3√ 3 ≈1.44, and so forth. Therefore, the number of coalescence processes that lead to a certain droplet size distribution of a formerly monodispersed emulsion can be calculated. This allows the simultaneous measurement of the stability of many drops and the emulsion droplets can be stored in bulk for deﬁned times, before taking a sample and measuring the number of coalescence events. Emulsions produced with mechanical emulsiﬁcation devices show rather broad droplet size distributions. 1. Introduction When evaluating the stability of such emulsions, either the complete droplet size distributions are compared, or characteristic values are taken from the distribution. The Sauter mean diameter, or x90,3, a characteristic value for the biggest droplets in the collective, which is responsible for creaming, among others, is often used as an example. p This work presents a comparison of the coalescence behavior of oil droplets from typical double-emulsion formulations in models of different size scales. The agreement of the models is demonstrated for oil droplet coalescence and the advantages and disadvan- tages of each system are discussed in terms of experimental effort and the validity of the results for real double emulsions. Using these experiments, the possibility of reducing the hydrophilic surfactant is discussed in terms of oil droplet stability. The results suggest that Colloids Interfaces 2022, 6, 12 4 of 17 lipophilic surfactants can help to stabilize oil droplets in W/O/W double emulsions when the concentration of the hydrophilic surfactant is too low for complete interface coverage. The ﬁndings on the stability of interfaces with multiple interfacial active compounds can also be transferred to other research ﬁelds. Enhanced oil recovery techniques, for exam- ple, also deal with the challenges of the break-up and coalescence of oil droplets within chemically complex systems [30–32]. 2. Materials and Methods 2.1. Materials The oil phase in the experiments was medium-chain triglycerides (MCT, Witarix 40/60), obtained from IOI Oleo GmbH, Hamburg, Germany. In the oil phase, the lipophilic surfactants PGPR from Evonik Dr. Straetmans GmbH, Hamburg, Germany and Span 80 from Carl Roth GmbH & Co. KG, Karlsruhe, Germany were dissolved at 1 wt%, respectively. Both lipophilic surfactants are widely used for the stabilization of W/O emulsion products. While Span 80 is a short-chained surfactant (MW = 429 g/mol) with a head-tail structure, PGPR is a much larger polymeric surfactant (MW ≈2500 g/mol), with hydrophilic and lipophilic groups distributed throughout the molecule. For the water phase, demineralized water was used with the short-chained (MW = 1280 g/mol) hydrophilic surfactant, Tween 40 (Carl Roth GmbH, Karlsruhe, Germany), added in different concentrations. Tween 40 was applied at 1 wt% as typical concentration of application and at 0.003 wt%, the averaged critical micelle concentration (cmc) of Tween 40 described in previous research [33–35]. Additionally, the stability of droplets without additional lipophilic surfactant and without Tween 40 was examined, resulting in nine different formulations for each experiment. For the interfacial tension measurements, microﬂuidics and single-droplet experi- ments, the MCT oil was puriﬁed according to the method described by Dopierala et al. [36] before mixing with surfactants, since the impurities in the unpuriﬁed oil could have a great inﬂuence due to the small interfacial area compared to the sample volume [7]. 2.2. Interfacial Tension Measurements The interfacial tension of interfaces with different combinations of water and oil phases was determined using the pendant drop method (OCA 15 LJ, DataPhysics Instruments GmbH, Filderstadt, Germany) at a constant temperature of 23 ◦C. The interfacial tensions were obtained after an equilibration time of 60 min. All measurements were performed three times. 2.3. Single-Droplet Experiments (DCTA) The measurement setup for obtaining the coalescence time of an oil droplet at an oil/water interface was ﬁrst published by Taboada et al. [24] and is described in detail in their study. Oil droplets with 5 µL volume were formed using a Hamilton syringe with a repeating dispenser (Hamilton Company, Reno, NV, USA). The droplet detached from the tip of the bent needle and ﬂoated to the oil/water interface. The measurement time is deﬁned as the time between the ﬁrst contact of the droplet at the interface and its coalescence. For each surfactant system, measurements were repeated for at least 30 droplets using at least three different cuvettes. Since the measured coalescence times of single droplets scatter statistically, the distributions are plotted in boxplot diagrams [7]. The median value t50 is used for comparing different surfactant systems. 2.4. Microﬂuidic Emulsions Regardless of the combination of different water and oil phases and their different interfacial tensions, oil droplets could be set to the same diameter (d = 144 μm; coefficient of variation (CV) < 4%) by adjusting the flow rates of both phases. The third cannula was used to empty the residual air in the setup prior to the production of microfluidic emulsions and to purge the system after the process. During the production of the monodispersed droplets, it was not in operation. T b ild h l ill i i h d d f b i d Fi d The oil phase flowed inside the conical capillary with the smaller tapered end (d1 = 45 µm), while the continuous water phase ﬂowed through the outer capillary in the same direction. The co-ﬂow stream passed compellingly through the second capillary (d2 = 310 µm) with the larger tapered end. There, oil droplets with highly monodisperse size distribution were formed. Regardless of the combination of different water and oil phases and their different interfacial tensions, oil droplets could be set to the same diameter (d = 144 µm; coefﬁcient of variation (CV) < 4%) by adjusting the ﬂow rates of both phases. The third cannula was used to empty the residual air in the setup prior to the production of microﬂuidic emulsions and to purge the system after the process. During the production of the monodispersed droplets, it was not in operation. To build the setup, glass capillaries with tapered ends were fabricated. First, a round glass capillary (outer diameter: 1 mm, World Precision Instruments, Sarasota, FL, USA) was heated and cyclically pulled using a Micropipette puller (P-1000, Sutter Instrument, Novato, CA, USA). The tapered ends of the capillaries were polished to the desired diam- eter. In this study, two capillaries with diameter of the orifice of 45 μm and 310 μm were used. Next, the capillaries with tapered ends were assembled in an outer capillary (inner diameter: 1.05 mm, Boro Square, Atlantic International Technologies Inc., Rockaway, NJ, USA) and coaxially aligned using a microscope. The distance between the tapered ends of the inner capillaries was set to 75 μm. Syringe pumps (LEGATO® 100, KD Scientific, Hol- liston, MA, USA) were used to pump in the phases at specific flow rates. This process was captured live through a high-speed camera (DMK 33UX273, The Imaging Source Europe GmbH, Bremen, Germany). 2.4. Microﬂuidic Emulsions Microﬂuidic emulsions were produced by a glass capillary system with coaxial glass capillaries. Figure 2 shows a draft of the setup. The construction used here was developed for the production of double emulsions, since three phases can be pumped into the chip simultaneously, and was used in previous studies [37,38]. In this study, the chip is used for single emulsion in co-ﬂow break-up, with an additional downstream ﬂow constriction, as described by Dewandre et al. [39]. The advantage of this setup for the production of Colloids Interfaces 2022, 6, 12 5 of 17 p is used 5 of 17 p is used single emulsions compared to counter ﬂow is that there is no need for complex surface modiﬁcations of the capillary tips to adjust the wettability [39]. as esc ibe by e a e e a [39] e a a age o is se up o e p o uc io o single emulsions compared to counter flow is that there is no need for complex surface modifications of the capillary tips to adjust the wettability [39]. Figure 2. Draft (a) and micrograph (b) of the used microfluidic chip. The three cannulas are con- nected to syringe pumps, controlling the flow-rates. The left and right capillary have a diameter of 45 µm and 310 µm, respectively. Figure 2. Draft (a) and micrograph (b) of the used microﬂuidic chip. The three cannulas are connected to syringe pumps, controlling the ﬂow-rates. The left and right capillary have a diameter of 45 µm and 310 µm, respectively. (a) (b) Figure 2. Draft (a) and micrograph (b) of the used microfluidic chip. The three cannulas are con- nected to syringe pumps, controlling the flow-rates. The left and right capillary have a diameter of 45 µm and 310 µm, respectively. Figure 2. Draft (a) and micrograph (b) of the used microﬂuidic chip. The three cannulas are connected to syringe pumps, controlling the ﬂow-rates. The left and right capillary have a diameter of 45 µm and 310 µm, respectively. The oil phase flowed inside the conical capillary with the smaller tapered end (d1 = 45 µm), while the continuous water phase flowed through the outer capillary in the same direction. The co-flow stream passed compellingly through the second capillary (d2 = 310 µm) with the larger tapered end. There, oil droplets with highly monodisperse size distri- bution were formed. 2.4. Microﬂuidic Emulsions p p To build the setup, glass capillaries with tapered ends were fabricated. First, a round glass capillary (outer diameter: 1 mm, World Precision Instruments, Sarasota, FL, USA) was heated and cyclically pulled using a Micropipette puller (P-1000, Sutter Instrument, Novato, CA, USA). The tapered ends of the capillaries were polished to the desired diameter. In this study, two capillaries with diameter of the oriﬁce of 45 µm and 310 µm were used. Next, the capillaries with tapered ends were assembled in an outer capillary (inner diameter: 1.05 mm, Boro Square, Atlantic International Technologies Inc., Rockaway, NJ, USA) and coaxially aligned using a microscope. The distance between the tapered ends of the inner capillaries was set to 75 µm. Syringe pumps (LEGATO® 100, KD Scientiﬁc, Holliston, MA, USA) were used to pump in the phases at speciﬁc ﬂow rates. This process was captured live through a high-speed camera (DMK 33UX273, The Imaging Source Europe GmbH, Bremen, Germany). 2.5. Measurement of Droplet Diameter of the Microfluidic Emulsions 2.5. Measurement of Droplet Diameter of the Microﬂuidic Emulsions n0 = nt ∑ i=0 d3 i d3 0 𝑛0 Table A1), the igure 3 are giv n0 = nt ∑ i=0 d3 i d3 0 𝑛0 Table A1), the igure 3 are giv (1) ated (a) (b) (c) Figure 3. Exemplary micrographs of microfluidic double emulsions after different storage times. Each droplet is labelled with its diameter in pixels. (a) Emulsion droplets directly after production, the emulsion is monodispersed. (b) More stable formulation after three months. (c) More instable emulsion after storage of one day. Figure 3. Exemplary micrographs of microﬂuidic double emulsions after different storage times. Each droplet is labelled with its diameter in pixels. (a) Emulsion droplets directly after production, the emulsion is monodispersed. (b) More stable formulation after three months. (c) More instable emulsion after storage of one day. (b) (c) (a) (b) (a) (c) Figure 3. Exemplary micrographs of microfluidic double emulsions after different storage times. Each droplet is labelled with its diameter in pixels. (a) Emulsion droplets directly after production, the emulsion is monodispersed. (b) More stable formulation after three months. (c) More instable emulsion after storage of one day. Figure 3. Exemplary micrographs of microﬂuidic double emulsions after different storage times. Each droplet is labelled with its diameter in pixels. (a) Emulsion droplets directly after production, the emulsion is monodispersed. (b) More stable formulation after three months. (c) More instable emulsion after storage of one day. 2.6. Top-Down Single and Double Emulsions For the double emulsions, an inner emulsion with (𝑚𝑊1/𝑚𝑂 = 1/9) was produced i h i di (IKA® i LAB® IKA® W k G bH & C KG S f G The number of occurred coalescence events per 100 droplets c is then calculated from the remaining number of droplets nt after a storage time: agic LAB , IKA Werke GmbH & Co. KG, Staufen, Ger ng to a maximum peripheral speed of 25.1 m/s for 5 min. spersed in the outer water phase with (𝑚𝑊1/𝑂/𝑚𝑊2 = c = n0 −nt n0 × 100 (2) min. = (2) 1/9) using a colloid mill (IKA® magic LAB®, IKA®-Werke GmbH & Co. KG, Staufen, Ger- many) for 5 min, with a gap width between the rotor and the stator of 0.318 mm. The In the Appendix information (Table A1), the measured diameters and calculated number of coalescence events from Figure 3 are given. 2.6. Top-Down Single and Double Emulsions 2.5. Measurement of Droplet Diameter of the Microfluidic Emulsions 2.5. Measurement of Droplet Diameter of the Microﬂuidic Emulsions Microﬂuidic emulsions were stored in rolled rim bottles preﬁlled with continuous phase for either one day or for three months and then characterized for the stability of the droplets against coalescence. The microﬂuidic emulsions were pipetted on a microscope slide (Marienfeld Superior, Lauda, Germany). Due to the density difference, the oil droplets migrated to the center of the liquid surface and formed a hexagonally ordered group. Photographs were taken using a digital SLR camera (EOS 700D, Canon, Tokyo, Japan) equipped with a macro lens (Canon EF 100 mm 1:2.8 USM, Canon, Tokyo, Japan). For each combination of surfactants and each storage time, several microscope slides were Colloids Interfaces 2022, 6, 12 6 of 17 l drop- ameter prepared so that at least 1200 droplets could be analyzed. On the microscope slide itself, no coalescence was observed. how many coalescence events per 100 droplets occurred, the initial droplet number n0 of the diameter d0 that resulted in nt droplets of the diameters di can be calculated by the formula (1): prepared so that at least 1200 droplets could be analyzed. On the microscope slide itself, no coalescence was observed. y p p , p the diameter d0 that resulted in nt droplets of the diameters di can be calculated by the formula (1): Figure 3 shows three exemplary pictures of microﬂuidic emulsions after different num- bers of coalescence events. Each droplet is labelled with its diameter in pixels, determined using a MATLAB code [40]. In Figure 3a, the emulsion still has a monomodal droplet size distribution. In Figure 3b, some of the droplets coalesced and the droplet diameter increased. In Figure 3c, the break-down of the emulsion is further progressed. To calculate how many coalescence events per 100 droplets occurred, the initial droplet number n0 of the diameter d0 that resulted in nt droplets of the diameters di can be calculated by the Formula (1): 3 ( ) 𝑛0 = ∑ⅆ𝑖 3 ⅆ0 3 𝑛𝑡 𝑖=0 (1) The number of occurred coalescence events per 100 droplets c is then calculated from the remaining number of droplets nt after a storage time: 𝑐= 𝑛0 − 𝑛𝑡 𝑛0 × 100 (2) n0 = nt ∑ i=0 d3 i d3 0 (1) 𝑛0 Table A1), the measured diameters and calculated igure 3 are given. 3. Results 3.1. Interfacial Tension Measurements 3.1. Interfacial Tension Measurements The interfacial tension is a sensitive value with which to measure changes at interfaces. While it cannot be directly linked to the stability of the interface or the corresponding emulsion, it can show whether surfactants are adsorbed at an interface or not [7]. Table 1 gives the interfacial tension values after a 60 min equilibration time for all nine formulations examined in this work. Table 1. Interfacial tensions of systems with different combinations of water and oil phase after 60 min equilibration time. The nine surfactant combinations shown here are used for all experiments. Table 1. Interfacial tensions of systems with different combinations of water and oil phase after 60 min equilibration time. The nine surfactant combinations shown here are used for all experiments. Table 1. Interfacial tensions of systems with different combinations of water and oil phase after 60 min equilibration time. The nine surfactant combinations shown here are used for all experiments. Water Phase Oil Phase Interfacial Tension γ in mN/m 1 wt% Tween 40 Pure MCT oil 5.6 ± 0.2 1 wt% PGPR 0.6 ± 0.1 1 wt% Span 80 3.5 ± 0.1 0.003 wt% Tween 40 Pure MCT oil 10.6 ± 0.2 1 wt% PGPR 1.7 ± 0.1 1 wt% Span 80 4.2 ± 0.0 Pure water Pure MCT oil 25.5 ± 0.4 1 wt% PGPR 3.1 ± 0.0 1 wt% Span 80 7.2 ± 0.1 Comparing all the values with 1 wt% Tween 40 in the water phase, both additional lipophilic surfactants decreased the interfacial tension further. This means that the ad- ditional adsorption of lipophilic surfactants to the interface occurred, which changed the properties of the interface. The same was observed for 0.003 wt% Tween 40 in the water phase. p The comparison between the PGPR of Span 80 at different Tween 40 concentrations shows differences between the formulations as well. Coming from measurements against pure water, the interfacial tension decreased with increasing Tween 40 concentrations, independently of the lipophilic surfactant in the oil phase. This shows that, here as well, the lipophilic surfactants did not adsorb at the interface alone, but the interfacial properties were a result of the combined adsorption. Therefore, all the examined formulations are different in interfacial composition and, accordingly, different droplet stabilities are to be expected. 2.6. Top-Down Single and Double Emulsions For the double emulsions, an inner emulsion with (mW1/mO = 1/9) was produced with a gear-rim disperser (IKA® magic LAB®, IKA®-Werke GmbH & Co. KG, Staufen, Germany) at 15,000 rpm, corresponding to a maximum peripheral speed of 25.1 m/s for 5 min. This W1/O emulsion was dispersed in the outer water phase with (mW1/O/mW2 = 1/9) using a colloid mill (IKA® magic LAB®, IKA®-Werke GmbH & Co. KG, Staufen, Germany) for 5 min, with a gap width between the rotor and the stator of 0.318 mm. The rotational speed was set to 15,000 rpm, which corresponds to a maximum peripheral speed of 25.1 m/s. For the single emulsion, the same second emulsiﬁcation step was performed with the surfactant-oil solutions as the disperse phase. The droplet size distributions were measured using static laser diffraction (Horiba LA-950, Retsch Technology GmbH, Haan, Germany). Emulsions of each combination of water and oil phases were produced in duplicate and each sample was measured in Colloids Interfaces 2022, 6, 12 7 of 17 7 of 17 triplicate after different storage times. Images of the double emulsions were obtained with a microscope (Eclipse Ci-L, Nikon, Shinagawa, Tokyo, Japan). triplicate after different storage times. Images of the double emulsions were obtained with a microscope (Eclipse Ci-L, Nikon, Shinagawa, Tokyo, Japan). 3. Results 3.2. Single Droplet Experiment Beginning with the left three boxes, droplets stabilized by 1 wt% Tween 40 are shown. For all the oil phases, with PGPR or Span 80 or without additional lipophilic surfactant, the coalescence times lay between 100 and 1000 s. Compared to other measurements with this measurement setup, the droplets were moderately stable. In other studies, some drop- lets were so well stabilized that no coalescence was observed at all within 30 min [24]. Here, the additional lipophilic surfactants increased the coalescence times slightly. The interface with two surfactants adsorbed increased the stability, or at least did not decrease it, as was found for the coalescence of water droplets [7,26]. Beginning with the left three boxes, droplets stabilized by 1 wt% Tween 40 are shown. For all the oil phases, with PGPR or Span 80 or without additional lipophilic surfactant, the coalescence times lay between 100 and 1000 s. Compared to other measurements with this measurement setup, the droplets were moderately stable. In other studies, some droplets were so well stabilized that no coalescence was observed at all within 30 min [24]. Here, the additional lipophilic surfactants increased the coalescence times slightly. The interface with two surfactants adsorbed increased the stability, or at least did not decrease it, as was found for the coalescence of water droplets [7,26]. When the Tween 40 concentration was reduced to 0.003 wt% (value given as the sur- factants’ cmc [33–35]), the stability of the droplets decreased, when no additional surfac- tant was added, by a factor of ten. The value of the interfacial tension measured after 60 min (10.6 mN/m instead of 5.6 mN/m) already indicates that the interface was occupied by fewer Tween 40 molecules at this concentration. This concentration effect can also be superimposed by slower adsorption times at concentrations around cmc [41]. Lower sur- factant concentrations consequently lead to faster coalescence of single droplets [42]. The influence of additional lipophilic surfactant was more pronounced compared to the higher Tween 40 concentration. Both PGPR and Span 80 increased the stability of the droplets significantly. An explanation for this could be the lipophilic surfactant filling the free space at the interface left by the absent amount of Tween 40. The full occupation of the interface by a mixed surfactant layer showed a similar stability to the surfactant layer oc- cupied by more Tween 40. 3.2. Single Droplet Experiment In Figure 4, the coalescence time distributions for the nine examined surfactant combi- nations are plotted in the form of a box plot diagram. The higher the coalescence time, the more stable the droplets against coalescence. Colloids Interfaces 2022, 6, 12 Colloids Interfaces 2022, 6, x FO 8 of 17 8 of 18 8 of 17 8 of 18 Figure 4. Coalescence time of single droplets in box plot diagram form. The box shows the middle quartiles, while the whiskers mark the upper and the down quartile. Additionally, the outliers and mean value are plotted. On the x-axis, the concentration of Tween 40 is varied. In the oil phase, either PGPR, Span 80 or no lipophilic surfactant were added. With decreasing concentrations of Tween 40, the stability of oil droplets decreases. Additional lipophilic surfactants can help to stabi- lize the water droplets. Figure 4. Coalescence time of single droplets in box plot diagram form. The box shows the middle quartiles, while the whiskers mark the upper and the down quartile. Additionally, the outliers and mean value are plotted. On the x-axis, the concentration of Tween 40 is varied. In the oil phase, either PGPR, Span 80 or no lipophilic surfactant were added. With decreasing concentrations of Tween 40, the stability of oil droplets decreases. Additional lipophilic surfactants can help to stabilize the water droplets. Figure 4. Coalescence time of single droplets in box plot diagram form. The box shows the middle quartiles, while the whiskers mark the upper and the down quartile. Additionally, the outliers and mean value are plotted. On the x-axis, the concentration of Tween 40 is varied. In the oil phase, either PGPR, Span 80 or no lipophilic surfactant were added. With decreasing concentrations of Tween 40, the stability of oil droplets decreases. Additional lipophilic surfactants can help to stabi- lize the water droplets. Figure 4. Coalescence time of single droplets in box plot diagram form. The box shows the middle quartiles, while the whiskers mark the upper and the down quartile. Additionally, the outliers and mean value are plotted. On the x-axis, the concentration of Tween 40 is varied. In the oil phase, either PGPR, Span 80 or no lipophilic surfactant were added. With decreasing concentrations of Tween 40, the stability of oil droplets decreases. Additional lipophilic surfactants can help to stabilize the water droplets. 3.2. Single Droplet Experiment p When the Tween 40 concentration was reduced to 0.003 wt% (value given as the surfactants’ cmc [33–35]), the stability of the droplets decreased, when no additional surfactant was added, by a factor of ten. The value of the interfacial tension measured after 60 min (10.6 mN/m instead of 5.6 mN/m) already indicates that the interface was occupied by fewer Tween 40 molecules at this concentration. This concentration effect can also be superimposed by slower adsorption times at concentrations around cmc [41]. Lower surfactant concentrations consequently lead to faster coalescence of single droplets [42]. The inﬂuence of additional lipophilic surfactant was more pronounced compared to the higher Tween 40 concentration. Both PGPR and Span 80 increased the stability of the droplets signiﬁcantly. An explanation for this could be the lipophilic surfactant ﬁlling the free space at the interface left by the absent amount of Tween 40. The full occupation of the interface by a mixed surfactant layer showed a similar stability to the surfactant layer occupied by more Tween 40. p y As expected, without any surfactant in the system, the droplets were completely un- stable and the coalescence time dropped to below ten seconds. Again, the lipophilic sur- factants increased the stability of the droplets. Still, there was a difference between the values without Tween 40 and with Tween 40 at cmc, showing that lipophilic surfactants alone cannot sufficiently stabilize oil droplets and that the stability of the combinations of Tween 40 at cmc with lipophilic surfactants resulted from mixed adsorption at the inter- As expected, without any surfactant in the system, the droplets were completely unstable and the coalescence time dropped to below ten seconds. Again, the lipophilic surfactants increased the stability of the droplets. Still, there was a difference between the values without Tween 40 and with Tween 40 at cmc, showing that lipophilic surfactants alone cannot sufﬁciently stabilize oil droplets and that the stability of the combinations of Tween 40 at cmc with lipophilic surfactants resulted from mixed adsorption at the interface. Tween 40 at cmc with lipophilic surfactants resulted from mixed adsorption at the inter face. 3.3. Microﬂuidic Emulsions f The stability of the ⅆ= The stability of the d = 144 µm microﬂuidic droplets after one day and after three months is shown in Figure 5. Compared to other studies on the coalescence stability of microﬂuidic droplets, three months is a rather long storage time. Most other studies focus on coalescence on the microﬂuidic chip, where droplets are examined for some seconds or a few minutes [29,43–45]. The external storage of the droplets offers the advantage of examining more stable formulations that do not show any instabilities in the microﬂuidic channel. Additionally, the long observation time allows observations near or at equilibrium state at the interfaces, while short time stability strongly depends on the adsorption kinetics of the surfactants [20,46]. Approximately, the number of coalescence events between one day and ninety days doubled for all the formulations. This means that the coalescence rate decreased drastically over the storage period. This behavior was in agreement with the ﬁlm drainage time for hard spheres and was observed in a similar form by Krebs et al. [47] for 100 µm drops. y p y months is shown in Figure 5. Compared to other studies on the coalescence stability of microfluidic droplets, three months is a rather long storage time. Most other studies focus on coalescence on the microfluidic chip, where droplets are examined for some seconds or a few minutes [29,43–45]. The external storage of the droplets offers the advantage of examining more stable formulations that do not show any instabilities in the microfluidic hannel. Additionally, the long observation time allows observations near or at equilib- ium state at the interfaces, while short time stability strongly depends on the adsorption kinetics of the surfactants [20,46]. Approximately, the number of coalescence events be- ween one day and ninety days doubled for all the formulations. This means that the coa- escence rate decreased drastically over the storage period. This behavior was in agree- ment with the film drainage time for hard spheres and was observed in a similar form by Krebs et al. [47] for 100 µm drops. Figure 5. Number of coalescence events within the microfluidic emulsions (d = 144 µm; CV < 3%) after one day and after three months. On the x-axis, the concentration of Tween 40 is varied. In the oil phase either 1 wt% PGPR 1 wt% Span 80 or no lipophilic surfactant were added Figure 5. 3.2. Single Droplet Experiment Even though the determination of coalescence times on single droplets is subject to large statistical deviations, this experiment shows interesting tendencies: in principle, the Even though the determination of coalescence times on single droplets is subject to large statistical deviations, this experiment shows interesting tendencies: in principle, the presence of an additional lipophilic emulsiﬁer at the interface of oil droplets helps to stabilize them better against coalescence. This is particularly important when there is too Colloids Interfaces 2022, 6, 12 9 of 17 o sta- 9 of 17 o sta- little hydrophilic emulsiﬁer in the system to stabilize the oil droplets alone. Especially at very low concentrations of hydrophilic emulsiﬁer (0.003% Tween 40), the longer resistance of the droplets to coalescence is assured just as well by the simultaneous presence of Span 80 or PGPR as by the sole application of 1% Tween 40. This offers interesting possibilities for double emulsions to reduce hydrophilic emulsiﬁers, which are known to destabilize inner water droplets. very low concentrations of hydrophilic emulsifier (0.003% Tween 40), the longer re- sistance of the droplets to coalescence is assured just as well by the simultaneous presence of Span 80 or PGPR as by the sole application of 1% Tween 40. This offers interesting possibilities for double emulsions to reduce hydrophilic emulsifiers, which are known to destabilize inner water droplets. 3 3 Mi fl idi E l i 3.3. Microﬂuidic Emulsions f The stability of the ⅆ= Number of coalescence events within the microﬂuidic emulsions (d = 144 µm; CV < 3%) after one day and after three months. On the x-axis, the concentration of Tween 40 is varied. In the oil phase, either 1 wt% PGPR, 1 wt% Span 80 or no lipophilic surfactant were added. igure 5. Number of coalescence events within the microfluidic emulsions (d = 144 µm; CV < 3%) fter one day and after three months. On the x-axis, the concentration of Tween 40 is varied. In the il phase either 1 wt% PGPR 1 wt% Span 80 or no lipophilic surfactant were added Figure 5. Number of coalescence events within the microﬂuidic emulsions (d = 144 µm; CV < 3%) after one day and after three months. On the x-axis, the concentration of Tween 40 is varied. In the oil phase, either 1 wt% PGPR, 1 wt% Span 80 or no lipophilic surfactant were added. p p p p For the microfluidic emulsion droplets with 1 wt% Tween 40, Figure 5 shows good stability values. Droplets of increased diameter were found in the samples but less than 1 out of 100 droplets coalesced within 1 day and less than 2 droplets coalesced after three h h h f l f d ff For the microﬂuidic emulsion droplets with 1 wt% Tween 40, Figure 5 shows good stability values. Droplets of increased diameter were found in the samples but less than 1 out of 100 droplets coalesced within 1 day and less than 2 droplets coalesced after three months. Between the three formulations, no signiﬁcant differences were seen. months. Between the three formulations, no significant differences were seen. At a reduced Tween 40 concentration of 0.003 wt%, the number of coalescence events ncreased. In the sample without lipophilic surfactant, approximately a quarter of the droplets coalesced within three months. With additional lipophilic surfactants, fewer co- alescence events were observed the number was still below 7 coalesced droplets per 100 At a reduced Tween 40 concentration of 0.003 wt%, the number of coalescence events increased. In the sample without lipophilic surfactant, approximately a quarter of the droplets coalesced within three months. With additional lipophilic surfactants, fewer coalescence events were observed the number was still below 7 coalesced droplets per 100 after 3 months. The two lipophilic surfactants showed a comparable stability within the measurement accuracy. 3.3. Microﬂuidic Emulsions f The stability of the ⅆ= The additional adsorption of PGPR and Span 80 obviously enhanced the droplets’ stability. Colloids Interfaces 2022, 6, 12 10 of 17 ously en Without Tween 40, the stability of the oil droplets was no longer given. Without lipophilic surfactant, complete phase separation was observed after 15 min and the max- imum value of 100 coalescence events per 100 droplets was achieved. The additional lipophilic surfactants helped with the stabilization, but a large proportion of the droplets still coalesced. In these experiments, the sample with PGPR was signiﬁcantly more stable than the sample with Span 80. While the lipophilic surfactants were also able to enhance stability without additional hydrophilic surfactant, the stability was strongly decreased in comparison with the samples with Tween 40. ophilic surfactant, complete phase separation was observed after 15 min and the maxi mum value of 100 coalescence events per 100 droplets was achieved. The additional lipo philic surfactants helped with the stabilization, but a large proportion of the droplets stil coalesced. In these experiments, the sample with PGPR was significantly more stable than the sample with Span 80. While the lipophilic surfactants were also able to enhance sta bility without additional hydrophilic surfactant, the stability was strongly decreased in comparison with the samples with Tween 40. Regarding the increasing error bars for measurement points with over 25 coalescenc events per 100 droplets the suitability of this method for very unstable formulations mus Regarding the increasing error bars for measurement points with over 25 coalescence events per 100 droplets, the suitability of this method for very unstable formulations must be limited. Since the method assumes spherical droplets on the microscope slide, the deformation of huge droplets under gravity increased the error, at which point, many coalescence events occurred. Additionally, the number of measured droplets decreased drastically, especially since most droplets still had their initial diameter, while some droplets increased in size drastically (see Figure 3). With 1 droplet with over 50 coalescence events leading to it (see Table A1), the statistical errors from obtaining the sample with a certain volume increased. events per 100 droplets, the suitability of this method for very unstable formulations mus be limited. Since the method assumes spherical droplets on the microscope slide, the de formation of huge droplets under gravity increased the error, at which point, many coa lescence events occurred. 3.4. Top-Down O/W Single Emulsions and W/O/W Double Emulsions p g Single and double emulsions were produced with a colloid Single and double emulsions were produced with a colloid mill with identical process conditions and were measured for their droplet sizes directly after production and after two weeks of storage time. In Figure 6, the characteristic value x90,3 of the droplet size distributions of the single emulsions (a) and of the double emulsions (b) are shown. Double emulsions without lipophilic surfactants could not be produced, since the inner emulsion separated instantly. The x90,3 values for these formulations are therefore not plotted in Figure 6b. cess conditions and were measured for their droplet sizes directly after production and after two weeks of storage time. In Figure 6, the characteristic value x90,3 of the droplet siz distributions of the single emulsions (a) and of the double emulsions (b) are shown. Dou ble emulsions without lipophilic surfactants could not be produced, since the inner emul sion separated instantly. The x90,3 values for these formulations are therefore not plotted in Figure 6b. (a) (b) Figure 6. Changes in droplet size over storage time for O/W emulsions (a) and W/O/W emulsion (b) with the same W2 and O phases. On the x-axis, the concentration of Tween 40 is varied. In th oil phase, either PGPR, Span 80 or no lipophilic surfactant were added. Double emulsions withou lipophilic surfactant could not be produced, since they would instantly result in single emulsions therefore, they are not shown. 0 20 40 60 80 100 120 140 160 180 200 droplet size x90,3 in µm Tween 40 concentration 1 wt% 0.003 wt% 0 wt% 0 20 40 60 80 100 120 140 160 180 200 droplet size x90,3 in µm without lipophilic surfactant after production two weeks storage with 1 wt% PGPR after production two weeks storage with 1 wt% Span 80 after production two weeks storage Tween 40 concentration 1 wt% 0.003 wt% 0 wt% Figure 6. Changes in droplet size over storage time for O/W emulsions (a) and W/O/W emulsions (b) with the same W2 and O phases. On the x-axis, the concentration of Tween 40 is varied. In the oil phase, either PGPR, Span 80 or no lipophilic surfactant were added. Double emulsions without lipophilic surfactant could not be produced, since they would instantly result in single emulsions; therefore, they are not shown. 3.3. Microﬂuidic Emulsions f The stability of the ⅆ= Additionally, the number of measured droplets decreased dras tically, especially since most droplets still had their initial diameter, while some droplet increased in size drastically (see Figure 3). With 1 droplet with over 50 coalescence event leading to it (see Table A1), the statistical errors from obtaining the sample with a certain volume increased. 3 4 Top Down O/W Single Emulsions and W/O/W Double Emulsions 3.4. Top-Down O/W Single Emulsions and W/O/W Double Emulsions p g Single and double emulsions were produced with a colloid When the emulsions were stored for two weeks, differences between the addition and non-addition of lipophilic surfactant became more pronounced. Both lipophilic surfactants enhanced the coalescence stability during storage and only small changes in droplet size occurred during storage. The same trends were seen for the double emulsions: with Span 80, the initial droplet size was smaller than with PGPR and the droplet sizes did not change over time with either lipophilic surfactant. ty during the process. For the single emulsions, the droplet sizes after production were imilar, whether PGPR or no lipophilic surfactant was added. Additional Span 80 led to maller droplets. When the emulsions were stored for two weeks, differences between the ddition and non-addition of lipophilic surfactant became more pronounced. Both lipo- philic surfactants enhanced the coalescence stability during storage and only small hanges in droplet size occurred during storage. The same trends were seen for the double mulsions: with Span 80, the initial droplet size was smaller than with PGPR and the droplet sizes did not change over time with either lipophilic surfactant. Without Tween 40, the stability of the oil droplets strongly decreased without lipo- p g p p Without Tween 40, the stability of the oil droplets strongly decreased without lipophilic surfactants; the same was observed when PGPR was added. The droplet sizes increased after production and were highly unstable after two weeks. Span 80 alone was also able to stabilize oil droplets 20 µm in diameter. For the double emulsions, the trend was more pronounced. With PGPR, the oil droplets were completely unstable; with Span 80, the droplets were still below 20 µm in diameter. The apparent reduction in droplet size for the samples with PGPR was due to the breakdown of droplets larger than 100 µm in the measurement device, which can lead to deviations. Without Tween 40, the stability of the oil droplets strongly decreased without lipo- philic surfactants; the same was observed when PGPR was added. The droplet sizes in- reased after production and were highly unstable after two weeks. Span 80 alone was lso able to stabilize oil droplets 20 µm in diameter. For the double emulsions, the trend was more pronounced. With PGPR, the oil droplets were completely unstable; with Span 0, the droplets were still below 20 µm in diameter. 3.4. Top-Down O/W Single Emulsions and W/O/W Double Emulsions p g Single and double emulsions were produced with a colloid (b) 0 20 40 60 80 100 120 140 160 180 200 droplet size x90,3 in µm without lipophilic surfactant after production two weeks storage with 1 wt% PGPR after production two weeks storage with 1 wt% Span 80 after production two weeks storage Tween 40 concentration 1 wt% 0.003 wt% 0 wt% (a) 0 20 40 60 80 100 120 140 160 180 200 droplet size x90,3 in µm Tween 40 concentration 1 wt% 0.003 wt% 0 wt% (a) (b) Figure 6. Changes in droplet size over storage time for O/W emulsions (a) and W/O/W emulsion (b) with the same W2 and O phases. On the x-axis, the concentration of Tween 40 is varied. In th oil phase, either PGPR, Span 80 or no lipophilic surfactant were added. Double emulsions withou lipophilic surfactant could not be produced, since they would instantly result in single emulsion therefore, they are not shown. Figure 6. Changes in droplet size over storage time for O/W emulsions (a) and W/O/W emulsions (b) with the same W2 and O phases. On the x-axis, the concentration of Tween 40 is varied. In the oil phase, either PGPR, Span 80 or no lipophilic surfactant were added. Double emulsions without lipophilic surfactant could not be produced, since they would instantly result in single emulsions; therefore, they are not shown. With 1 wt% Tween 40, the oil droplets for both emulsions were below 10 µm and did not change their size within two weeks. Additional lipophilic surfactant neither changed With 1 wt% Tween 40, the oil droplets for both emulsions were below 10 µm and did not change their size within two weeks. Additional lipophilic surfactant neither changed the initial droplet size nor changed the stability during storage. Furthermore, no differences between single and double emulsions were seen. With 0.003 wt% Tween 40, the droplet sizes were increased directly after the process for all the formulations. This might either have been inﬂuenced by changes in interfacial tensions (see Section 3.1) and, thus, decreased break-up, or by reduced coalescence stability Colloids Interfaces 2022, 6, 12 11 of 17 ocess facial 11 of 17 ocess facial during the process. For the single emulsions, the droplet sizes after production were similar, whether PGPR or no lipophilic surfactant was added. Additional Span 80 led to smaller droplets. 3.4. Top-Down O/W Single Emulsions and W/O/W Double Emulsions p g Single and double emulsions were produced with a colloid The apparent reduction in droplet size or the samples with PGPR was due to the breakdown of droplets larger than 100 µm in he measurement device, which can lead to deviations. To finally e aluate the suitability of the chosen surfactant combinations for the pro To ﬁnally evaluate the suitability of the chosen surfactant combinations for the produc- tion of double emulsions, micrographs of the double emulsion are shown in Figure 7. The double emulsions were obtained directly after production and presented on the microscope slide without dilution. Longer storage times or dilution led to severe coalescence of the oil droplets for the emulsions without Tween 40 for both lipophilic surfactants. To finally evaluate the suitability of the chosen surfactant combinations for the pro- duction of double emulsions, micrographs of the double emulsion are shown in Figure 7. The double emulsions were obtained directly after production and presented on the mi- roscope slide without dilution. Longer storage times or dilution led to severe coalescence f the oil droplets for the emulsions without Tween 40 for both lipophilic surfactants. Figure 7. Micrographs of the double emulsions produced with 1 wt% PGPR or Span 80 and different Tween 40 concentrations directly after production. While the emulsions with PGPR as lipophilic surfactant showed encapsulated droplets and, therefore, a double-emulsion structure, the double emulsion with Span 80 had no inner droplets left. For both lipophilic surfactants, the oil droplet size decreased with increasing concentrations of Tween 40. Figure 7. Micrographs of the double emulsions produced with 1 wt% PGPR or Span 80 and different Tween 40 concentrations directly after production. While the emulsions with PGPR as lipophilic surfactant showed encapsulated droplets and, therefore, a double-emulsion structure, the double emulsion with Span 80 had no inner droplets left. For both lipophilic surfactants, the oil droplet size decreased with increasing concentrations of Tween 40. igure 7. Micrographs of the double emulsions produced with 1 wt% PGPR or Span 80 and different ween 40 concentrations directly after production. While the emulsions with PGPR as lipophilic urfactant showed encapsulated droplets and, therefore, a double-emulsion structure, the double mulsion with Span 80 had no inner droplets left. For both lipophilic surfactants, the oil droplet size ecreased with increasing concentrations of Tween 40. Figure 7. Micrographs of the double emulsions produced with 1 wt% PGPR or Span 80 and different Tween 40 concentrations directly after production. 4. Discussion In this study, the effect of reducing the concentration of the hydrophilic surfactant on the stability of oil droplets in double-emulsion formulations was studied using different model experiments. p Interfacial tension measurements indicated that for all the surfactant combinations examined in this study, the composition of the interface differed, which resulted in different interfacial tensions. The inﬂuence of the interface composition on the stability of oil droplets was examined with model experiments of different size scales. The median coalescence time of single droplets at an interface varied between 3.5 s and 1200 s for the examined formulations. In principle, the presence of an additional lipophilic emulsiﬁer at the interface of oil droplets increased the coalescence times for formulations with strongly decreased concentrations of hydrophilic surfactant. The wide scattering of the coalescence times prevented us from proving the statistical signiﬁcance of the differences between the two lipophilic surfactants. The number of coalescence events in single emulsions produced by a microﬂuidic device showed values between less than 1 coalesced droplet per 100 droplets to 100 coalescence events per 100 droplets within minutes. Compared to the single-droplet experiment, the examination of microﬂuidic double emulsions increased the number of examined droplets from 30 to over 1000, allowing the performance of an improved statistical analysis. Finally, the droplet sizes of the single and double emulsions produced by mechanical emulsiﬁcation were analyzed and the growth during storage was discussed. The value x90,3 was chosen for comparison as a relevant parameter for the stability of an emulsion, since the biggest droplets were the most likely to lead to phase separation via creaming. The micrographs of the double emulsions emphasized the need for the complete examination of the double-emulsion system before recommending a suitable formulation. All the experiments suggested that the presence of a lipophilic surfactant helps to stabilize oil droplets even at very low concentrations of hydrophilic surfactant. Even though a similar behavior for PGPR and Span 80 was found concerning the stability of single oil droplets, the micrographs of the double-emulsion droplets revealed that the encapsulated water droplets were not stable when Span 80 was used as the lipophilic surfactant. This emphasizes the necessity of the different experiments proposed: while the model experiments can help to improve mechanistic understanding, only research on real double emulsion reveals interactions between all the possible instability mechanisms as they are equally important. 3.4. Top-Down O/W Single Emulsions and W/O/W Double Emulsions p g Single and double emulsions were produced with a colloid While the emulsions with PGPR as lipophilic surfactant showed encapsulated droplets and, therefore, a double-emulsion structure, the double emulsion with Span 80 had no inner droplets left. For both lipophilic surfactants, the oil droplet size decreased with increasing concentrations of Tween 40. In the top row, double emulsions stabilized with PGPR are shown. With decreasing Tween 40 concentration, the oil droplet sizes decreased, as discussed above. The droplets were all grey, with some visible inner droplets, which means the double emulsion struc- ure was intact. The Span 80-stabilized inner water droplets were no longer visible; the oil droplets were brighter, indicating that there were no inner water droplets. Pictures with In the top row, double emulsions stabilized with PGPR are shown. With decreasing Tween 40 concentration, the oil droplet sizes decreased, as discussed above. The droplets were all grey, with some visible inner droplets, which means the double emulsion structure was intact. The Span 80-stabilized inner water droplets were no longer visible; the oil droplets were brighter, indicating that there were no inner water droplets. Pictures with higher magniﬁcation conﬁrmed the absence of inner droplets. Although many studies using Span 80 for double emulsions are available [48–50], in our study, no double emulsion could be produced with this lipophilic surfactant. Colloids Interfaces 2022, 6, 12 12 of 17 12 of 17 4. Discussion While the method allows an optimum of mechanistic understanding to be gained, the method will be only used by research groups focusing on microﬂuidics. With the establishment of a suitable microﬂuidic process through which to produce double emulsions with multiple inner droplets, the complete break-down of a double-emulsion formulation can also be observed [51]. Accordingly, all three coalescence mechanisms can be tracked at once, eliminating the disadvantage that arises from using single emulsions for the description of double emulsions, namely that the optimizing of the stability against one coalescence mechanism might result in a worse stability against the other two. In the case of single emulsions produced in laboratory-scale emulsiﬁcation machines, the proximity to the application is good, as production and measuring instruments are Table 2. Comparison of the effects of the three different measurement methods on the different Tween 40 concentrations with addition of PGPR. For the microﬂuidic experiment, the values after one day for the emulsiﬁcation experiments directly after production are given. Tween 40 Concentration in wt% Median Coalescence Time of Single Droplets t50 in s Coalescence Events in Microﬂuidic Emulsion per 100 Droplets Emulsion Droplet Size x90,3 in µm Double Emulsions Oil Droplet Size x90,3 in µm 0 109 13.7 46.9 170.8 0.003 298 2.42 32.6 45.1 1 530 0.69 5.88 5.35 Table 2. Comparison of the effects of the three different measurement methods on the different Tween 40 concentrations with addition of PGPR. For the microﬂuidic experiment, the values after one day for the emulsiﬁcation experiments directly after production are given. Table 2. Comparison of the effects of the three different measurement methods on the different Tween 40 concentrations with addition of PGPR. For the microﬂuidic experiment, the values after one day for the emulsiﬁcation experiments directly after production are given. The general trend found in all the experiments is that a lower Tween 40 concentration results in increased coalescence. For the single-droplet experiment, an increase in the median coalescence time by a factor 5 was found when the Tween 40 concentration was increased from 0 wt% to 1 wt%. In the microﬂuidic experiment, the same Tween 40 concentration change led to a more than 10 fold decreased number of coalescence events. The increase in x90,3 of single and double emulsions was changed by a factor of 8 and 30, respectively. 4. Discussion The comparison shows that the same trends were seen in the different model experiments; however, the effects occurred to varying degrees. 4. Discussion When comparing the information obtained from the different experiments, this study showed that at a typical application concentration of Tween 40 (1 wt%), the presence of additional lipophilic surfactants had no effect on the stability of the oil droplets. With a reduced Tween 40 concentration (0.003 wt% ~ cmc), the lipophilic surfactants increased the stability of the oil droplets against coalescence. Working without Tween 40 resulted in a strong decrease in oil droplet stability, conﬁrming that lipophilic surfactants alone cannot stabilize O/W emulsions for longer than a day. In summary, Tween 40 provides satisfactory stability of oil droplets, even at low concentrations, when additional lipophilic surfactants such as PGPR or Span 80 are present. This opens up the possibility of reducing the concentration of Tween 40 in double emulsions drastically and still retaining relatively stable oil droplets. Since short-chained hydrophilic surfactants can destabilize encapsulated water droplets at high concentrations [7,18], the reduction of hydrophilic surfactant may offer the possibility of increased overall double-emulsion stability. Additionally, to reduce the concentration of surfactants as far as possible is a desirable goal from both ecological and economic viewpoints. p The changes in droplet stability were found to be similar in all three experiments at different size scales: single droplets (1000 µm), microﬂuidic O/W emulsions (150–500 µm) and (W/)O/W emulsions (1–50 µm). Table 2 shows the measured values obtained with the different experiments. For the sake of clarity, only the results of Tween 40 with PGPR after the short storage period are shown and discussed. Comparable trends were found after longer storage times and with Span 80 (see Tables A2–A4). The chosen storage times for Colloids Interfaces 2022, 6, 12 13 of 17 13 of 17 the microﬂuidic experiment (90 days) and the mechanically emulsiﬁed samples (14 days) differed. Because of the different droplet sizes, the time needed to develop signiﬁcant differences between the samples differed. the microﬂuidic experiment (90 days) and the mechanically emulsiﬁed samples (14 days) differed. Because of the different droplet sizes, the time needed to develop signiﬁcant differences between the samples differed. the microﬂuidic experiment (90 days) and the mechanically emulsiﬁed samples (14 days) differed. Because of the different droplet sizes, the time needed to develop signiﬁcant differences between the samples differed. Table 2. Comparison of the effects of the three different measurement methods on the different Tween 40 concentrations with addition of PGPR. 4. Discussion For the microﬂuidic experiment, the values after one day for the emulsiﬁcation experiments directly after production are given. Tween 40 Concentration in wt% Median Coalescence Time of Single Droplets t50 in s Coalescence Events in Microﬂuidic Emulsion per 100 Droplets Emulsion Droplet Size x90,3 in µm Double Emulsions Oil Droplet Size x90,3 in µm 0 109 13.7 46.9 170.8 0.003 298 2.42 32.6 45.1 1 530 0.69 5.88 5.35 The general trend found in all the experiments is that a lower Tween 40 concentration results in increased coalescence. For the single-droplet experiment, an increase in the median coalescence time by a factor 5 was found when the Tween 40 concentration was increased from 0 wt% to 1 wt%. In the microﬂuidic experiment, the same Tween 40 concentration change led to a more than 10 fold decreased number of coalescence events. The increase in x90,3 of single and double emulsions was changed by a factor of 8 and 30, respectively. The comparison shows that the same trends were seen in the different model experiments; however, the effects occurred to varying degrees. 5. Conclusions All the methods studied generally work for the screening of surfactant combina- tions for double emulsions. They show how an additional surfactant or a change in its concentration affects the stability of oil droplets against coalescence. However, each experi- ment has its advantages and disadvantages and allows different effects to be illuminated more directly. Interfacial tension measurements give information as to whether both surfactants adsorb simultaneously at the interface, without giving any information as to whether this affects droplet stability. Single-droplet coalescence experiments allow the prediction of stability problems that may arise from speciﬁc surfactant combinations. No special laboratory equipment is needed to carry them out. However, this technique is very prone to distortion by small con- centrations of impurities. Additionally, only rough approximations can be made, unless a huge number of repetitions are performed. The method is therefore rather time-consuming. The observation of droplets produced by microﬂuidics unites the advantage of work- ing with highly deﬁned systems (droplet sizes) with the possibility of measuring hundreds of droplets simultaneously. Once the microﬂuidic process is established for the model system of interest, expanding the experiments to a wider variety of surfactant combinations works effectively. Still, handling microﬂuidic processes is challenging and the equipment is not widely available to research laboratories. 5. Conclusions All the methods studied generally work for the screening of surfactant combina- tions for double emulsions. They show how an additional surfactant or a change in its concentration affects the stability of oil droplets against coalescence. However, each experi- ment has its advantages and disadvantages and allows different effects to be illuminated more directly. Interfacial tension measurements give information as to whether both surfactants adsorb simultaneously at the interface, without giving any information as to whether this affects droplet stability. Single-droplet coalescence experiments allow the prediction of stability problems that may arise from speciﬁc surfactant combinations. No special laboratory equipment is needed to carry them out. However, this technique is very prone to distortion by small con- centrations of impurities. Additionally, only rough approximations can be made, unless a huge number of repetitions are performed. The method is therefore rather time-consuming. The observation of droplets produced by microﬂuidics unites the advantage of work- ing with highly deﬁned systems (droplet sizes) with the possibility of measuring hundreds of droplets simultaneously. Once the microﬂuidic process is established for the model system of interest, expanding the experiments to a wider variety of surfactant combinations works effectively. Still, handling microﬂuidic processes is challenging and the equipment is not widely available to research laboratories. While the method allows an optimum of mechanistic understanding to be gained, the method will be only used by research groups focusing on microﬂuidics. With the establishment of a suitable microﬂuidic process through which to produce double emulsions with multiple inner droplets, the complete break-down of a double-emulsion formulation can also be observed [51]. Accordingly, all three coalescence mechanisms can be tracked at once, eliminating the disadvantage that arises from using single emulsions for the description of double emulsions, namely that the optimizing of the stability against one coalescence mechanism might result in a worse stability against the other two. In the case of single emulsions produced in laboratory-scale emulsiﬁcation machines, the proximity to the application is good, as production and measuring instruments are Colloids Interfaces 2022, 6, 12 14 of 17 14 of 17 widely available. Furthermore, the transfer to the real system is easier. However, the (broad) distribution of droplet sizes in these systems makes an interpretation complex. Additionally, there is no possibility of tracking the other coalescence mechanism in double emulsions, such as W1–W2 coalescence, while the other methods can be adapted for this coalescence effects as well. 5. Conclusions Although the methods introduced in this research are highly effective at describing the one coalescence mechanism for which they are designed (O–O), the ﬁnal step in formulation optimization remains the testing of real double emulsions. Each reduction in complexity might also reduce an important, yet unknown, effect on the stability. While the models discussed in this paper can help to establish mechanistic understand- ing and support users with double-emulsion instability troubleshooting, the last step is conﬁrmation in a real system. Besides oil droplet size, measured in this study, a microscopic picture should also be taken, and the encapsulation efﬁciency or release rate should be measured to assess the quality of the formulation. Author Contributions: Conceptualization, N.L. and H.P.K.; formal analysis, N.L. and C.Y.; funding acquisition, H.P.K.; methodology, N.L. and C.Y.; supervision, H.P.K.; visualization, N.L. and C.Y.; writing—original draft, N.L. and C.Y.; writing—review and editing, H.P.K. 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: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding authors. Acknowledgments: The authors want to thank Lydia Schütz for her assistance in the laboratory. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Appendix A Table A1. Exemplary table for the analysis of coalescence events from the micrographs from Figure 3. Each droplet diameter was measured in pixels and the number of coalescence events necessary for this droplet size was calculated and normalized to the initial number of droplets necessary for this volume. Diameter in Pixels Figure 3a Figure 3b Figure 3c ci Number of Droplets Found 19 0 185 171 128 20 0.2 1 2 1 21 0.4 0 0 1 22 0.6 0 0 0 23 0.8 0 0 1 24 1.0 0 2 3 25 1.3 0 1 0 26 1.6 0 0 0 27 1.9 0 0 0 28 2.2 0 0 0 28 2.6 0 0 0 30 2.9 0 0 1 Colloids Interfaces 2022, 6, 12 15 of 17 Table A1. Cont. Diameter in Pixels Figure 3a Figure 3b Figure 3c ci Number of Droplets Found 31 3.3 0 0 0 32 3.8 0 1 1 33 4.2 0 0 0 34 4.7 0 0 0 35 5.3 0 0 1 36 5.8 0 0 1 37 6.4 0 0 0 38 7.0 0 0 0 39 7.6 0 0 0 40 8.3 0 0 0 41 9.0 0 0 0 42 9.8 0 0 0 43 10.6 0 0 0 44 11.4 0 0 0 45 12.3 0 0 0 46 13.2 0 0 0 47 14.1 0 1 0 48 15.1 0 0 0 49 16.2 0 0 1 50 17.2 0 0 0 51 18.3 0 0 1 Number coalescence events per 100 droplets 0.1 10.8 28.8 Table A2. Comparison of the effect of the three different measurement methods on the example of different Tween 40 concentrations the with addition of PGPR. For the microﬂuidic experiment, the values are from after 90 days and for the emulsiﬁcation experiments, from after 14 days. Tween 40 Concentration in wt% Median Coalescence Time of Single Droplets t50 in s Coalescence Events in Microﬂuidic Emulsion per 100 Droplets Emulsion Droplet Size x90,3 in µm Double Emulsions Oil Droplet Size x90,3 in µm 0 109 26.2 90.5 133.5 0.003 298 5.38 37.8 50.5 1 530 2.37 6.40 5.98 Table A3. Comparison of the effect of the three different measurement methods on the example of different Tween 40 concentrations with the addition of Span 80. For the microﬂuidic experiment, the values are from after one day. Appendix A Inﬂuence of Hydrophilic Surfactants on the W1–W2 Coalescence in Double Emulsion Systems Investigated by Single Droplet Experiments. Colloids Interfaces 2021, 5, 21. [CrossRef] 7. Leister, N.; Karbstein, H.P. Inﬂuence of Hydrophilic Surfactants on the W1–W2 Coalescence in Double Emulsion Systems Investigated by Single Droplet Experiments. Colloids Interfaces 2021, 5, 21. [CrossRef] g y g p p f 8. Ficheux, M.-F.; Bonakdar, L.; Leal-Calderon, F.; Bibette, J. Some Stability Criteria for Double Emulsions. Langmuir 1998, 14, 2702–2706. [CrossRef] 9. Pawlik, A.; Cox, P.W.; Norton, I.T. Food grade duplex emulsions designed and stabilised with different osmotic pressures. J. Colloid Interface Sci. 2010, 352, 59–67. [CrossRef] 10. Sela, Y.; Magdassi, S.; Garti, N. Release of markers from the inner water phase of W/O/W emulsions stabilized by silicone based polymeric surfactants. J. Control. Release 1995, 33, 1–12. [CrossRef] 11. Oppermann, A.K.L.; Noppers, J.M.E.; Stieger, M.; Scholten, E. Effect of outer water phase composition on oil droplet size and yield of (w1/o/w2) double emulsions. Food Res. Int. 2018, 107, 148–157. [CrossRef] [PubMed] 12. Leister, N.; Pfaff, D.; Karbstein, H.P. Coalescence of Inner Water Droplets in Double Emulsions Due through Oil. Chem. Ing. Tech. 2021, Early View. [CrossRef] 13. Dickinson, E. Double Emulsions Stabilized by Food Biopolymers. Food Biophys. 2011, 6, 1–11. [CrossRe ll l l b l f / / 13. Dickinson, E. Double Emulsions Stabilized by Food Biopolymers. Food Biophys. 2011, 6, 1–11. [CrossRef] 14. Hattrem, M.N.; Dille, M.J.; Seternes, T.; Draget, K.I. Macro- vs. micromolecular stabilisation of W/O/W-emulsions. Food Hydrocoll. 2014, 37, 77–85. [CrossRef] 15. Neumann, S.M.; Wittstock, N.; van der Schaaf, U.S.; Karbstein, H.P. Interactions in water in oil in water double emulsions: Systematical investigations on the interfacial properties and emulsion structure of the outer oil in water emulsion. Colloids Surf. A Physicochem. Eng. Asp. 2018, 537, 524–531. [CrossRef] 16. Gülseren, ˙I.; Corredig, M. Interactions at the interface between hydrophobic and hydrophilic emulsiﬁers: Polyglycerol polyrici- noleate (PGPR) and milk proteins, studied by drop shape tensiometry. Food Hydrocoll. 2012, 29, 193–198. [CrossRef] 17. Tamnak, S.; Mirhosseini, H.; Tan, C.P.; Tabatabaee Amid, B.; Kazemi, M.; Hedayatnia, S. Encapsulation properties, release behavior and physicochemical characteristics of water-in-oil-in-water (W/O/W) emulsion stabilized with pectin–pea protein isolate conjugate and Tween 80. Food Hydrocoll. 2016, 61, 599–608. [CrossRef] j g y 18. Kanouni, M.; Rosano, H.; Naouli, N. Preparation of a stable double emulsion (W1/O/W2): Role of the interfacial ﬁlms on the stability of the system. Adv. Appendix A The values for the emulsiﬁcation experiments directly after production are also given. Tween 40 Concentration in wt% Median Coalescence Time of Single Droplets t50 in s Coalescence Events in Microﬂuidic Emulsion per 100 Droplets Emulsion Droplet Size x90,3 in µm Double Emulsions Oil Droplet Size x90,3 in µm 0 124 22.2 15.1 11.6 0.003 1165 1.32 15.9 14.6 1 330 0.810 6.59 5.36 Table A1. Cont. Table A2. Comparison of the effect of the three different measurement methods on the example of different Tween 40 concentrations the with addition of PGPR. For the microﬂuidic experiment, the values are from after 90 days and for the emulsiﬁcation experiments, from after 14 days. Table A3. Comparison of the effect of the three different measurement methods on the example of different Tween 40 concentrations with the addition of Span 80. For the microﬂuidic experiment, the values are from after one day. The values for the emulsiﬁcation experiments directly after production are also given. Colloids Interfaces 2022, 6, 12 16 of 17 16 of 17 Table A4. Comparison of the effect of the three different measurement methods on the example of different Tween 40 concentrations with the addition of Span 80. For the microﬂuidic experiment, the values are from after 90 days and for the emulsiﬁcation experiments, they are from after 14 days. Tween 40 Concentration in wt% Median Coalescence Time of Single Droplets t50 in s Coalescence Events in Microﬂuidic Emulsion per 100 Droplets Emulsion Droplet Size x90,3 in µm Double Emulsions Oil Droplet Size x90,3 in µm 0 124 54.7 19.7 18.7 0.003 1165 5.57 20.6 18.4 1 330 1.60 6.88 5.64 Table A4. Comparison of the effect of the three different measurement methods on the example of different Tween 40 concentrations with the addition of Span 80. For the microﬂuidic experiment, the values are from after 90 days and for the emulsiﬁcation experiments, they are from after 14 days. References 5. Muschiolik, G.; Dickinson, E. Double Emulsions Relevant to Food Systems: Preparation, Stability, and Applications. Compr. Rev. Food Sci. Food Saf. 2017, 16, 532–555. [CrossRef] 5. Muschiolik, G.; Dickinson, E. Double Emulsions Relevant to Food Systems: Preparation, Stability, and Applications. Compr. Rev. Food Sci. Food Saf. 2017, 16, 532–555. [CrossRef] 6. 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Vladisavljevi´c, G.T.; Kobayashi, I.; Nakajima, M. Production of uniform droplets using membrane, microchannel and microﬂuidic emulsiﬁcation devices. Microﬂuid Nanoﬂuid 2012, 13, 151–178. [CrossRef] ﬂ ﬂ 22. Neumann, S.M.; Scherbej, I.; van der Schaaf, U.S.; Karbstein, H.P. Investigations on the inﬂuence of osmotic active substances on the structure of water in oil emulsions for the application as inner phase in double emulsions. Colloids Surf. A Physicochem. Eng. Asp. 2018, 538, 56–62. [CrossRef] 22. Neumann, S.M.; Scherbej, I.; van der Schaaf, U.S.; Karbstein, H.P. Investigations on the inﬂuence of osmotic active substances on the structure of water in oil emulsions for the application as inner phase in double emulsions. Colloids Surf. A Physicochem. Eng. Asp. 2018, 538, 56–62. [CrossRef] p 23. Neumann, S.M.; van der Schaaf, U.S.; Schuchmann, H.P. The Diffusion and Coalescence Time Analyzer (DCTA): A novel experimental setup for investigating instability phenomena in double emulsions. Food Struct. 2017, 12, 103–112. [CrossRef] p 23. Neumann, S.M.; van der Schaaf, U.S.; Schuchmann, H.P. The Diffusion and Coalescence Time Analyzer (DCTA): A novel experimental setup for investigating instability phenomena in double emulsions. Food Struct. 2017, 12, 103–112. [CrossRef] 17 of 17 17 of 17 Colloids Interfaces 2022, 6, 12 24. Taboada, M.L.; Leister, N.; Karbstein, H.P.; Gaukel, V. Inﬂuence of the Emulsiﬁer System on Breakup and Coalescence of Oil Droplets during Atomization of Oil-In-Water Emulsions. ChemEngineering 2020, 4, 47. [CrossRef] p g g g 25. Won, J.Y.; Krägel, J.; Makievski, A.V.; Javadi, A.; Gochev, G.; Loglio, G.; Pandolﬁni, P.; Leser, M.E.; Gehin-Delval, C.; Miller, R. Drop and bubble micro manipulator (DBMM)—A unique tool for mimicking processes in foams and emulsions. Colloids Surf. A Physicochem. Eng. Asp. 2014, 441, 807–814. [CrossRef] y g p 26. Neumann, S.M.; van der Schaaf, U.S.; Karbstein, H.P. Appendix A Investigations on the relationship between interfacial and single droplet experiments to describe instability mechanisms in double emulsions. Colloids Surf. A Physicochem. Eng. Asp. 2018, 553, 464–471. [CrossRef] 27. Umbanhowar, P.B.; Prasad, V.; Weitz, D.A. Monodisperse Emulsion Generation via Drop Break Of Langmuir 2000, 16, 347–351. [CrossRef] 27. Umbanhowar, P.B.; Prasad, V.; Weitz, D.A. Monodisperse Emulsion Generation via Drop Break Off in a Coﬂowing Stream. Langmuir 2000, 16, 347–351. [CrossRef] h h h d h d Langmuir 2000, 16, 347 351. [CrossRef] 28. Shah, R.K.; Shum, H.C.; Rowat, A.C.; Lee, D.; Agresti, J.J.; Utada, A.S.; Chu, L.-Y.; Kim, J.-W.; Fernandez-Nieves, A.; Martinez, C.J.; et al. Designer emulsions using microﬂuidics. Mater. Today 2008, 11, 18–27. [CrossRef] 28. Shah, R.K.; Shum, H.C.; Rowat, A.C.; Lee, D.; Agresti, J.J.; Utada, A.S.; Chu, L.-Y.; Kim, J.-W. 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[CrossRef] Appendix A Microﬂuidic investigation of the coalescence susceptibility f i bili d l i Eff f i id i l l F d H d ll 102 105610 [C R f] p p p y [ ] 47. Krebs, T.; Ershov, D.; Schroen, C.G.P.H.; Boom, R.M. Coalescence and compression in centrifuged emulsions studied with in situ optical microscopy. Soft Matter 2013, 9, 4026. [CrossRef] 47. Krebs, T.; Ershov, D.; Schroen, C.G.P.H.; Boom, R.M. Coalescence and compression in centrifuged emul optical microscopy. Soft Matter 2013, 9, 4026. [CrossRef] 48. Qi, X.; Wang, L.; Zhu, J. Water-in-oil-in-water double emulsions: An excellent delivery system for improving the oral bioavailability of pidotimod in rats. J. Pharm. Sci. 2011, 100, 2203–2211. [CrossRef] 49. Michaut, F.; Hébraud, P.; Perrin, P. Amphiphilic polyelectrolyte for stabilization of multiple emulsions. Polym. Int. 2003, 52, 594–601. [CrossRef] 50. Dinarvand, R.; Moghadam, S.H.; Sheikhi, A.; Atyabi, F. 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https://openalex.org/W4245438682	https://www.researchsquare.com/article/rs-27322/latest.pdf	English	null	Psychological distress and internet-related behaviors between schoolchildren with and without overweight during the COVID-19 outbreak	Research Square (Research Square)	2,020	cc-by	6,611	Psychological distress and internet-related behaviors between schoolchildren with and without overweight during the COVID-19 outbreak Chao-Ying Chen Hong Kong Polytechnic University https://orcid.org/0000-0002-9632-6659 I-Hua Chen Minnan Normal University Kerry O’Brien Monash University https://orcid.org/0000-0003-3145-6038 Janet D Latner University of Hawaii at Manoa Chung-Ying Lin (  cylin36933@gmail.com ) Hong Kong Polytechnic University https://orcid.org/0000-0002-2129-4242 Research Article Keywords: stigma, overweight, obesity, COVID-19, anxiety, depression, internet, stress Posted Date: May 7th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-27322/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on January 25th, 2021. See the published version at https://doi.org/10.1038/s41366-021-00741-5. Psychological distress and internet-related behaviors between schoolchildren with and without overweight during the COVID-19 outbreak Chao-Ying Chen Hong Kong Polytechnic University https://orcid.org/0000-0002-9632-6659 I-Hua Chen Minnan Normal University Kerry O’Brien Monash University https://orcid.org/0000-0003-3145-6038 Janet D Latner University of Hawaii at Manoa Chung-Ying Lin (  cylin36933@gmail.com ) Hong Kong Polytechnic University https://orcid.org/0000-0002-2129-4242 Research Article Keywords: stigma, overweight, obesity, COVID-19, anxiety, depression, internet, stress Posted Date: May 7th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-27322/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on January 25th, 2021. See the published version at https://doi.org/10.1038/s41366-021-00741-5. Research Article Version of Record: A version of this preprint was published on January 25th, 2021. See the published version at https://doi.org/10.1038/s41366-021-00741-5. Page 1/15 Abstract Objective: The novel coronavirus disease 2019 (COVID-19) pandemic, and its resulting social policy changes, may result in psychological distress among schoolchildren with overweight. This study thus aimed to (1) compare psychological distress (including fear of COVID-19 infection, stress, anxiety, and depression), perceived weight stigma, and problematic internet-related behaviors between schoolchildren with and without overweight; (2) assess whether perceived weight stigma and problematic internet-related behaviors explained psychological distress. Methods: Schoolchildren (n=1 357; mean age=10.7 years) with overweight (n=236) and without overweight (n=1 121) completed an online survey assessing their fear of COVID-19 infection, stress, anxiety, depression, perceived weight stigma, problematic smartphone application use, problematic social media use, and problematic gaming. Results: Schoolchildren with overweight had significantly higher levels of COVID-19 infection fear, stress, depression, perceived weight stigma, and problematic social media use than those without overweight. Regression models showed that perceived weight stigma and problematic internet-related behaviors were significant predictors of psychological distress among schoolchildren with overweight. Conclusion: Strategies to manage perceived weight stigma and problematic internet-related behaviors may have a positive influence on mental health among schoolchildren with overweight under health- threatening circumstances, such as the current COVID-19 pandemic. Introduction The rapid growth of novel coronavirus disease 2019 (COVID-19) infection worldwide (>3.4 million COVID- 19 infections) has caused more than 230 000 deaths as of May 1, 2020.1 The absence of an effective treatment means that healthcare providers have paid high level attention to people who are particularly vulnerable to the COVID-19. This includes those with chronic disease (e.g., diabetes, coronary heart disease), compromised immune systems, and those with overweight (including obesity). Patients with overweight may disproportionately develop severe comorbidities after COVID-19 infection.2 This suggestion is supported by previous work investigating the relationship between influenza and overweight. Specifically, the altered immune response and sedentary lifestyle that influences the physiological health contribute to poorer clinical outcomes for patients with overweight following influenza.3 Recent evidence from France found that the severity of COVID-19 infection was positively related to patients’ body mass index (BMI). That is, patients with a higher BMI, especially those with a BMI higher than 35kg/m2, are more likely to require support from invasive mechanical ventilators.4 Because of the health consequences mentioned above, people with overweight may additionally suffer from fears about their health and associated psychological distress. Specifically, people with overweight who feel they are more vulnerable to COVID-19 related morbidity and mortality may also display greater Page 2/15 Page 2/15 psychological distress. Moreover, lifestyle and living patterns have by necessity changed during the COVID-19 outbreak. For example, many governments have implemented social distancing and lockdowns laws to prevent expansion of COVID-19 infection.5,6 Mainland China has implemented temporary school closures and required primary schoolchildren to study online at home for several months during the COVID-19 outbreak. Such changes in lifestyles may have negative consequence on individuals’ mental health. Prior research has shown that major life events, such as the loss of a parent or friend can be associated with psychological distress, and people with overweight can be more vulnerable to such life stressors than their counterparts with normal-weight.7 Accordingly, children with overweight may encounter quite different levels of psychological distress and associated behavioral outcomes due to COVID-19 than children without overweight. Introduction Even during pre-pandemic times, children with overweight experience greater psychological distress and impaired psychosocial outcomes due to weight stigma (weight bias).8 Research shows that people with overweight have a higher chance of being bullied and teased than those without overweight.8 These stigmatizing experiences and the ongoing fear and perception of weight stigma can lead to significant psychological distress.9 One major lifestyle and behavioral change that has occurred during school closures is increased internet usage by schoolchildren. Schoolchildren are very likely to engage in more internet-related activities for educational, entertainment, and social-engagement purposes during periods of physical distancing and isolation. However, there can be unintended negative consequences if internet behaviors are not monitored or constrained. For example, a study across eight Asian and European countries found more problematic internet use to be associated with higher levels of depressive and anxiety symptoms.10 Moreover, increased severity of gaming disorder and social media addiction may lead to psychological and sleep problems.11 During the unprecedented period of COVID-19 self-isolation and distancing an increase home-based online activities is likely, and it seems important to examine the relationship between internet-related activities and psychological distress in schoolchildren with overweight during the pandemic. There is little research examining whether weight status and weight stigma may influence perceptions of vulnerability to infectious diseases, and associated psychological distress. And whether problematic internet related behaviors during the COVID-19 lockdown are associated with greater fear of COVID-19 infection, and psychological distress. The present study recruited a large sample of schoolchildren with and without overweight from mainland China to examine the relationships between weight status, weight stigma, problematic internet and social media use, and psychological distress (including fear of COVID- 19) during the COVID-19 lockdown. Methods Participants, procedure, and ethical concerns Page 3/15 The study was part of an ongoing longitudinal project assessing problematic internet-related behaviors among primary school children. This research was approved by the ethics committee of the Hong Kong Page 3/15 Page 3/15 Polytechnic University (IRB ref: HSEARS20190718001). The longitudinal project routinely assesses primary school children’s characteristics, problematic internet-related behaviors (smartphone applications, social media, and gaming), perceived weight stigma, and psychological distress across a one-year period. During the period of the longitudinal project, the COVID-19 outbreak occurred in mainland China and the primary schoolchildren were instructed to stay at home for online learning. Consequently, an item on fear of COVID-19 infection was incorporated into the longitudinal project (see Instruments section for details) to assess the extent of children’s fear related to COVID-19. The present study was cross-sectional, with questionnaire data from the same wave that included the COVID-19 question analyzed. The survey was conducted online with the distribution of the survey facilitated by teachers from three primary schools in Sichuan, China. The online survey was generated by the research team and checked by the teachers to ensure that the survey hyperlink worked. Schoolteachers then sent the hyperlink to their students for participation. The first page of the online survey stated the objectives and participants’ rights, and sought consent from the students and one parent. Following the provision of participation consent from students and one of their parents, access to the survey proper was opened. Eligibility of the primary schoolchildren was defined the following inclusion criteria (i) they could read and understand the survey, which was written in Chinese and (ii) they owned and used at least one smartphone with internet access. The participants were excluded if they misperceived their weight status; that is, their perceived weight status did not fit with their body mass index (BMI) defined weight status. The reason for the exclusion criterion is because prior research shows that misperception of weight status is a strong predictor of children’s psychological distress, and we did not wish for this association to confound the present study.12-14 Measures Fear of COVID-19 infection. Using a visual analogue scale (from 0 [not at all afraid] to 10 [completely afraid]), a question “Are you afraid of being infected by COVID-19?” was used to understand to what extent participants fear the COVID-19 infection. Depression, Anxiety, Stress Scale-21 (DASS-21). Using 21 items rated on a 4-point Likert scale (ranging from 0 [did not apply to me at all] to 3 [applied to me very much or most of the time]), the DASS-21 assesses three types of psychological distress: stress (7 items), anxiety (7 items), and depression (7 items). Example items for the DASS-21 are “I found it hard to wind down” (for stress), “I was aware of dryness of my mouth” (for anxiety), and “I could not seem to experience any positive feeling at all” (for depression). The item scores are summated in each subscale to indicate the level of psychological distress, where higher scores indicate a higher level of depression, anxiety, or stress. The psychometric properties of the DASS-21 and the Chinese DASS-21 have been found to be good.15,16 The present study found that the Chinese DASS-21 had good internal consistency (α = 0.82 for stress subscale; 0.79 for anxiety subscale; and 0.84 for depression subscale). Page 4/15 Perceived weight stigma. A 10-item measure was used to assess perceived weight stigma. Participants responded to items such as “people act as if you are inferior because of your weight” using a dichotomous scale (0 = No and 1 = Yes). Item scores were summated to indicate the level of perceived weight stigma (scores ranging from 0-10), with higher scores indicating a higher level of perceived weight stigma. The linguistic validity and internal consistency of the Chinese perceived weight stigma items have been found to be satisfactory.17 The present study also showed that the Chinese perceived weight stigma items had good internal consistency (α = 0.83). Smartphone Application-Based Addiction Scale (SABAS). The six-item SABAS was used to assess level of problematic smartphone application use. The SABAS was designed based on the addiction component model criteria (i.e., salience, mood modification, tolerance, withdrawal conflict and relapse) proposed by Griffiths.18,19 Participants responded to items such as “During the past week, my smartphone is the most important thing in my life” using a Likert scale ranging from 1 (strongly disagree) to 6 (strongly agree). Measures The item scores are summed to indicate the level of problematic smartphone application use, where a higher score indicates a higher level of problematic use. The psychometric properties of the SABAS and the Chinese SABAS have been found to be satisfactory.20-24 The present study also showed that the Chinese SABAS had good internal consistency (α = 0.88). Bergen Social Media Addiction Scale (BSMAS). The BSMAS assesses the level of problematic social media use with six items rated on a 5-point Likert scale ranging from 1 (very rarely) to 5 (very often). The scale was developed based on the addiction component model criteria proposed by Griffiths.18,19 The following is an example item from the BSMAS “How often during the last week have you spent a lot of time thinking about social media or planned use of social media?”. Item scores are summated to indicate the level of problematic social media use, where a higher score indicate a higher level of problematic use. The psychometric properties of the BSMAS and Chinese BSMAS have been found to be satisfactory.25-28 The present study also showed that the Chinese BSMAS had good internal consistency (α = 0.88). Internet Gaming Disorder Scale-Short Form (IGDS9-SF). The nine-item IGDS9-SF assesses the level of disordered internet gaming behavior. The scale items were designed based on the nine criteria indicating internet gaming disorder [IGD] in the Diagnostic and statistical manual of mental disorders, fifth edition (DSM-5).29 Items such as “During last week, do you feel more irritability, anxiety or even sadness when you try to either reduce or stop your gaming activity?” were presented to participants who responded using a 5-point Likert scale ranging from 1 (never) to 5 (very often). The item scores are summed to indicate the level of problematic gaming, where higher scores indicating higher levels of problematic gaming. The psychometric properties of the IGDS9-SF have been found to be good.30-36 The present study also showed that the Chinese IGDS9-SF had good internal consistency (α = 0.926). Time spent on internet-related behaviors. Time spent on internet-related behaviors was assessed with the question: “How much time did you spend on each of the following internet-related behaviors daily?”. Participants were asked to indicate time spent on each of the following internet-related behaviors in the Page 5/15 past week: smartphone, social media, and gaming. The time spent on each internet-related behavior was then converted into hours per day in use. Data analysis Independent t-tests were used to detect the significant differences between the group with overweight and that without overweight in their psychological distress, perceived weight stigma, problematic internet- related behaviors, and time spent on internet-related behaviors. In addition, multivariate linear regression models were used to examine the associations between psychological distress (i.e., fear of COVID-19 infection, stress, anxiety, and depression), perceived weight stigma, and problematic internet-related behaviors (including problematic smartphone application use, problematic social media use, and problematic gaming). We divided the participants into two groups (participants with overweight and without overweight) to conduct the regression models for each group. Age and gender were adjusted in all the regression models. Moreover, a regression model using all participants (i.e., including those with and without overweight) was used to examine the associations between weight status, psychological distress, perceived weight stigma, and problematic internet-related behaviors. Measures Weight status. The schoolchildren self-reported their height in cm and weight in kg. BMI was then calculated to determine whether a schoolchild belongs to a group with overweight or a group without overweight.37 In addition to reporting height and weight, each schoolchild was asked to report their self- perceived weight status using the question: “What do you think your weight status is?” Answer choices included “Very thin”, “Thin”, “Normal weight”, “Overweight” , or “Obese”. Then, the self-perceived weight status was reclassified into two categories of “overweight” or “nonoverweight”. Results Table 1 presents participants’ characteristics among schoolchildren with overweight, (n = 236; 132 boys), and without overweight (n = 1 121; 557 boys), and noneligible participants (n = 657; 318 boys). Table 2 shows the means and SDs for psychological distress, perceived weight stigma, problematic internet- related behaviors, and time spent on internet-related behaviors between the two groups. As can be seen in Table 2, schoolchildren with overweight had significantly higher levels of COVID-19 infection fear, stress, and depression than did those without overweight. Schoolchildren with overweight also had significantly higher levels of perceived weight stigma than did those without overweight. With regards to problematic internet-related behaviors, schoolchildren with overweight had significantly higher levels of problematic social media use than did those without overweight. There were no significant differences between overweight and non-overweight in time spent on internet-related behaviors. Regression models for schoolchildren without overweight (Table 3) indicated that perceived weight stigma was a significant predictor in models, with higher levels of weight stigma associated with greater fear of COVID-19 infection, and higher levels of stress, anxiety, and depression. Problematic smartphone, Page 6/15 social media, and gaming use were also all significant positive predictor in stress, anxiety and depression, but not COVID-19 fear. Regression models for schoolchildren with overweight (Table 3) indicated that perceived weight stigma was a significant predictor in fear of COVID-19 infection at the p<0.10 level, but not at the conventional 0.05 level. Weight stigma was a significant predictor of stress, anxiety, and depression. With higher levels of weight stigma associated with higher levels of stress, anxiety, and depression. Similar to non- overweight, problematic smartphone and social media application use were also significant positive predictor in stress, anxiety and depression, but not fear of COVID-19 infection. Problematic gaming was not a significant predictor of COVID-19 infection fear, but was a significant predictor of stress, and only reached the 0.10 level of significance for anxiety and depression. Regression models for schoolchildren regardless of weight status (Table 3) indicated that overweight was a significant predictor in fear of COVID-19 infection. Weight stigma was a significant predictor of all types of psychological distress. With higher levels of weight stigma associated with higher levels of fear of COVID-19 infection, stress, anxiety, and depression. Additionally, problematic smartphone, social media, and gaming use were also all significant positive predictor in stress, anxiety and depression, but not COVID-19 fear. Discussion Consistent with previous findings,8,12-14 the present study found that schoolchildren with overweight had higher levels of psychological distress and perceived weight stigma than their counterparts with normal- weight. Moreover, schoolchildren with overweight had greater problematic social media use than did those without overweight. Regression models indicated that greater perceived weight stigma was associated with greater psychological distress in schoolchildren regardless of their weight status. Moreover, problematic smartphone application use and problematic social media use were associated schoolchildren’s stress, anxiety, and depression, regardless of their weight status. Among schoolchildren without overweight, problematic gaming was associated with higher levels of stress, anxiety, and depression, which is in line with previous findings.10,11 However, among those with overweight, problematic gaming was associated with lower levels of stress, anxiety, and depression. There are several explanations for higher psychological distress in schoolchildren with overweight than their peers without overweight. One of the major reasons might be because of the perceived weight stigma in this population. This speculation is supported by the recent meta-analysis conclusion that perceived weight stigma is significantly associated with depression and anxiety in people with overweight.9 Apart from perceived weight stigma, schoolchildren with overweight might be afraid of the serious health outcomes resulting from COVID-19 infection.4 In the present study, schoolchildren with overweight demonstrated more fear of being affected by COVID-19 than did those without overweight. However, the present study did not investigate specific reasons why they fear COVID-19 infection. Page 7/15 Page 7/15 Therefore, the speculation of fearing serious health outcomes resulted from COVID-19 infection needs corroboration from future studies. Moreover, the present study revealed that problematic social media use was more severe in schoolchildren with overweight than those without overweight. Thus, the higher level of psychological distress may come from a greater level of problematic social media use, as prior evidence showing the positive association between psychological distress and problematic social media use.11 Problematic social media use in children with overweight requires additional attention during prolonged school suspension after COVID-19 outbreak. Addiction or prolonged use of social media has been found to be a great risk of overweight.38,39 People with overweight and resultant stigmatizing experiences may be more likely to use social media for virtual interaction instead of real interpersonal activities, and they might be more avoidant of live social interactions, possibly due to anxiety or fear of stigma. Discussion They may also heavily rely on social media to search information relating to overweight, which may increase the risk of problematic internet use.40,41 Therefore, schoolchildren with overweight might heavily depend on social media because they want to search COVID-19-related information and seek emotional support during the current pandemic period. Unfortunately, such dependence on social media, even when not increasing significantly the time spent on social media, may trigger the development of psychological distress, especially for schoolchildren with overweight in the present study. In our results, perceived weight stigma was an important factor contributing to psychological distress regardless of weight status. Children with high perceived weight stigma might fear that they will not receive high quality health care should they get COVID-19. However, because weight stigma is closely associated with anxiety and depression, the association of weight stigma with fear of COVID infection may simply reflect higher anxiety and poorer mood in those who are stigmatized. Interestingly, the association between perceived weight stigma and the fear of COVID-19 infection was significant in schoolchildren without overweight but only significant at the 0.10 level in those with overweight. The relationship between perceived weight stigma and fear of COVID-19 infection among schoolchildren without overweight is in line with prior evidence showing associations between weight stigma and psychological distress among individuals without overweight.43 Surprisingly, problematic gaming seemed to be a protective factor against psychological distress in schoolchildren with overweight. Specifically, increased problematic use of gaming was significantly associated with less stress, anxiety, and depression. It is possible that the feature of hiding one’s identity (i.e., without revealing real information) during gaming may ease psychological distress for schoolchildren with overweight. Contrary to gaming, social media usually needs to reveal some personal information and may expose weight information. Thus, for schoolchildren with overweight who suffered from weight stigma, anonymous gaming could be an anxiety-alleviating activity, which may possibly override the more typical negative influence of problematic online behaviors on psychological well- being.11 Moreover, gaming does not offer a platform for sharing COVID-19-related information while social media does. Considering the potential impact of COVID-19 on psychological distress, gaming being.11 Moreover, gaming does not offer a platform for sharing COVID-19-related information while social media does. Considering the potential impact of COVID-19 on psychological distress, gaming Page 8/15 Page 8/15 engagement may protect schoolchildren with overweight. Future research might explore the potential benefits of certain gaming activities in specific populations. Conclusion In conclusion, schoolchildren with overweight suffered from stress, depression, perceived weight stigma, and fear of COVID-19 infection during the COVID-19 outbreak period. They also demonstrated problematic social media use. Perceived weight stigma was positively associated with fear of COVID-19 infection, while problematic use of social media and smart phones were also correlated with increased psychological distress, regardless of weight status. However, problematic gaming appeared to be associated with protective effects in schoolchildren with overweight. Therefore, strategies to manage perceived weight stigma may have a positive influence on mental health under health-threatening circumstances, such as the current COVID-19 pandemic. Future studies are warranted on the causes, correlates, and amelioration of fears related to public health crises including COVID-19. Finally, it would be important to develop and test the efficacy of psychological support and monitoring programs for internet-related activities among schoolchildren with overweight. Discussion There are some limitations in the present study. First, all the data including demographics (e.g., body weight and height), psychological constructs, perceived weight stigma, and internet-related behaviors are self-reported. Hence, some information may be susceptible to memory-related bias or social desirability bias. Second, the societal perception of discrimination toward people with overweight varies across countries. Therefore, the perceived weight stigma and resulting consequences during this health crisis may also be different. That is to say, the results should be generalized to other populations with caution. Lastly, some important confounding variables could not be controlled in the present study. Specifically, schoolchildren’s screen behaviors are highly associated with their parents’ screen behaviors.44,45 However, we did not collect information regarding parents’ screen use. References 1. World Health Organization. Coronavirus disease (COVID-2019): Situation report-85. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200504-covid-19-sitrep- 105.pdf?sfvrsn=4cdda8af_2. Accessed May 5, 2020. 1. World Health Organization. Coronavirus disease (COVID-2019): Situation report-85. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200504-covid-19-sitrep- 105.pdf?sfvrsn=4cdda8af_2. 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Measurement invariance of the Internet Gaming Disorder Scale–Short-Form (IGDS9-SF) between the United States of America, India and the United Kingdom. Page 11/15 Page 11/15 Tables Table 1. Participants’ characteristics F or χ2 (p-value) M (SD) or n (%) Page 12/15 Non-OW OW Noneligible childrena n = 1121 n = 236 n = 657 Age (year) 10.67 (1.11) 10.78 (0.93) 10.75 (1.06) 1.74 (0.18) Gender (boy) 557 (49.7%) 132 (55.9%) 318 (48.4%) 4.04 (0.13) Height (cm) 144.47 (9.00) 149.80 (8.81) 145.20 (9.64) 32.95 (<0.001) Weight (kg) 35.94 (10.51) 58.88 (22.33) 51.66 (19.06) 342.52 (<0.001) Body mass index (kg/m2) 17.09 (4.11) 26.02 (8.90) 24.35 (8.08) 374.95 (<0.001) Ethnicity (Han) 1106 (98.7%) 235 (99.6%) 647 (98.5%) 1.69 (0.43) Current sick (yes) 18 (1.6%) 6 (2.5%) 7 (1.1%) 2.57 (0.28) a Noneligible children were those who misperceived their weight status; 123 perceived themselves overweight but were classified as nonoverweight according to body mass index; 534 perceived themselves as nonoverweight but were classified asoverweight according to body Non-OW OW Noneligible childrena n = 1121 n = 236 n = 657 Age (year) 10.67 (1.11) 10.78 (0.93) 10.75 (1.06) 1.74 (0.18) Gender (boy) 557 (49.7%) 132 (55.9%) 318 (48.4%) 4.04 (0.13) Height (cm) 144.47 (9.00) 149.80 (8.81) 145.20 (9.64) 32.95 (<0.001) Weight (kg) 35.94 (10.51) 58.88 (22.33) 51.66 (19.06) 342.52 (<0.001) Body mass index (kg/m2) 17.09 (4.11) 26.02 (8.90) 24.35 (8.08) 374.95 (<0.001) Ethnicity (Han) 1106 (98.7%) 235 (99.6%) 647 (98.5%) 1.69 (0.43) Current sick (yes) 18 (1.6%) 6 (2.5%) 7 (1.1%) 2.57 (0.28) a Noneligible children were those who misperceived their weight status; 123 perceived themselves overweight but were classified as nonoverweight according to body mass index; 534 perceived themselves as nonoverweight but were classified asoverweight according to body mass index. Table 2. Comparisons of psychological distress and internet-related behaviors between groups M (SD)/n t (p-value) Non-OW OW Fear of COVID-19 infection 4.00 (3.64)/1042 5.08 (9.03)/210 2.87 (0.004) Stress 9.00 (2.77)/1121 9.61 (3.04)/236 2.85 (0.005) Anxiety 8.15 (2.12)/1121 8.39 (2.07)/236 1.58 (0.12) Depression 8.26 (2.42)/1121 8.62 (2.61)/236 1.97 (0.049) Perceived weight stigma 0.92 (1.78)/1121 1.30 (2.12)/236 2.59 (0.01) Problematic smartphone application use 12.43 (6.17)/1121 12.77 (6.25)/236 0.78 (0.44) Problematic social media use 8.90 (3.81)/1121 9.61 (4.12)/236 2.56 (0.01) Problematic gaming 12.82 (5.41)/1121 13.10 (5.49)/236 0.72 (0.47) Time spent on smartphone 2.29 (2.60)/1121 2.57 (2.78)/236 1.48 (0.14) Time spent on social media 1.11 (2.03)/1121 1.20 (1.65)/236 0.65 (0.52) Time spent gaming 0.92 (2.00)/1121 1.02 (1.95)/236 0.72 (0.47) Table 2. Comparisons of psychological distress and internet-related behaviors between groups Table 3. a Noneligible children were those who misperceived their weight status; 123 perceived themselves overweight but were classified as nonoverweight according to body mass index; 534 perceived themselves as nonoverweight but were classified asoverweight according to body mass index. Non-OW = group with nonoverweight; OW = group with overweight. Non-OW = group with nonoverweight; OW = group with overweight. Non-OW = group with nonoverweight; OW = group with overweight. Tables Regression models investigating relationships between internet-related behaviors and psychological distress amongchildren with or without overweight Coefficient (SE)/ Standardized coefficient Coefficient (SE)/ Standardized coefficient Page 13/15 Fear of COVID-infection Stress Anxiety Fear of COVID-infection Depression Anxiety Group without overweight Age -0.016 (0.101)/ -0.005 -0.033 (0.061)/ -0.013 0.017 (0.048)/ 0.009 -0.072 (0.055)/ -0.033 Gender (Ref: boys) -0.372 (0.229)/ -0.051 -0.141 (0.139)/ -0.025 0.116 (0.109)/ 0.027 -0.089 (0.124)/ -0.018 Perceived weight stigma 0.279 (0.065)/ 0.138* 0.426 (0.040)/ 0.274* 0.382 (0.032)/ 0.320* 0.398 (0.036)/ 0.293* PSAU -0.010 (0.027)/ -0.017 0.101 (0.017)/ 0.226*** 0.032 (0.013)/ 0.092* 0.044 (0.015)/ 0.113 PSMU 0.050 (0.04)/ 0.052 0.068 (0.024)/ 0.094 0.049 (0.019)/ 0.088 0.085 (0.022)/ 0.134* PG 0.018 (0.031)/ 0.027 0.086 (0.019)/ 0.168* 0.086 (0.015)/ 0.221* 0.087 (0.017)/ 0.194*** R2 (Adj. R2) 0.030 (0.024) 0.335 (0.331) 0.298 (0.294) 0.307 (0.303) Group with overweight Age 0.485 (0.668)/ 0.050 -0.370 (0.161)/ -0.113* -0.143 (0.114)/ -0.064 -0.303 (0.144)/ -0.108* Gender -1.798 (1.291)/ -0.099 0.014 (0.311)/ 0.002 0.062 (0.219)/ 0.015 0.021 (0.277)/ 0.004 Perceived weight stigma 0.592 (0.312)/ 0.141# 0.346 (0.077)/ 0.241* 0.345 (0.054)/ 0.353* 0.442 (0.068)/ 0.359* PSAU -0.093 (0.150)/ -0.618 0.178 (0.036)/ 0.362* 0.107 (0.026)/ 0.323* 0.129 (0.032)/ 0.309* PSMU -0.232 (0.213)/ -0.106 0.287 (0.053)/ 0.388* 0.130 (0.037)/ 0.259* 0.157 (0.047)/ 0.248* PG 0.286 (0.182)/ 0.175 -0.115 (0.044)/ -0.208 -0.055 (0.031)/ -0.147# -0.067 (0.039)/ -0.140# R2 (Adj. R2) 0.047 (0.019) 0.451 (0.436) 0.410 (0.394) 0.404 (0.388) All participants Weight (Ref: non overweight) 0.297 (0.125)/ 0.067* 0.108 (0.055)/ 0.043# 0.008 (0.042)/ 0.004 0.035 (0.049)/ 0.016 Age 0.024 (0.128)/ 0.005 -0.068 (0.058)/ -0.026 0.003 (0.044)/ 0.002 -0.094 (0.051)/ -0.042 Gender (Ref: boys) -0.615 (0.286)/ -0.062* -0.097 (0.128)/ -0.017 0.121 (0.098)/ 0.029 -0.056 (0.113)/ -0.011 Perceived weight stigma 0.326 (0.079)/ 0.122* 0.420 (0.036)/ 0.275* 0.377 (0.027)/ 0.330* 0.412 (0.032)/ 0.310* PSAU -0.021 (0.034)/ -0.027 0.113 (0.015)/ 0.246* 0.044 (0.012)/ 0.129* 0.058 (0.013)/ 0.145* PSMU 0.008 (0.049)/ 0.007 0.105 (0.022)/ 0.143* 0.061 (0.017)/ 0.112* 0.097 (0.020)/ 0.154* PG 0.057 (0.039)/ 0.062 0.055 (0.018)/ 0.105 0.064 (0.014)/ 0.165* 0.062 (0.016)/ 0.138*** R2 (Adj. R2) 0.032 (0.026) 0.348 (0.344) 0.309 (0.305) 0.320 (0.316) # p<0.1; * p<0.05; p<0.01; * p<0.001 Disclosure: The authors declared no conflict of interest. Correspondence: C.-Y. Lin, PhD, Department of Rehabilitation Sciences, Faculty of Health and Social Sciences, The Hong Kong Polytechnic University, 11 Yuk Choi Rd, Hung Hom, Hong Kong. E-mail: cylin36933@gmail.com; Tel: 852-2766-6755; Fax: 852-2330-8656 Page 14/15 Conflict of interests: All the authors declare that there is no conflict of interest. Declarations Disclosure: The authors declared no conflict of interest. Page 14/15 Conflict of interests: All the authors declare that there is no conflict of interest. Page 14/15 Funding resources: None. Funding resources: None. Funding resources: None. Page 15/15
W2144154023.txt	https://zenodo.org/records/1528354/files/article.pdf	de		Nachweis von Metallgiften	Analytical and bioanalytical chemistry/Analytical & bioanalytical chemistry	1,909	public-domain	429	4. Auf gerichtliche Chemic beztigliche. 519 Zum Nachweis von Blaus~ure. G. C a 1 v i and M. M a 1 a c a r n e ~) machen darauf aufmerksam, dass in F~ulnis ttbergegangene Leicheuteile. namentlich aber sauer reagierende Untersuchungsobjekte. wie Erbrochenes, zu einer wesentlich schnelleren Zersetzung der Cyanalkalien durch die Luft beitragen. Ein Zusatz yon 95-prozentigem Alkohol dagegen h~tlt die Zersetzung wesentlich auf, so dass bei in diesem aufbewahrten Leichenteilen Blausiture noch uach einem Monat nachgewiesen werdeD konnte. N a c h w e i s yon MetaUgiften. Eine schnelle Methode zum Nachweis yon Metallgiften in Leichenteilen haben (3. D. L a n d e r und H. W. W i n t er'2) ausgearbeitet. Sie richteten ihr Augenmerk haupts~tchlich darauf, mit m0glichst wenig Substanz auszukommen und nur geringe Mengen an Reagenzien anzuwenden. Dcshalb teilen sic auch ihr Untersuchungsmaterial in zwei Teile und prtifen den einen T e i l unter Benutzung des R e i n s c h ' s c h e n Verfahrens auf Arsen, Antimon, Wismut ~and Quecksilber. Den anderen Teil extrahieren sie mit 50-prozentiger Salpeters~ure unter Hinzuftigen yon ein wenig Schwefelsiiure uad erhalten in dem Auszug Blei, QueCksilber. Wismut, Kupfer, Zink m~d CArom. Wird die Schwefelshure weggelassen, so finder man hier auch Baryum. Das Extrahieren geschieht in der Weise. dass die zu untersvchende Substanz (etwa 1 0 g ) mit dem Siiuregemisch ( 4 0 c c m 50-prozentige Salpetersi~ure und 5 10 ccm konzel~trierte Schwefels~ture) in einer Schale bis zum Entweichen yon braunen D~tmpfen mit kleiuer Flamme erhitzt. dann mit Wasser verdtinnt und filtriert wird In dem F i l t r a t ktinnen die einzelnen Metalle nach bekannten Methoden identifiziert werden. Mit der filr den ersten Teil der Untersuchungsobjekte angewandten R e i n s c h 'schen Methode war es den Verfassern gelungen, noch 0,05 mg Arsen und O ~ l m g Antimon in 120g, dagegen n u r t rng Quecksilber in 60 g organischer Substanz nachzuweisen. Den Nachweis von {~uecksilber in L e i c h e n t e i l e n fiihrt C. L o m b a r d o'3) auf folgende Weise. Als Reagens benutzt er eine LSsung, die 1 2 g Zinnchlorttr, 25 ccm Salzs~iure uud 75 ccm Wasser enth~tlt. Von dieser ftigt er 5 ccm zu 5 cc,t einer aus den Leichenteilen her- 1) Giorn. Farm. CAirn. ~6, 5; dutch Zeitschr. f. Unters. d. Nahrungs: und Genussmittel 17, 208. 2) The Analyst 88. 450. 8) Arch. d. Farmacol. sperim. 7, 400: dutch Chem. Zentralblatt 79, H. 1788.
https://openalex.org/W2884068272	https://researchonline.ljmu.ac.uk/id/eprint/9007/1/Clark_et_al-2018-Sports_Medicine_-_Open.pdf	English	null	Contemporary perspectives of core stability training for dynamic athletic performance: a survey of athletes, coaches, sports science and sports medicine practitioners	Sports medicine - open/Sports medicine - Open	2,018	cc-by	9,536	Clark, DR, Lambert, MI and Hunter, AM Contemporary perspectives of core stability training for dynamic athletic performance: a survey of athletes, coaches, sports science and sports medicine practitioners. LJMU Research Online Article Citation (please note it is advisable to refer to the publisher’s version if you intend to cite from this work) Clark, DR, Lambert, MI and Hunter, AM (2018) Contemporary perspectives of core stability training for dynamic athletic performance: a survey of athletes, coaches, sports science and sports medicine practitioners. Sports Medicine Open, 4 (1). 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Sports Medicine - Open (2018) 4:32 https://doi.org/10.1186/s40798-018-0150-3 Open Access Contemporary perspectives of core stability training for dynamic athletic performance: a survey of athletes, coaches, sports science and sports medicine practitioners David R. Clark1* , Michael I. Lambert2 and Angus M. Hunter3 * Correspondence: d.r.clark@ljmu.ac.uk 1School of Sport and Exercise Sciences, Faculty of Science, Liverpool John Moore’s University, 102, 2 Moorfields, Liverpool L2 2BS, UK Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. * Correspondence: d.r.clark@ljmu.ac.uk 1School of Sport and Exercise Sciences, Faculty of Science, Liverpool John Moore’s University, 102, 2 Moorfields, Liverpool L2 2BS, UK Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Abstract Background: Core stability training has grown in popularity over 25 years, initially for back pain prevention or therapy. Subsequently, it developed as a mode of exercise training for health, fitness and sport. The scientific basis for traditional core stability exercise has recently been questioned and challenged, especially in relation to dynamic athletic performance. Reviews have called for clarity on what constitutes anatomy and function of the core, especially in healthy and uninjured people. Clinical research suggests that traditional core stability training is inappropriate for development of fitness for heath and sports performance. However, commonly used methods of measuring core stability in research do not reflect functional nature of core stability in uninjured, healthy and athletic populations. Recent reviews have proposed a more dynamic, whole body approach to training core stabilization, and research has begun to measure and report efficacy of these modes training. The purpose of this study was to assess extent to which these developments have informed people currently working and participating in sport. Methods: An online survey questionnaire was developed around common themes on core stability training as defined in the current scientific literature and circulated to a sample population of people working and participating in sport. Survey results were assessed against key elements of the current scientific debate. Results: Perceptions on anatomy and function of the core were gathered from a representative cohort of athletes, coaches, sports science and sports medicine practitioners (n = 241), along with their views on effectiveness of various current and traditional exercise training modes. Most popular method of testing and measuring core function was subjective assessment through observation (43%), while a quarter (22%) believed there was no effective method of measurement. Perceptions of people in sport reflect the scientific debate, and practitioners have adopted a more functional approach to core stability training. There was strong support for loaded, compound exercises performed upright, compared to moderate support for traditional core stability exercises. Half of the participants (50%) in the survey, however, still support a traditional isolation core stability training. Conclusion: Perceptions in applied practice on core stability training for dynamic athletic performance are aligned to a large extent to the scientific literature. Keywords: Core, Stability, Dynamic, Trunk, Athletic, Performance, Loaded, Functional, Compound, Exercise © The Author(s). Abstract 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Key points Key points articles [1, 6, 7, 12–14]. Reviewers have noted that re- search cannot progress this topic effectively until there is a standardised agreement on the anatomical structure and function of the core [1, 6, 7]. A further limitation re- ported by most reviewers is the absence of a valid and reliable test of core function [1, 12]. As a result most re- search on the topic is methodologically limited [12, 13] and therefore ineffective in confirming or challenging the concept and practice of CST for health and per- formance. A case has been made in the literature for a more functional definition of anatomy of the core, applicable to healthy and athletic populations [1, 8]. Similarly, it is proposed that the description of core function is revised to encompass normal healthy and athletic human movement [8]. Core stability training for healthy and athletic populations has recently been questioned and challenged in scientific literature. The narrow definition of both the anatomy, spinal region between pelvis and diaphragm, and the method of training the core through the isolation of muscles in this region does not relate to full body core function that characterises dynamic athletic performance. y The survey reveals that this is reflected in opinions of people working and participating in sport. Half of the participants identified the area between and including the pelvic and shoulder girdles as the core. Majority supported functional loaded exercises such farmer’s walk (87%) and barbell squats (84%) as effective exercises for the development of core stability. Several comprehensive reviews over the last decade have examined the research on the effectiveness of various CST methods for athletic performance [1, 6, 7, 12–14]. Reviews covered the variations in CST including instability training, trunk rotation exercises, functional training and exercise intensity. Martuscello et al. pro- posed a five core exercise classification system based on their review of the research [6]. The categories were traditional core exercise (sit-ups), core stability exercises (isometric plank), ball or device exercises (stability ball), free weight exercise (squat and deadlift) and noncore free weight exercise (upper body). In a recent study con- ducted in an applied performance sport setting, Spencer et al. proposed a comprehensive spinal exercise classifi- cation [2]. The classification incorporated static and dynamic exercises that were either functional or non-functional according to spinal displacement across four physical outcomes: mobility, motor control, work capacity and strength. Page 2 of 10 Page 2 of 10 Clark et al. Sports Medicine - Open (2018) 4:32 Key points Both studies [2, 6] clarify the range and nature of core stability exercises used in the literature and practice; however, there is concern that many core stability intervention studies are di- luted by other exercises and activities preventing a clear assessment of impact of CST [7, 12, 13]. Fur- thermore, in athletic populations, a reductionist ap- proach or selective activation to improve integrated function is unsubstantiated [1, 2, 7, 12]. Despite the support for a more functional approach, selected traditional core stability training methods do retain a certain amount of support; isometric plank exercise (56%) and unstable stability ball exercises (41%). Many respondents (42%) felt that core function should be measured subjectively through observation of sporting and or exercise performance. Trunk is the preferred name of the anatomical region for almost half (45%) the participants while 35% supported the term core. Methods The online survey questionnaire (Additional file 1) was developed around common themes on core stability as defined in the current scientific literature. The online survey was created and distributed using Bristol Online Survey (BOS) tool (Tower Hill, Bristol, UK). The ques- tionnaire comprised four sections: anatomy of the core, function of the core, methods of measuring core func- tion and methods of training the core. The survey con- cluded with general questions about the application of core strength training for dynamic athletic performance. y A limitation identified by Prieske et al. [12] was the lack of validity of tests used in most of the research. Trunk muscle strength in most studies was measured by timed isometric test (prone bridge) which, firstly, does not reflect force and velocity of movement of dynamic athletic activity [12]. Secondly, CST programmes in many of the studies incorporated prone plank or similar isometric exercises in the exercise intervention, which rendered timed isometric prone plank an inappropriate test of trunk muscle strength in these cases. Most reviews conclude there is not a valid method of measur- ing the effect of CST on trunk muscle strength within the context of improving dynamic athletic performance [1, 13, 14, 17, 25, 26]. As a result, many researchers have resorted to using conventional performance tests such as countermovement jump and sprint tests [12, 13, 27]. The survey question on the anatomy of the core is based on definitions in the literature. We used the defin- ition of local and global stabilization of intersegmental spine proposed by Bergmark (1989) [38]; the passive spinal column, active spinal muscles and neural control unit as described by Panjabi [39]; axial skeleton between pelvic and shoulder girdle including rib cage, spinal col- umn and associated muscle and nerves proposed by Behm et al. [8]; and lumbo-pelvic hip complex according to Faries and Greenwood [23]. Categories of exercises and selection criteria for CST used in the survey ques- tion were drawn from published studies that investigated muscle activation using these manipulations. The ques- tion around core strength and core stability were based on reviews of this topic [1, 7]. The first three levels of Martuscello’s [6] core exercise classification system appear to contravene the estab- lished overload training principle [28] when applied to an athletic population. Background The absence of a universally accepted definition of core stability (CS) is well noted in the scientific literature [1–8]. A number of these publications have proposed a defin- ition, focussing either on function, anatomical constitu- ents of the core or both. Several reviews have questioned and challenged core stability training (CST) for prevention and treatment of back pain [9–11] and for improvement of function and performance in healthy and athletic popu- lations [1, 5–7, 12–14]. There is a view [1, 7] that CST in its current form evolved from clinical research [15] in the 1990s. The application of a clinical exercise approach in healthy and athletic populations has been criticised, primarily on the basis that teaching an iso- lated muscle pattern in uninjured athletes is unfounded [6, 10, 16]. Despite this, CST as an intervention spread to all exercise disciplines across clinical, fitness and sports performance settings with significant commer- cial interest and support [14]. The proposed protection against injury and improved athletic performance from CST has been the subject of many research studies and review papers. Silfies et al. con- cluded that following a review of 11 studies, there was limited evidence to support the use of CST to prevent upper extremity injury and improve athletic performance [3]. The authors questioned whether performance in core stability tests reflected physical or athletic capability and level of conditioning, rather than solely core stabilization. Tests included the isometric front and side bridge, single-leg raise [10], star excursion test [11] and closed kinetic chain upper extremity stability test [12]. A Most review articles on this topic recognised that the application of traditional CST in healthy and athletic groups lack scientific justification [3, 7, 14, 17]. This re- sulted in a body of research investigating CST in healthy populations [18–22] along with aforementioned review Clark et al. Sports Medicine - Open (2018) 4:32 Page 3 of 10 Page 3 of 10 systematic review conducted by Prieske et al. [12] con- cluded that CST compared with no training or regular sports-specific training does improve trunk muscle strength measured predominantly by isometric plank. However, increases in trunk muscle strength only had a small effect on physical fitness and athletic performance measures in trained individuals. CST compared to al- ternative physical training methods in trained individ- uals had little impact on trunk muscle strength, physical fitness and athletic performance measures. Background Both studies strongly suggest that high levels of gen- eral fitness are associated with better performance in CS tests and therefore a lower risk of injury and bet- ter athletic performance test scores [3, 12]. athletic events. Researchers have begun to investigate trunk muscle activation in a number of dynamic, loaded free weight exercises to determine their suitability for the devel- opment of dynamic trunk strength and stability [29–37]. Surface electromyography methodology shows there is good evidence that loaded exercises performed in a stand- ing position are an effective method of overloading the trunk stabilization system in a dynamic manner. While several reviewers recognise this development [6, 7, 14], it is best summarised by Wirth et al. (2016), ‘… we recom- mend the use of classical strength-training exercises as these provide the necessary stimuli to induce the desired adaptations.’ The flawed foundations of CST for dynamic athletic performance have been exposed in the scientific litera- ture. Research is underway to better understand the most effective training methods for the development of trunk stability. The aim of this survey is to assess the current perspectives of CST in the applied sports setting to determine how well scientific literature informs these opinions. Our hypothesis is that opinions of those who work and participate in sport will reflect scientific debate on key core stability training topics. Separating the core into smaller local and larger global muscles has little bearing on core stability for dynamic movement in healthy people. In Lederman’s [10] words, this is an anatomical classification with no functional relevance. The role the core plays in stabilis- ing the body is dynamic and responsive to many postural challenges that occur in normal movement and complex, reactive environment of sport [14]. The concepts of core strength and core stability have been reviewed the literature [1, 5, 23]. Whether these are separate attributes [5] or whether core strength is required for core stability [23] re- main unresolved questions [1]. In this context, core stability is an integrated, functional motor task [7, 24] and training should reflect this according to movement pat- terns [14, 24], forces [7, 24] and torque and velocity [8, 24]. The survey was circulated using two methods: shared with the principal authors’ 700 LinkedIn connections and sent by email to 220 qualifying contacts. All recipi- ents were asked to share the survey with all their contacts that met the criteria of working or participating in sport. Methods Traditional low load core exer- cises, minimal range or isometric core stability exercises and ball/device exercises are all characterised by low force, low velocity and restricted range of movement. Hence, these do not represent training overload in prep- aration for activities that characterise most sports and A pilot survey was conducted using the postgraduate sports studies group (n = 20) at the University of Stirling. The questionnaire was modified according to feedback from the pilot survey. Approval for the study was granted Page 4 of 10 Clark et al. Sports Medicine - Open (2018) 4:32 Fig. 2 Responses to a series of questions on the effectiveness of selected categories of exercise in developing core stability for dynamic athletic performance. Data are reported as mean level of effectiveness with 95% CI. 1 = very effective, 5 = not effective at all. Significant differences p < 0.001: a vs c, d, e, f, g and h; b vs c, d, e, f, g and h; c vs d and f; d vs e, f and h; e vs f; f vs g and h; g vs h. CI: confidence interval by the local research ethics committee in accordance with the Helsinki Declaration (2013) [40]. Statistical analysis The data analysis was descriptive and frequency was pre- sented in the tables as number and percentage (n (%)). Data presented in Figs. 1, 2, 3 and 4 were analysed using Kruskal-Wallis test to assess support for each statement on 5-point Likert scale. Data presented as mean and 95% CI. Five-point scale is as follows: 1 = strongly agree or very effective and 5 = strongly disagree or not effect- ive at all. Significant differences were further analysed using Dunn’s multiple comparison post hoc test. Priori alpha level of significance was set at p < 0.05. Fig. 2 Responses to a series of questions on the effectiveness of selected categories of exercise in developing core stability for dynamic athletic performance. Data are reported as mean level of effectiveness with 95% CI. 1 = very effective, 5 = not effective at all. Significant differences p < 0.001: a vs c, d, e, f, g and h; b vs c, d, e, f, g and h; c vs d and f; d vs e, f and h; e vs f; f vs g and h; g vs h. CI: confidence interval Fig. 2 Responses to a series of questions on the effectiveness of selected categories of exercise in developing core stability for dynamic athletic performance. Data are reported as mean level of effectiveness with 95% CI. 1 = very effective, 5 = not effective at all. Significant differences p < 0.001: a vs c, d, e, f, g and h; b vs c, d, e, f, g and h; c vs d and f; d vs e, f and h; e vs f; f vs g and h; g vs h. CI: confidence interval recreational sport made up 15% while 9% were semi-professional in part-time paid roles. Results Participants Responses to all questions were analysed for all re- spondents (n = 241) and for each of the five demo- graphic groups. There were no differences between group responses and total cohort, so data are presented and discussed for the total cohort. Participants The online survey was completed by 241 respondents from a range of disciplines involved in sport (Table 1). The high- est return by employment group was received from strength and conditioning coaches (S&CC; 47%) followed by athletes and players (A&P; 17%) and sport medicine practitioners and physiotherapists (SM&P; 17%). A quarter of the cohort were involved in sport at university or school level (27%). A similar number (33%) were working in professional sport, either with full-time professional athletes (21%), or elite funded athletes in institutes of sport (12%). Volunteers working in The majority (87%) were qualified to degree level or higher, 40% had masters or MSc degrees and 12% had doctoral degrees. Most respondents (73%) re- ported to have a discipline specific professional quali- fication. Respondents reported to have been working in their specific discipline for an average of 8 years (range 0–36 years). Fig. 1 Reported support for a series of statements relating to core stability and core strength. Data are reported as mean level of agreement with 95% CI. 1 = strongly agree, 5 = strongly disagree. Significant differences p < 0.001: a vs b, a vs d, b vs d and c vs d. CI: confidence interval Fig. 1 Reported support for a series of statements relating to core stability and core strength. Data are reported as mean level of agreement with 95% CI. 1 = strongly agree, 5 = strongly disagree. Significant differences p < 0.001: a vs b, a vs d, b vs d and c vs d. CI: confidence interval Fig. 3 Responses to which criteria should inform exercise selection for the development of core stability for dynamic athletic performance. Data are reported as mean level of agreement with 95% CI. 1 = strongly agree, 5 = strongly disagree. Significant differences p < 0.05: a vs c, a vs d, b vs c, b vs d and c vs d. CI: confidence interval Fig. 3 Responses to which criteria should inform exercise selection for the development of core stability for dynamic athletic performance. Data are reported as mean level of agreement with 95% CI. 1 = strongly agree, 5 = strongly disagree. Significant differences p < 0.05: a vs c, a vs d, b vs c, b vs d and c vs d. CI: confidence interval Fig. Anatomy and name of the core In response to the question on the anatomical region that comprised the core, half of the respondents (50%) identified the region between and including the pelvic and shoulder girdles and associated muscles and nerves (Table 2). Approximately, a quarter of respondents (27%) identified the region between the diaphragm and pelvic floor and associated muscles and nerves as the core, while for 18%, this was the lumbar spine, pelvis, hip joints and related muscles and nerves. Interestingly, more participants (45%) felt that the region should be called the trunk while 35% supported the term core and 18% preferred torso. Core function and core stability training y g g The majority believed that core strength is required for stability (mean 1.9, 95% CI 1.8–2.0, p < 0.001) and far fewer agreed that these were separate attributes (mean 2.6, 95% CI 2.4–2.7, p < 0.001) (Fig. 1). Most participants disagreed with the statement that core strength was re- quired for athletic performance, but not everyday life (mean 3.9, 95% CI 3.7–4.0, p < 0.001). Data presented as number and percentage (n (%)) of all respondents. Italics represent the highest response for the column S&CC strength and conditioning coaches, A&P athletes and players, SM&P sports medicine practitioners and physiotherapists, SP&B sports physiologists and biomechanists SC sports coaches Data presented as number and percentage (n (%)) of all respondents. Italics represent the highest response for the column S&CC strength and conditioning coaches, A&P athletes and players, SM&P sports medicine practitioners and physiotherapists, SP&B biomechanists, SC sports coaches Methods of measuring core function Methods of measuring core function Respondents were asked to identify the most effective method of measuring core stability in a healthy, unin- jured person. Almost a quarter (22%) reported that there was no effective method to test core stability. A number (43%) of the respondents proposed subjective assessment of core stability through observation. Of these, 17% sug- gested observation of sport-specific movement or exer- cise technique and 26%, observation of ground-based loaded barbell exercises. Objective assessments were proposed by 32% and included the timed isometric plank (19%), functional movement screen (9%) and isometric trunk bracing with biofeedback (4%). Fig. 4 Responses to a series of statements relating to ground-based loaded free barbell exercises and trunk muscle activation. Data are reported as mean level of agreement with 95% CI. 1 = strongly agree, 5 = strongly disagree. Significant differences p < 0.001: a vs b, a vs d, b vs c, c vs d. CI: confidence interval p g p p g p ioning coaches, A&P athletes and players, SM&P sports medicine practitioners and physiotherapists, SP&B sports physiologists and h p p g ( ( )) p p g p S&CC strength and conditioning coaches, A&P athletes and players, SM&P sports medicine practitioners and physiotherapists, SP&B sp biomechanists, SC sports coaches Participants 3 Responses to which criteria should inform exercise selection for the development of core stability for dynamic athletic performance. Data are reported as mean level of agreement with 95% CI. 1 = strongly agree, 5 = strongly disagree. Significant differences p < 0.05: a vs c, a vs d, b vs c, b vs d and c vs d. CI: confidence interval Fig. 3 Responses to which criteria should inform exercise selection for the development of core stability for dynamic athletic performance. Data are reported as mean level of agreement with 95% CI. 1 = strongly agree, 5 = strongly disagree. Significant differences p < 0.05: a vs c, a vs d, b vs c, b vs d and c vs d. CI: confidence interval Fig. 1 Reported support for a series of statements relating to core stability and core strength. Data are reported as mean level of agreement with 95% CI. 1 = strongly agree, 5 = strongly disagree. Significant differences p < 0.001: a vs b, a vs d, b vs d and c vs d. CI: confidence interval Fig. 1 Reported support for a series of statements relating to core stability and core strength. Data are reported as mean level of agreement with 95% CI. 1 = strongly agree, 5 = strongly disagree. Significant differences p < 0.001: a vs b, a vs d, b vs d and c vs d. CI: confidence interval Clark et al. Sports Medicine - Open (2018) 4:32 Page 5 of 10 Page 5 of 10 Fig. 4 Responses to a series of statements relating to ground-based loaded free barbell exercises and trunk muscle activation. Data are reported as mean level of agreement with 95% CI. 1 = strongly agree, 5 = strongly disagree. Significant differences p < 0.001: a vs b, a vs d, b vs c, c vs d. CI: confidence interval Methods of measuring core function Methods of measuring core function The effectiveness of certain exercise categories on CST (Fig. 2) The exercise categories deemed most effective in devel- oping core stability for dynamic athletic performance were (Fig. 2) squats and Olympic lifts (mean 1.7, 95% CI, 1.6–1.8, p < 0.001) and farmers walk (mean 1.7, 95% CI Table 1 (A) Employment and (B) education information presented for all respondents (total and group) Total S&CC A&P SM&P SP&B SC All respondents 241 114 (47) 42 (17) 41 (17) 24 (10) 20 (8) A. Academic, university or school sport role 66 (27) 29 (12) 10 (4) 11 (5) 10 (4) 6 (2) Professional: full-time paid position, full-time paid athletes 50 (21) 37 (15) 0 (0) 9 (4) 3 (1) 1 (0) Volunteer, recreational club sport 35 (15) 4 (2) 21 (9) 6 (2) 2 (1) 2 (1) Elite professional: full-time paid position, funded, amateur athletes (Institute) 30 (12) 15 (6) 1 (0) 4 (2) 7 (3) 3 (1) Elite non-professional, part-time, regional or national athletes 30 (12) 16 (7) 5 (2) 7 (3) 0 (0) 2 (1) Semi-professional: paid part-time position 22 (9) 9 (4) 2 (1) 3 (1) 2 (1) 6 (2) Other 8 (3) 4 (2) 3 (1) 1 (0) 0 (0) 0 (0) B. MSc/Masters 96 (40) 51 (21) 7 (3) 20 (8) 13 (5) 5 (2) Degree/Hons 84 (35) 41 (17) 17 (7) 9 (4) 7 (3) 10 (4) PhD 28 (12) 10 (4) 2 (1) 10 (4) 4 (2) 2 (1) Diploma 27 (11) 9 (4) 13 (5) 2 (1) 0 (0) 3 (1) Other 6 (2) 3 (1) 3 (1) 0 (0) 0 (0) 0 (0) Data presented as number and percentage (n (%)) of all respondents. Italics represent the highest response for the column S&CC strength and conditioning coaches, A&P athletes and players, SM&P sports medicine practitioners and physiotherapists, SP&B sports physiologists and biomechanists, SC sports coaches Table 1 (A) Employment and (B) education information presented for all respondents (total and group) and (B) education information presented for all respondents (total and group) Clark et al. Sports Medicine - Open (2018) 4:32 Page 6 of 10 Page 6 of 10 Table 2 Responses to the question of what (A) anatomic region makes up the core and (B) which term best describes this anatomical region Total A. The effectiveness of certain exercise categories on CST (Fig. 2) The spine and the associated muscles and nerves 5 (2) The lumbar spine, pelvic and hip joints and associated muscles and nerves 43 (18) The region between and including the pelvic and shoulder girdles and associated muscles and nerves 120 (50) The region between and diaphragm and pelvic floor and associated muscles and nerves 65 (27) Other 8 (3) B. Torso 43 (18) Trunk 108 (45) Core 85 (35) Upper limb 0 (0) Other 5 (2) Data presented as number and percentage (n (%)) of all respondents. Italics represent the highest response (Fig. 4). Equally important in this form of resistance training was correct postural control (mean 2.0, 95% CI 1.9–2.2, p < 0.001). Slow controlled movement (mean 2.8, 95% CI 2.7–2.9, p < 0.001) and increases in velocity (mean 2.6, 95% CI 2.5–2. 8, p < 0.001) of strength train- ing exercises were not seen as important in eliciting trunk muscle activation in ground-based free barbell exercises. Finally, results for the general questions on the appli- cation of core stability exercises are presented on Table 3. Most participants (85%) felt that it was appropriate to include specific exercises to train core stability in healthy, uninjured individuals. Less than half (45%) felt that it was effective to exercise the core stabilisers in iso- lation, while a majority (65%) agreed that core stability is developed during normal progressive exercise training. Discussion Core stability training for healthy and athletic popula- tions has been scrutinised and challenged in recent years in scientific literature [6, 7, 10, 13, 41–43]. Descriptions of the core by anatomic structures are entirely dependent on the chosen definition of core function [1]. The original narrow definition presented in early re- search focussed on the spinal region between the dia- phragm and pelvis [44]. This approach identified muscular and neural dysfunction associated with back pain. Hence, core function was isolated to this region and proposed training intervention isolated the involved muscles. This approach did not transfer to healthy indi- viduals and athletes where core function is obviously at the centre of dynamic movement characterised by force and velocity through the length of the body [10]. Core stability described by Fletcher (2016), ‘…is the kinetic 1.6–1.9, p < 0.001). Conversely, support was moderate to low for traditional core stability exercises, namely sus- pended compound exercises (mean 2.2, 95% CI 2.1– 2.3, p < 0.001), isometric plank (mean 2.5, 95% CI, 2.4–2.6, p < 0.001), hanging leg raise (mean 2.8, 95% CI 2.7–2.9, p < 0.001) and instability abdominal exer- cises (mean 2.8, 95% CI 2.7–3.0, p < 0.001). Partici- pants identified two exercise categories that were more ineffective than effective; abdominal bracing (mean 3.2, 95% CI, 3.0–3.3, p < 0.001) and sit-ups (mean 3.7, 95% CI, 3.5–3.8, p < 0.001). The exercise selection criteria for effective CST (Fig. 3) Table 3 Answer to a series of questions about the application of core stability Table 3 Answer to a series of questions about the application of core stability Total Do you think it is necessary to include specific exercises to train core stability in a healthy, uninjured athlete’s exercise programme? Yes 206 (85) No 30 (12) Do not know 5 (1) Do you think it is possible to isolate and train the core stabilization system? Yes 120 (50) No 82 (34) Do not know 39 (16) Do you think it is effective to isolate and train the core stabilization system? Yes 89 (37) No 108 (45) Do not know 44 (18) Do you think that the core stability is automatically developed during normal, progressive exercise training? Yes 157 (65) No 67 (28) Do not know 17 (7) Data presented as number and percentage (n (%)) of all respondents. Italics represent the highest response for each question Correct movement pattern (mean 1.8, 95% CI 1.7–1.9, p < 0.001) was identified as most important exercise selec- tion criteria for development of core stability for dynamic athletic performance (Fig. 3). Exercises characterised by forces that were equal to or greater than the force in the sport or event, were supported by 60% of the cohort (mean 2.4, 95% CI 2.3–2.5, p < 0.05). Most were either un- decided or disagreed on the importance of velocity of movement (mean 2.6, 95% CI 2.5–2.8, p < 0.05) and sustained isometric contraction (mean 2.7, 95% CI 2.6–2.8, p < 0.05) in core stability exercises for athletic performance. Ground-based free barbell exercises and trunk muscle activation (Fig. 4) Most participants agreed that increases in external load in standing barbell exercises would increase trunk muscle activation (mean 2.0, 95% CI 1.9–2.1, p < 0.001) Data presented as number and percentage (n (%)) of all respondents. Italics represent the highest response for each question Clark et al. Sports Medicine - Open (2018) 4:32 Page 7 of 10 Page 7 of 10 term core strength relates to the overarching nature of the exercise, rather than the impact on or adaptation in the core stabilization system. link transferring torques between the upper and lower extremities in sporting actions’ [45]. Consequently, constituent anatomy of the core is described in the literature to reflect, i.e. region between and including pelvic and shoulder girdles and associated skeleton, muscles and nerves [1, 8]. Our survey results suggest this shift has permeated applied sports setting; half of the respondents agreed with this definition of the core while a quarter identified with the original de- scription, i.e. structures between diaphragm and pelvic floor including muscles and nerves. While core strength and core stability may well be viewed by some in our survey as separate entities, this has yet to be demonstrated scientifically [1]. The selec- tion of exercises used to develop core stability for healthy function can range from low load, minimal range of movement, abdominal bracing exercises to dynamic, loaded resistance exercises [6]. Research has not been able to identify and describe adaptations that occur in muscles responsible for stabilising the core as a conse- quence of different exercise modes [1, 12]. It is recog- nised though that effective core stability is the control of movement, including high force and high velocity movement, generated by interaction between axial and appendicular skeletons [5, 7, 8]. Most survey responses disagreed with the statement that core strength was re- quired for athletic performance, but not everyday life. This demonstrated alignment with the principle that core stability underpins both healthy function and dy- namic athletic performance. In effect core strength and core stability are synonyms and are used accordingly in the literature [1, 5, 23]. This is reflected in the survey question seeking to determine whether core stability and strength are separate attributes. Responses were mixed with just over half (57%) in agreement and the rest ei- ther undecided (16%) or in disagreement (27%). Ground-based free barbell exercises and trunk muscle activation (Fig. 4) Surveys have been used effectively to assess nutri- tion knowledge [46] and understanding of scientific training principles [47] in the workplace. Response rate to our survey (n = 241) was good in comparison to similar surveys which gathered information from both athletes (Wade et al., n = 57) [48] and people working in sport (Taylor et al., n = 28) [49], (Durell et al., n = 137) [47] and (Torres-McGehee et al., n = 579) [46]. Furthermore, the representative quality of our cohort is reflected by the spread of respondents, with 33% in full-time professional positions, either working with professional athletes (21%) or full-time Institute of sport athletes (12%). A quarter (27%) were involved in sport in an academic setting, either school or uni- versity and a quarter (27%) were in non-professional roles, either volunteering (15%) or part-time (12%). The majority were qualified to degree level (87%) and half had postgraduate degrees (52%). Most had an industry-specific qualification and on average were well experienced (mean 8 years) in their discipline. The cohort is therefore representative of people work- ing and participating in sport. Furthermore, they were reasonably well informed, indicating survey results that represent unbiased perceptions of the wider population. In our survey questions that assessed support for ex- ercise categories most effective in developing core sta- bility for dynamic athletic performance, there was clearly more support for functional, loaded exercises (Fig. 2). Squats and Olympic lifts and farmers walk that engage the full kinetic chain. Conversely support was moderate to low for traditional, non-functional core stability exercises, namely suspended compound exercises, isometric plank, hanging leg raise, and in- stability abdominal exercises. Two exercise categories, namely abdominal bracing and sit-ups, were regarded as ineffective rather than effective, The survey results therefore reflect the many reviews that highlighted a lack of evidence to support traditional CST for healthy individuals and recommended loaded, dynamic exercises that engage the full kinetic chain [1, 6, 7, 12–14, 45]. Our survey investigated perceptions around core stability and core strength (Fig. 1). The majority be- lieved that core strength is required for stability and far fewer agreed that these were separate attributes. In a comprehensive review Hibbs et al. [1] concluded that these two terms had yet to be clearly defined, in fact they failed to identify any characteristics that differen- tiated exercises for core strength and core stability. Conclusion The survey has provided evidence that a revised, more functional definition of core function and constituent anat- omy described in the literature is starting to be used in the practical setting. Almost half (45%) of the respondents pre- ferred trunk as the name for this anatomical region over core (35%). The absence of a valid objective method of measuring core function (22%) means that the most effect- ive way is through observation (43%) of exercise and ath- letic movement. A quarter (26%) proposed subjective assessment of movement in upright loaded resistance exer- cises as the most effective method of measuring core func- tion. This coincides with the strong shift in perceptions towards more functional approach to core stability training for dynamic athletic performance. Loaded exercises in an upright position, such as barbell squat and farmers walk, were viewed as effective training methods as proposed in the literature [7, 8, 14]. Core stability as an integrated, func- tional motor task [7], with training reflecting this according to movement patterns [14], forces [7], torque and velocity [8], appear to be guiding practice in the workplace accord- ing to the survey. These findings along with strong support for developing core stability through normal progressive exercise training, means we found in favour of our hy- pothesis. Some support remained for traditional CST through specific exercises (85%) and the isolation ap- proach (50%). Our findings lead to the following recom- mendations: Research to continue into efficacy of activating trunk stabilisers through selected sport specific and supplementary training modalities, including com- pound, loaded strength exercises. Continue to investigate The survey included a series of questions (yes/no/do not know) investigating perceptions on the application of CST for dynamic athletic performance (Table 3). Most (85%) of the cohort felt it necessary to include specific exercises to train core stability in healthy, uninjured ath- letes. With reference to traditional CST, two questions were asked; whether it was possible to isolate and train the core stabilization system, and whether this approach was effective. Half of the group believed that this was possible, 34% felt not and the rest were undecided (16%). The isolated training approach was regarded as not effective by 45%, and 37% were supportive. Prieske’s review highlighted growing evidence that specific, trad- itional CST is ineffective in healthy individual and ath- letes [12]. Ground-based free barbell exercises and trunk muscle activation (Fig. 4) These researchers reviewed studies that investigated core stability in response to loaded resistance exercises and traditional core stability exercises. A later system- atic review proposed a five-level core exercise classifi- cation system that progressed from traditional core exercises to noncore free weight exercises [6]. Inter- estingly the fourth classification level was free weight exercises defined as ‘dynamic, externally loaded, intent to activate lower body and core muscles’. Both these reviews suggest that the concept of strength in the Correct movement pattern was identified as most im- portant exercise selection criteria for development of core stability for dynamic athletic performance (Fig. 3). Exercises characterised by forces that were equal to or greater than force in the sport or event, were supported by 60% of the cohort. Most were either undecided or disagreed on whether velocity of movement and sus- tained isometric contraction were important in core sta- bility exercises for athletic performance. Kibler et al. (2006) accurately describes the exercise criteria for Clark et al. Sports Medicine - Open (2018) 4:32 Page 8 of 10 Page 8 of 10 Most survey respondents (65%) concurred with this by agreeing that core stability is developed through normal, progressive exercise training. The perception in applied practice conflicts with scientific literature with regards effectiveness of traditional core stability exercises for athletic performance. The majority (85%) of survey re- spondents believed that specific exercises were required to train core stability and half supported the use of exer- cises that isolated trunk stabilisers. effective CST: ‘integrated activation of multiple seg- ments’ providing ‘force generation’ that produces ‘inter- active movement’ characterised by ‘proximal stability and distal mobility’ [5]. Core stability development is therefore integral to all dynamic exercise training and sports specific movement, while quality of training effect is determined by specificity of movement, forces and velocity. y There is growing evidence in the literature that external load in free barbell exercises performed in a standing pos- ition is related to muscle activation of trunk stabilisers [29, 30, 33, 34, 37, 50]. Impact of this stimulus on core sta- bility in dynamic athletic performance is more difficult to demonstrate. In a recent systematic review, Prieske et al. (2016) reported a large effect for CST on trunk muscle strength measured by timed isometric plank, compared to no or only regular sports training [12]. Ground-based free barbell exercises and trunk muscle activation (Fig. 4) When compared to alternative training, such as whole-body strength train- ing, CST had a small sized effect on trunk muscle strength. CST had a small sized effect on muscle strength (e.g. Squat 1RM), a medium sized effect on muscle power (e.g. countermovement jump) and a small sized effect on athletic performance (e.g. 5000 m run time). They con- cluded that CST for healthy individuals, in the absence of any other fitness training, would increase trunk muscle strength. However, when combined with other training, such as whole-body strength training, CST is not effective. They also propose that increases in trunk muscle strength from CST, has limited effect on physical fitness and athlete performance in trained individuals. Findings from the sur- vey indicate that this information has begun to inform ap- plied practice (Fig. 4). Most agreed that increases in external load in standing barbell exercises would increase trunk muscle activation. Equally important in this form of resistance training was correct postural control. A limitation of the survey was the method of recruit- ing participants through email and direct messaging on an online professional community platform (LinkedIn). Emails and notifications may have been filtered to spam or junk folders and not reached intended participants. Participants were directed to an online survey, which may have served as a deterrent. Despite this, the number and quality of participants was good in comparison to similar surveys. A further limitation may well have been the inconsistency of prevailing terminology around the topic of CST and broader area of exercise and fitness. Steps were taken to adhere to the most commonly used terms from the scientific literature in the survey. Conclusion They also that reported that regular sports training and commonly used supplementary training, such as whole-body strength training, presents superior stimuli, that adhere to the overload training principle [28], for development of core stability in this population. Page 9 of 10 Clark et al. Sports Medicine - Open (2018) 4:32 Page 9 of 10 the transfer of training induced trunk muscle activation to functional performance, specifically functional stability. 3. Silfies SP, Ebaugh D, Pontillo M, Butowicz CM. Critical review of the impact of core stability on upper extremity athletic injury and performance. Braz J Phys Ther. 2015;19:360–8. 4. Key J. “The core”: understanding it, and retraining its dysfunction. J Bodyw Mov Ther. 2013;17(4):541–59. 5. Kibler WB, Press J, Sciascia A. The role of core stability in athletic function. Sports Med. 2006;36(3):189–98. 6. Martuscello JM, Nuzzo JL, Ashley CD, Campbell BI, Orriola JJ, Mayer JM. Systematic review of core muscle activity during physical fitness exercises. J Strength Cond Res. 2013;27:1684–98. 7. Wirth K, Hartmann H, Mickel C, Szilvas E, Keiner M, Sander A. Core stability in athletes: a critical analysis of current guidelines. Sport Med. 2016:1–14. 8. Behm DG, Drinkwater EJ, Willardson JM, Cowley PM. The use of instability to train the core musculature. Appl Physiol Nutr Metab. 2010;35:91–108. 9. Briggs MS, Givens DL, Best TM, Chaudhari AM. Lumbopelvic neuromuscular training and injury rehabilitation: a systematic review. Clin J Sport Med. 2013;23:160–71. 10 Lederman E The myth of core stability J Bodyw Mov Ther 2010;14:84 98 3. Silfies SP, Ebaugh D, Pontillo M, Butowicz CM. Critical review of the impact of core stability on upper extremity athletic injury and performance. Braz J Phys Ther. 2015;19:360–8. 3. Silfies SP, Ebaugh D, Pontillo M, Butowicz CM. Critical review of the impact of core stability on upper extremity athletic injury and performance. Braz J Phys Ther. 2015;19:360–8. 3. Silfies SP, Ebaugh D, Pontillo M, Butowicz CM. Critical review of the impact of core stability on upper extremity athletic injury and performance. Braz J Phys Ther. 2015;19:360–8. 4. Key J. “The core”: understanding it, and retraining its dysfunction. J Bodyw Mov Ther. 2013;17(4):541–59. 4. Key J. “The core”: understanding it, and retraining its dysfunction. J Bodyw Mov Ther. 2013;17(4):541–59. Competing interests 21. Edwards S, Austin A, Bird SP. The role of the trunk control in athletic performance of a reactive change-of-direction task. J Strength Cond Res. 2016;31(1):126–39. Competing interests David Clark, Mike Lambert and Angus Hunter declare that they have no competing interests. David Clark, Mike Lambert and Angus Hunter declare that they have no competing interests. 22. Tong TK, Wu S, Nie J. Sport-specific endurance plank test for evaluation of global core muscle function. Phys Ther Sport. 2014;15(1):58–63. 22. Tong TK, Wu S, Nie J. Sport-specific endurance plank test for evaluation of global core muscle function. Phys Ther Sport. 2014;15(1):58–63. Funding 12. Prieske O, Muehlbauer T, Granacher U. The role of trunk muscle strength for physical fitness and athletic performance in trained individuals: a systematic review and meta-analysis. Sport Med. 2016;46(3):401–19. No funding was received for any stage of this research from design, data collection, analysis, interpretation and preparation for publication. 13. Reed CA, Ford KR, Myer GD, Hewett TE. The effects of isolated and integrated “core stability” training on athletic performance measures: a systematic review. Sports Med. 2012;42:697–706. Availability of data and materials Appendix will include the survey questionnaire, and survey master results document will be made available and labelled Additional file 1. 14. Willardson JM. Core stability training: applications to sports conditioning programs. J Strength Cond Res. 2007;21:979–85. Ethics approval and consent to participate 18. Stanton R, Reaburn PR, Humphries B. The effect of short-term Swiss ball training on core stability and running economy. J Strength Cond Res. 2004; 18:522–8. Approval for the study was granted by the local research ethics committee in accordance with the Helsinki Declaration (2013) [40]. Approval for the study was granted by the local research ethics committee in accordance with the Helsinki Declaration (2013) [40]. 19. Shinkle J, Nesser TW, Demchak TJ, McMannus DM. Effect of Core strength on the measure of power in the extremities. J Strength Cond Res. 2012;26:373–80. Abbreviations g 7. Wirth K, Hartmann H, Mickel C, Szilvas E, Keiner M, Sander A. Core stability in athletes: a critical analysis of current guidelines. Sport Med. 2016:1–14. g 7. Wirth K, Hartmann H, Mickel C, Szilvas E, Keiner M, Sander A. Core stability in athletes: a critical analysis of current guidelines. Sport Med. 2016:1–14. 1RM: 1 Repetition maximum; CST: Core stability training; MSc: Master of Science; S&CC: Strength and conditioning coaches; A&P: Athletes and players; SM&P: Sport medicine practitioners and physiotherapists; SP&B: Sport physiologists and Biomechanists; SC: Sport coaches 8. Behm DG, Drinkwater EJ, Willardson JM, Cowley PM. The use of instability to train the core musculature. Appl Physiol Nutr Metab. 2010;35:91–108. 9. Briggs MS, Givens DL, Best TM, Chaudhari AM. Lumbopelvic neuromuscular training and injury rehabilitation: a systematic review. Clin J Sport Med. 2013;23:160–71. Author details 1 25. Okada T, Huxel KC, Nesser TW. Relationship between Core stability, functional movement, and Performance. J Strength Cond Res. 2010;25(1):252–61. 25. Okada T, Huxel KC, Nesser TW. Relationship between Core stability, functional movement, and Performance. J Strength Cond Res. 2010;25(1):252–61. 1School of Sport and Exercise Sciences, Faculty of Science, Liverpool John Moore’s University, 102, 2 Moorfields, Liverpool L2 2BS, UK. 2Division of Exercise Science and Sports Medicine, Department of Human Biology, University of Cape Town, Cape Town, South Africa. 3Physiology, Exercise and Nutrition Research Group, Faculty of Health Sciences and Sport, University of Stirling, Stirling, UK. 1School of Sport and Exercise Sciences, Faculty of Science, Liverpool John Moore’s University, 102, 2 Moorfields, Liverpool L2 2BS, UK. 2Division of Exercise Science and Sports Medicine, Department of Human Biology, 3 26. Akuthota V, Ferreiro A, Moore T, Fredericson M. Core stability exercise principles. Curr Sports Med Rep. 2008;7(1). https://doi.org/10.1519/JSC. 0000000000002144. 26. Akuthota V, Ferreiro A, Moore T, Fredericson M. Core stability exercise principles. Curr Sports Med Rep. 2008;7(1). https://doi.org/10.1519/JSC. 0000000000002144. 27. Hartmann H, Wirth K, Klusemann M. Analysis of the load on the knee joint and vertebral column with changes in squatting depth and weight load. Sports Med. 2013;43:993–1008. 27. Hartmann H, Wirth K, Klusemann M. Analysis of the load on the knee joint and vertebral column with changes in squatting depth and weight load. Sports Med. 2013;43:993–1008. Received: 26 March 2018 Accepted: 5 July 2018 Received: 26 March 2018 Accepted: 5 July 2018 Received: 26 March 2018 Accepted: 5 July 2018 28. Hellebrandt FA. Application of the overload principle to muscle training in man. Am J Phys Med. 1958;37(5):278–83. 28. Hellebrandt FA. Application of the overload principle to muscle training in man. Am J Phys Med. 1958;37(5):278–83. 29. Clark D, Lambert MI, Hunter AM. Reliability of trunk muscle electromyography in the loaded back squat exercise. Int J Sports Med. 2016;37(6):448–56. 29. Clark D, Lambert MI, Hunter AM. Reliability of trunk muscle electromyography in the loaded back squat exercise. Int J Sports Med. 2016;37(6):448–56. Acknowledgements The authors want to thank John Taylor for his assistance in developing the questionnaire and his suggestions for the circulation of the survey. 10. Lederman E. The myth of core stability. J Bodyw Mov Ther. 2010;14:84–98. 11. Smith BE, Littlewood C, May S. An update of stabilisation exercises for low back pain: a systematic review with meta-analysis. BMC Musculoskelet Disord. 2014;15:416. Publisher’s Note 23. Faries MD, Greenwood M. Core training: stabilizing the confusion. Strength Cond J. 2007;29(2):10. 23. Faries MD, Greenwood M. Core training: stabilizing the confusion. Strength Cond J. 2007;29(2):10. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 24. McGill S. Core training: evidence translating to better performance and injury prevention. Strength Cond J. 2010;32(3):33–46. 24. McGill S. Core training: evidence translating to better performance and injury prevention. Strength Cond J. 2010;32(3):33–46. Additional file 1: Core Stability Survey. (PDF 103 kb) 6. Martuscello JM, Nuzzo JL, Ashley CD, Campbell BI, Orriola JJ, Mayer JM. Systematic review of core muscle activity during physical fitness exercises. J Strength Cond Res. 2013;27:1684–98. Consent for publication Survey instructions informed participants of the details of the research study, completion and submission implied consent. 20. Gottschall JS, Mills J, Hastings B. Integration core exercises elicit greater muscle activation than isolation exercises. J Strength Cond Res. 2012; 27(3):590–6. Authors’ contributions d d d 15. Hodges PW, Richardson C. Inefficient muscle stabilization of the lumbar spine associated with low back pain: a motor control evaluation of transversus abdominis. Spine (Phila Pa 1976). 1996;21:2640–50. DC conceived and designed survey with assistance from JT (Acknowledgements) and ML. DC managed the data collection, survey circulation, data collation, analysis and interpretation. Final analysis and interpretation for publication was done by DC with assistance from ML and AH. All authors read and approved the final manuscript. 16. Allison GT, Morris SL. Transversus abdominis and core stability: has the pendulum swung? Br J Sports Med. 2008;42:930–1. 17. Akuthota V, Nadler SF. Core strengthening - focused review. Arch Phys Med Rehabil. 2004;85:86–92. Additional file 5. Kibler WB, Press J, Sciascia A. The role of core stability in athletic function. Sports Med. 2006;36(3):189–98. 5. Kibler WB, Press J, Sciascia A. The role of core stability in athletic function. Sports Med. 2006;36(3):189–98. Additional file 1: Core Stability Survey. (PDF 103 kb) References 30. Clark DR, Lambert MI, Hunter AM. Trunk muscle activation in the back and hack squat at the same relative loads. J Strength Cond Res. 2017;1. https:// doi.org/10.1519/JSC.0000000000002144. 1. Hibbs AE, Thompson KG, French D, Wrigley A, Spears I. Optimizing performance by improving core stability and core strength. Sport Med. 2008;38:995–1008. 31. Bressel E, Willardson JM, Thompson B, Fontana FE. Effect of instruction, surface stability, and load intensity on trunk muscle activity. J Electromyogr Kinesiol. 2009;19:e500–4. 2. Spencer S, Wolf A, Rushton A. Spinal-exercise prescription in sport: classifying physical training and rehabilitation by intention and outcome. J Athl Train. 2016;51(8):613–28. 2. Spencer S, Wolf A, Rushton A. Spinal-exercise prescription in sport: classifying physical training and rehabilitation by intention and outcome. J Athl Train. 2016;51(8):613–28. 31. Bressel E, Willardson JM, Thompson B, Fontana FE. Effect of instruction, surface stability, and load intensity on trunk muscle activity. J Electromyogr Kinesiol. 2009;19:e500–4. Page 10 of 10 Clark et al. Sports Medicine - Open (2018) 4:32 32. Willardson JM, Fontana FE, Bressel E. Effect of surface stability on core muscle activity for dynamic resistance exercises. Int J Sports Physiol Perform. 2009;4:97–109. 33. Nuzzo JL, McCaulley GO, Cormie P, Cavill MJ, McBride JM. Trunk muscle activity during stability ball and free weight exercises. J Strength Cond Res. 2008;22:95–102. 34. Hamlyn N, Behm DG, Young WB. Trunk muscle activation during dynamic weight-training exercises and isometric instability activities. J Strength Cond Res. 2007;21:1108–12. 35. Comfort P, Pearson SJ, Mather D. An electromyographical comparison of trunk muscle activity during isometric trunk and dynamic strengthening exercises. J Strength Cond Res. 2011;25(1):149–54. 36. Fletcher IM, Bagley A. Changing the stability conditions in a back squat: the effect on maximum load lifted and erector spinae muscle activity. Sport Biomech. 2014;13(4):380–90. 37. Aspe RR, Swinton PA. Electromyographic and kinetic comparison of the back squat and overhead squat. J Strength Cond Res. 2014;28:2828–36. 38. Bergmark A. Stability of the lumbar spine. A study in mechanical engineering. Acta Orthop Scand Suppl. 1989;230(230):1–54. 39. Panjabi MM. The stabilizing system of the spine. Part I. Function, dysfunction, adaptation, and enhancement. J Spinal Disord. 1992;5:383–9. 40. World Medical Association. World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. JAMA. 2013;310(20):2191–4. 41. Hibbs AE, Thompson KG, French DN, Hodgson D, Spears IR. Peak and average rectified EMG measures: which method of data reduction should be used for assessing core training exercises? Clark et al. Sports Medicine - Open (2018) 4:32 References J Electromyogr Kinesiol. 2011;21:102–11. 42. Prieske O, Muehlbauer T, Borde R, Gube M, Bruhn S, Behm DG, et al. Neuromuscular and athletic performance following core strength training in elite youth soccer: role of instability. Scand J Med Sci Sport. 2016;26(1):48–56. 43. Willardson J. Core stability training for healthy athletes: a different paradigm for fitness professionals. Strength Cond J. 2007;29(6):42–9. 44. Hodges P, Cresswell A, Thorstensson A. Preparatory trunk motion accompanies rapid upper limb movement. Exp Brain Res. 1999;124(1):69–79. 45. Fletcher BI. Myths and reality : training the torso. Prof Strength Cond. 2014; 33:25–30. 46. Torres-McGehee TM, Pritchett KL, Zippel D, Minton DM, Cellamare A, Sibilia M. Sports nutrition knowledge among collegiate athletes, coaches, athletic trainers, and strength and conditioning specialists. J Athl Train. 2012;47(2):205–11. 47. Durell D, Pujol T, Barnes J. A survey of the scientific data and training methods utilized by collegiate strength and conditioning coaches. J Strength Cond Res. 2003;17(2):368–73. 48. Wade S, Pope Z, Simonson S. How prepared are college freshmen athletes for the rigors of college strength and conditioning? A survey of college strength and conditioning coaches. J Strength Cond Res. 2014;28(10):2746–53. 49. Taylor K-L, Chapman DW, Cronin JB, Newton MJ, Gill N. Fatigue monitoring in high performance sport: a survey of current trends. J Aust Strength Conditoning. 2012;20(1):12–23. 50. McBride JM, Larkin TR, Dayne AM, Haines TL, Kirby TJ. Effect of absolute and relative loading on muscle activity during stable and unstable squatting. Int J Sports Physiol Perform. 2010;5:177–83.
https://openalex.org/W4234514275	https://www.oatext.com/pdf/CCRR-1-116.pdf	English	null	Preserve patient's confidentiality in primary care	Clinical case reports and reviews.	2,015	cc-by	1,883	Case discussion Solving of any medical ethical dilemma should be verified through eight values, but unconditional perfect solutions are not present always. However, it can give an idea of how to grip a particular ethical dilemma, at the same time; it will offer a useful outline for understanding conflicts. The ethical values are (Beneficence ; Non-maleficence; Autonomy; Justice; Dignity; Truthfulness, honesty, and informed consent) [4,5]. The General Medical Council (GMC) has stated that: Confidentiality is central to trust between doctors and patients. Without assurances about confidentiality, patients may be reluctant to seek medical attention or to give doctors the information to provide the good care [8]. The confidentiality element is one of the important foundation stones for supporting “trusted therapeutic relationship” between patients and their doctors. It can be requested by the concerned patients, additionally there is inherent physician’s obligation that patient’s information is permanently kept confidentially. However, that confidentiality is not outright in all situations. In Bahrain, breach of confidentiality rule was issued in Decree law no. 7, article 126 (1989). Which give permission for giving patient’s information at “compulsory legally” required or when there is a significant risk of serious harm to the others if confidentiality is maintained [6]. Maintaining confidentiality is part of the “good faith” that exists between doctor and patient [11]. Ignoring patients’ confidentiality would lose their trust, and might prevent them from seeking help when needed. Confidentiality will preserve personal dignity, prevents information misuse, and protects patient’s autonomous decision, [12] they may not divulge significant information that would support their diagnosis and management [9]. The consequences of breach patient’s confidentiality definitely will harm both the individual patient and overall trust in the medical profession [12]. Therefore, physicians should obey the ethical principles by preventing harm and try for benefiting patients. Confidentiality in the medical setting refers to “the principle of keeping information in state of secured and secret from others” [7]. It is patient’s right, and should be maintained even after patient’s death [8]. The physician’s breach confidentiality may be acceptable, only when pregnancy scenarios was happened in under age group (≤ 16 years) [13] or there was history of sexual abuse/ sexual rape [14]. Breaches of confidentiality in routine practice at primary care services don’t follow into the standard scenario of disclosure for sensitive clinical information; many may be unintentional or related to lack of knowledge of the relevant legal and professional ethical requirements. Introduction The morality/ethics are an essential need for any profession, particularly medicinal profession. Many of the “Western and Eastern medical ethical schemes” were driven from Hippocratic Oath [1]. “Thomas Percival”(1794) was setting up the “English Constitution for the Doctor” in medical ethics; he provided physicians with fresh standard of conduct [2]. The brand new version of “Constitution of the American Medical Association” was developed in 1980 with influential seven items, while the upgraded version was developed in 2001 using nine significant items [3]. Case study 25 years old, foreigner housemaid female was presented to primary care clinic complaining from delayed in her menses for the last two month, worried that she might be pregnant. A pregnancy test was positive and examination confirmed a pregnancy of 10 weeks. The physician reported the case to the police for illegal pregnancy from her boyfriend. Abstract The law defines the confidentiality as a balance to public interests than a “right” afforded to the person, which contrary conflict with the medical definition. Breaches of patient confidentiality are happened in certain situations (e.g. disclosure to protect others, police cases, notifiable diseases, and patient’s fitness to drive). Protection Society” were from primary care physicians who mostly related to the confidential dilemma [10]. Preserve patient’s confidentiality in primary care i Basem Abbas Al Ubaidi* Consultant Family Physician, North Muharaq Health Centre, Kingdom of Bahrain Basem Abbas Al Ubaidi* Consultant Family Physician, North Muharaq Health Centre, Kingdom of Bahrain Clinical Case Reports and Reviews Case Study ISSN: 2059-0393 ISSN: 2059-0393 Clin Case Rep Rev, 2015 doi: 10.15761/CCRR.1000116 Correspondence to: Basem Abbas Al Ubaidi, ABFM, MHPEd, Consultant Family Physician, North Muharaq Health Centre, Kingdom of Bahrain, E-mail: bahmed1@health.gov.bh Correspondence to: Basem Abbas Al Ubaidi, ABFM, MHPEd, Consultant Family Physician, North Muharaq Health Centre, Kingdom of Bahrain, E-mail: bahmed1@health.gov.bh Received: March 10, 2015; Accepted: April 01, 2015; Published: April 05, 2015 Received: March 10, 2015; Accepted: April 01, 2015; Published: April 05, 2015 Received: March 10, 2015; Accepted: April 01, 2015; Published: April 05, 2015 References • If there is risk of death from serious crime. • If there is risk of death from serious crime. 1. http://www.pbs.org/wgbh/nova/body/hippocratic-oath-today.html. 1. http://www.pbs.org/wgbh/nova/body/hippocratic-oath-today.html. • If the patient is victim of neglect or abuse (physical, sexual or emotional abuse). 2. http://wikipedia.atpedia.com/en/articles/m/e/d/Medical_ethics.html. 2. http://wikipedia.atpedia.com/en/articles/m/e/d/Medical_ethics.html. 3. Percival, Thomas. Medical ethics, 49-57 esp section 8 pg.52. 3. Percival, Thomas. Medical ethics, 49-57 esp section 8 pg.52. 3. Percival, Thomas. Medical ethics, 49-57 esp section 8 pg.52. • To facilitate medical research. 4. Shanawani H, Lowe KN (2005) Is Greenacres (SNF) the place to be? Virtual Mentor [serial on the Internet]. 2005 [cited 2005 July 24]; 7 (7). 4. Shanawani H, Lowe KN (2005) Is Greenacres (SNF) the place to be? Virtual Mentor [serial on the Internet]. 2005 [cited 2005 July 24]; 7 (7). • For education/secondary uses of information that benefit society over time. 5. Veatch RM (2000) The basics of bioethics. 2nded. Upper Saddle River (NJ): Prentice Hall. 5. Veatch RM (2000) The basics of bioethics. 2nded. Upper Saddle River (NJ): Prentice Hall. • If there is lack of patient mental capacity to give consent (permanent or temporary). 6. http://www.bahrainlaw.net/post1295.html. 6. http://www.bahrainlaw.net/post1295.html. 7. BMA (1999) Confidentiality and disclosure of health information. Physician duty of confidentiality should continue even after the patient has died, except in certain circumstances (to write death certificates, when a parent asks for information about the circumstances and causes of a child’s death) [8,10,16]. 8. General Medical Council (2009) Confidentiality: protecting and providing information 2009. 9. Slowther A (2010) Confidentiality in primary care: ethical and legal considerations. InnovAiT 3: 753–759. 10. General Medical Council. Confidentiality (2009a) London: GMC. Accessed via www. gmc-uk.org/guidance/ethical_guidance/confidentiality.asp. Physicians have a duty to tell the police immediately, when the patient is brought with history or suspicion injuries of gunshot or knife wounds, suicidal attempt, prepared to use weapons, domestic violence and suspicion of the crime/assault/road traffic accidents [3,8,10,15]. 11. O’Neill O (2002) Licence to decieve. Reith lectures. 12. Souhami R, Chalmers S, Collins R, Luker K, Newton J, et al. (2006) Personal data for the public good: using health information in medical research. London: The Academy of Medical Sciences. In Bahrain, we need more legislative laws on how should physicians maintain and when can they reveal patient’s confidentiality? When should physician have legal obligation to report the case to the police 13. (1984) Gillickv West Norfolk and Wisbech Area Health Authority. Recommendation: • To formulate a new practical outline “Ethical Curriculum” to Family Practice Residency Programmed (FPRP). • To set tools on how does tutor in FPRP assist medical ethics teaching process? Check how much do students gain their ethical competencies? Breach of patient’s confidentiality should be considered in certain situations: • If there are legal requirements to disclose information (infectious cases). • To have primary care handbook presents an “ethics primer” for family physicians and general practitioners (GPs). • If it is asking by various regulatory bodies or by a judge or presiding officer to a court. • To have user-friendly, valid, and reliable guidelines with common primary care ethical dilemma discussion. • To have hotline calls from primary care physician to “Medical Ethical Committee” to discuss confidential dilemma. • For sharing patient’s information within the healthcare team. • If patient came in an emergency situation. • If patient came in an emergency situation. • To have more legislative laws on how should physicians maintain and when a physician should reveal the patient confidentiality? • If there is request from the patient’s insurers. • To verify patient’s medical certificate/report. Case discussion A clear understanding of the duty of physicians towards patients’ confidentiality and how it is applied in the regular primary care practice is an important competence for all general practitioners (GPs) [9]. The rape rule was arranged in Bahrain’s Decree-Law No. 15/1976 Thirds of the calls which had been received by the “Medical Clin Case Rep Rev, 2015 doi: 10.15761/CCRR.1000116 Volume 1(3): 42-44 Al Ubaidi BA (2015) Preserve patient’s confidentiality in primary care version of the Penal Code on the show article 344 – that any person has to be punished by imprisonment for a victim not reach the sixteenth under age, or without self-possessed consent of the victim at any adult age, that was unsuitable with our presented case [15]. department in police cases? When should report to General Directorate of Traffic Office? ; If the patient is diagnosed with a medical condition that could impair fitness to drive [8,10,16]. Key points: Any autonomous patient has the right to make verbal/written consent, when possible about who can share patient’s facts, even when information is shared in the benefit to the patient/sponsor care. Eventually, any clinician who shares patient’s information with others, without patient’s consent, does not respect the patient’s autonomy and will have behaved in a morally questionable way [10]. • Recognizing and maintaining a duty of confidentiality can bring particular challenges in the context of primary care setting. • Physicians need to be aware of their statutory obligations to disclose information and the limits of this obligation. • When there is a significant risk of serious harm to others, if information is not shared (the duty to protect or worn may override the duty of confidentiality). However, the duty of confidentiality is not obsolete; and there are many ethical justifications for breaching confidentiality; corresponding in our case study because of harm to the sponsor’s contract interest, nonetheless we should take in consideration patient’s interest (patient’s right). It is legitimate to constrain individual freedom, if that result in harm to others. Nevertheless, personal information should not be disclosed to a third party (e.g. solicitors or police officer) without the patients express their consent. • Medical ethics guidelines that equip primary care physician the necessary competence to solve ethical dilemma. • Medical ethics guidelines that equip primary care physician the necessary competence to solve ethical dilemma. Al Ubaidi BA (2015) Preserve patient’s confidentiality in primary care Clin Case Rep Rev, 2015 doi: 10.15761/CCRR.1000116 14. Ablashi DV, Zompetta C, Lease C, Josephs SF, Balachandra N, Komaroff AL, et al. (1991) Human herpesvirus 6 (HHV6) and chronic fatigue syndrome (CFS). Can Dis Wkly Rep17 (suppl 1E): 33-40. [Crossref] Al Ubaidi BA (2015) Preserve patient’s confidentiality in primary care 15. http://ministryofethics.co.uk/index.php?p=6&q=7. 16. http://www.legalaffairs.gov.bh/LegislationSearchDetails.aspx?id=4069. Copyright: ©2015 Al Ubaidi BA. 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 Case Rep Rev, 2015 doi: 10.15761/CCRR.1000116 References All Engl Law Rep1985: 533-59. [Crossref] Clin Case Rep Rev, 2015 doi: 10.15761/CCRR.1000116 Volume 1(3): 42-44 Copyright: ©2015 Al Ubaidi BA. 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 Case Rep Rev, 2015 doi: 10.15761/CCRR.1000116 Volume 1(3): 42-44
https://openalex.org/W2955763550	https://europepmc.org/articles/pmc6627195?pdf=render	English	null	Modelling the Effect of Compliance with Nordic Nutrition Recommendations on Cardiovascular Disease and Cancer Mortality in the Nordic Countries	Nutrients	2,019	cc-by	10,507	Received: 23 May 2019; Accepted: 21 June 2019; Published: 25 June 2019 Abstract: The objective of this study is to estimate the number of deaths attributable to cardiovascular diseases and diet-related cancers that could be prevented or delayed in the Nordic countries, i.e., Sweden, Denmark, Finland, Norway, and Iceland, if adults adhere to the Nordic Nutrition Recommendations (NNR). A sex- and age-group speciﬁc epidemiological macro-simulation model was used to estimate the preventable deaths due to the diﬀerences between country speciﬁc actual intake and recommended intake of changes in food components. Data included in the model are a baseline scenario (actual dietary intake), a counterfactual scenario (recommended intake), and age-and sex-speciﬁc mortality for cardiovascular and diet-related cancer diseases, together with the total population risk of a speciﬁc year. Monte Carlo analyses with 5000 iterations were performed to produce the 95% uncertainty intervals. The model predicts that Iceland would beneﬁt the most by adhering to the NNR, followed by Finland. In all the Nordic countries, the highest beneﬁt would be achieved by adhering to the fruits and vegetable intakes, except Denmark, where a lower recommended intake of salt would provide the highest beneﬁt. For men, fruits and vegetables could have saved more lives compared to other dietary components for all the Nordic countries, while for women, dietary ﬁber was the most prominent factor, except in Iceland. The Nordic Council should consider policies for promoting healthy eating according to the needs of each country. Keywords: Nordic diet; Nordic countries; dietary guidelines; macro simulation model; cardiovascular diseases; recommended intake; health Beneﬁt Sanjib Saha 1,* , Jonas Nordström 2,3, Irene Mattisson 4, Peter M. Nilsson 5,6 and Ulf-G Gerdtham 1,7 1 Health Economics Unit, Department of Clinical Science (Malmö), Lund University, SE-22381 Lund, Sweden ulf.gerdtham@med.lu.se 1 Health Economics Unit, Department of Clinical Science (Malmö), Lund University, SE-22381 Lund, Sweden; ulf.gerdtham@med.lu.se ulf.gerdtham@med.lu.se 2 School of Economics and Management, Agrifood Economics Centre, Lund University, SE-22007 Lund, Sweden; jonas.nordstrom@agrifood.lu.se 3 Department of Food and Resource Economics University of Copenhagen DK 1958 Frederiksberg C 2 School of Economics and Management, Agrifood Economics Centre, Lund University, SE-22007 Lund, Sweden; jonas.nordstrom@agrifood.lu.se 2 School of Economics and Management, Agrifood Economics Centre, Lund University, SE-22007 Lund, Sweden; jonas.nordstrom@agrifood.lu.se 2 School of Economics and Management, Agrifood Economics Centre, Lund University, SE-22007 Lund, Sweden; jonas.nordstrom@agrifood.lu.se 3 Department of Food and Resource Economics, University of Copenhagen, DK-1958 Frederiksberg C, Denmark 4 National Food Agency, SE-75126 Uppsala, Sweden; evairene@live.se 5 Department of Internal Medicine, Skane University Hospital, SE-20502 Malmo, Sweden; peter.nilsson@med.lu.se p 6 Department of Clinical Sciences (Malmo), Lund University, SE-20502 Malmo, Sweden 7 Department of Economics, Lund University, SE-22363 Lund, Sweden * Correspondence: sanjib.saha@med.lu.se; Tel.: +46-(0)-40-391424; Fax: +46-(0)-46-2224118 Received: 23 May 2019; Accepted: 21 June 2019; Published: 25 June 2019 Received: 23 May 2019; Accepted: 21 June 2019; Published: 25 June 2019 nutrients nutrients Modelling the Eﬀect of Compliance with Nordic Nutrition Recommendations on Cardiovascular Disease and Cancer Mortality in the Nordic Countries S jib S h 1 * J N d t ö 2 3 I M tti 4 P t M Nil 5 6 d Sanjib Saha 1,* , Jonas Nordström 2,3, Irene Mattisson 4, Peter M. Nilsson 5,6 and Ulf-G Gerdtham 1,7 1. Introduction For several decades, the Nordic countries have collaborated to establish dietary guidelines. The Nordic Nutrition Recommendations (NNRs) are based on research data from epidemiological studies and laboratory studies by a panel of experts. These recommendations are applicable to all the Nordic countries: Sweden, Denmark, Finland, Norway, and Iceland [1]. The decision to develop www.mdpi.com/journal/nutrients Nutrients 2019, 11, 1434; doi:10.3390/nu11061434 www.mdpi.com/journal/nutrients 2 of 17 Nutrients 2019, 11, 1434 joint NNRs by the Nordic countries emerged not only from the geographical location of the Nordic countries but also from the similarities shared in dietary habits and similarities in the prevalence of diet-related diseases, such as cardiovascular diseases (CVDs), obesity, and Type 2 Diabetes (T2D) [2]. The NNR include reference values for total energy intake and recommendations on macronutrients as a percentage of total energy intake, daily intakes of vitamins and minerals, as well as intake of ﬁber and salt, together with recommendations on physical activity [1]. The latest NNR was developed in 2012, when about 100 scientists from all ﬁve Nordic countries were involved in developing recommendations [1]. The primary aim of the NNR 2012 was to present the scientiﬁc background of the recommendations and their applications. A secondary aim was to function as a basis for national recommendations, i.e., food-based dietary guidelines that are adopted by the individual Nordic countries. Following these, the Nordic countries have developed their own dietary recommendations. However, studies exploring how well the Nordic population adheres to dietary recommendations are limited. For example, there is a large gap between the actual dietary practice and recommended intake in Sweden [3]. The SYSDIET study found that 65% of the study participants who were from Sweden, Finland, Denmark, and Iceland did not meet the recommendations for saturated fatty acids, polyunsaturated fatty acids (PUFA), dietary ﬁber, and sodium [4]. In Norway, less than 40% of the adolescents adhere to recommendations for frequencies of eating fruits, vegetables, added sugar, and ﬁsh [5]. Adherence to dietary recommendation is also low for the general Danish population [6]. The number of deaths from chronic diseases and/or the incidences of chronic diseases that could be prevented or avoided by changing the dietary intake of the Nordic population according to the recommendations of the NNR 2012 are still unidentiﬁed. Furthermore, the dietary components that could provide the highest beneﬁcial health eﬀects in the respective countries are unknown. 2.1. Recommended Intake The NNR 2012 [1] was used as a recommended intake for this study. Each Nordic country has modiﬁed the NNR 2012 to ﬁt the food culture and ability of the consumer. The Swedish National Food Agency (Livsmedelsverket) published the revised version of the National Food-based Dietary Guidelines in 2015 (Swedish: Hitta ditt sätt) [15]. The ministry of Food, Agriculture, and Fisheries of Denmark published the oﬃcial dietary guidelines (Danish: De Oﬃcielle Kostråd) for the Danish population in September 2013 [16]. The Norwegian Directorate of Health published a revised version of the Guidelines in 2014, named “Norwegian guidelines on diet, nutrition, and physical activity, 2014” (Norwegian: Anbefalinger om kosthold, ernæring, og fysisk aktivitet, 2014) [17]. The National Nutrition Council led the development of Finish Recommendations together with various stakeholders, Finnish nutrition recommendations 2014 (Finnish: Terveyttä ruoasta. Suomalaiset ravitsemussuositukset 2014) [18]. The oﬃcial name of the dietary recommendations of Iceland is Dietary guidelines, for adults and children from two years of age (Icelandic: Ráðleggingar um mataræði fyrir fullorðna og börn frá tveggja ára aldri), which was published in 2014 [19]. The guidelines were developed by an expert group, including professionals from academia and the Directorate of Health. For the ease of comparison, we used the same recommendations for each country. The NNR provides the recommendation for energy yielding nutrients as a range. For example, 10%–20% of the energy should come from monounsaturated fatty acids (MUFA). For this study, we required exact targets for consumption, so we converted these ranges into the best values. Furthermore, the NNR guidelines combine fruits and vegetable recommendations into one (i.e., 500 g/day), but we have separated the intake by 250 g for each component in this study. 1. Introduction Since most of the top determinants of the burden of disease are diet-related such, as CVDs, T2D, obesity, and numerous cancers [7], it is thus important to identify and measure the health beneﬁts (losses) that can be obtained (avoided) if the Nordic populations adhere to the NNR 2012. Such knowledge could guide policymakers to prioritize interventions to allocate scarce resources strategically, since these diseases contribute to a substantial economic burden and create health inequalities [8,9] in these societies. Simulation models are suitable for integrating results from observational studies where diﬀerent experimental studies, such as randomized controlled trials (RCTs), are diﬃcult to conduct [10,11]. This is more suitable for diet-related intervention/policies, where it is impractical or unethical to estimate, for example, the eﬀect of the proposed taxes on saturated fat intake by RCTs followed over, say, 50 years. This would mean randomizing people, shops, or areas to face increases in food prices. A simulation model is a helpful tool that can combine all the available evidence to estimate a scenario where a head-to-head comparison is impossible to perform. Simulation models use a collection of mathematical equations to quantify the relationships between proposed or hypothetical interventions and speciﬁc outcomes of interests [12]. Moreover, simulation models pose many advantages over RCTs, for example, linking intermediate clinical endpoint to ﬁnal outcomes, e.g., linking changes in blood pressure to hypertension-associated diseases. This enables policymakers to make decisions in the absence of reliable data using realistic assumptions [13]. The aim of this study is ﬁrstly to quantify health beneﬁts by means of deaths related to CVDs and cancer that could be prevented or delayed if the Nordic population could follow the Nordic nutrient recommendations using a simulation model. Secondly, the aim is to perform an inter-country comparison to identify which dietary components would provide the highest health beneﬁts for each country population. Thirdly, this study aims to observe heterogeneity in the quantiﬁed beneﬁts based on age and gender. Nutrients 2019, 11, 1434 3 of 17 2. Materials and Methods We compared the recommended nutrient intake with the actual dietary intake of the country-speciﬁc Nordic population. A validated and transparent macrosimulation model, the PRIME (Preventable Risk Integrated ModEl) [14], was used to estimate the cardiovascular and diet-related cancer death toll that could be prevented or delayed for the populations of Nordic countries in a year. 2.2. Actual Intake The diet in the Nordic countries is characterized by a higher consumption of animal, processed, and sweetened foods, including non-alcoholic beverages and soft drinks [20]. The food consumption patterns in the Nordic countries are also high in dairy and bread [21]. Typically dominating grains are ray, barley wheat, and oats, which are rich in dietary ﬁbers [22]. Saying that, inter-country diﬀerences do exist in terms of dietary intake. The actual average dietary intake of Nordic populations was obtained from the most recent dietary surveys conducted in each country. In Table 1, we present the details based on the dietary surveys of Sweden [23], Denmark [24], Finland [25], Norway [26], and Iceland [27]. These surveys were used to estimate population intake of energy, fruits and vegetables, ﬁber, salt and fats, which includes total fats, saturated fats, PUFA, MUFA, and cholesterol, stratiﬁed by age and sex. It is not surprising to note that the national dietary surveys vary with respect to sample size, age group, participation rate, as well as the methodology to collect the dietary intakes. Except for Finland, all countries had invited sample representatives to the country. However, the participation rate varied from 36% to 68.8%, where Sweden had the lowest and Iceland had the highest rate of participation. The methods to collect the dietary information also varied. Only Norway and Iceland used a similar method (2 × 24 h recall together with a Food Frequency Questionnaire). The food intake was converted into specific nutrient intakes using country-specific nutrient databases, which are also presented in Table 1. The actual mean intakes of nutrients is presented in Table 2, together with the recommended intake from the NNR 2012 that was used in this study. However, details of the data used as model inputs are presented in the Supplementary Materials (Annex 1). 4 of 17 4 of 17 Nutrients 2019, 11, 1434 Table 1. National Dietary Survey across the Nordic Countries. Table 1. National Dietary Survey across the Nordic Countries. Abbreviations: FFQ, Food Frequency Questionnaire; h, hour 2.2. Actual Intake Country Survey Name Survey Year Country Represent Sample Age Invited Sample Sample Size Participation Rate Dietary Methodology Nutrient Reference Database Sweden [23] Riksmaten 2010–2011 Swedish Adults Dietary Survey 2010–2011 Yes 18–80 5000 1797 36% 4 day food diary (consecutive) The food database—Livsmedelsverket http://www7.slv.se/ SokNaringsinnehall Denmark [24] Danish National Survey of Diet and Physical Activity (DANSDA) 2011–2013 2011 –2013 Yes 4–75 7253 3946 54.4% 7 day diary (consecutive) Danish Food Composition Databank http://www.foodcomp.dk/v7/ fcdb_default.asp Finland [25] The National FINDIET 2012 survey (FINRISK) 2012 No 25–74 3268 1708 52% 48 h recall National Food Composition Database-Fineli https://ﬁneli.ﬁ/ﬁneli/en/index Norway [26] Norwegian national diet survey NORKOST3 2010–2011 Yes 18–70 5000 1787 37% 2 × 24 h recall and FFQ The Norwegian Food Composition Table http://www.matportalen.no/ Iceland [27] The Diet of Icelanders—a national dietary survey 2010–2011 2010–2011 Yes 18–80 2000 1312 68.6% 2 × 24 h recall and FFQ Icelandic Database of Food Ingredients (ÍSGEM); Public Health Institute for Raw Materials in the Icelandic Market http://www.matis.is/neytendur/ leit-i-isgem-gagnagrunni/ Abbreviations: FFQ, Food Frequency Questionnaire; h, hour. 5 of 17 Nutrients 2019, 11, 1434 Table 2. Mean dietary component intake versus recommended intake (RI) for men and women in Nordic Countries. 2.2. Actual Intake Food/Nutrient RI * Sweden Denmark Norway Finland Iceland Men (n = 792) Women (n = 1005) Men (n = 1494) Women (n = 1552) Men (n = 862) Women (n = 925) Men (n = 795) Women (n = 913) Men (n = 632) Women (n = 6 80) Fruits (g/day) 250 105.0 (3.97) 147.0 (3.53) 162.30 (3.74) 209.0 (3.58) 162.2 (5.06) 188.0 (4.63) 102.8 (5.05) 146.2 (5.54) 102.0 (4.73) 136.0 (4.69) Vegetables (g/day) 250 169.0 (3.69) 182.0 (3.09) 190.33 (3.09) 204.5 (2.85) 156.2 (3.62) 153.4 (3.41) 83.0 (3.45) 92.6 (2.74) 121.0 (4.25) 110.0 (3.57) Fiber (g/day) 25–35 (30) 21.30 (0.29) 18.80 (0.22) 28.83 (0.23) 20.83 (0.17) 26.6 (0.37) 22.2 (0.27) 22.0 (0.35) 20.6 (0.28) 17.8 (0.32) 15.83 (0.24) Salt (g/day) 6 8.84 (0.10) 6.78 (0.063) 10.96 (0.08) 8.04 (0.06) 9.05 (0.12) 6.25 (0.08) 8.76 (0.11) 6.38 (0.07) 9.46 (0.15) 6.48 (0.09) Total fat (%E) 25–40 (40) 34.0 (1.21) 34.40 (0.20) 36.33 (0.14) 35.83 (0.13) 34.0 (0.25) 34.2 (0.24) 35.84 (0.28) 35.14 (0.26) 36.53 (0.28) 35.43 (0.27) Saturated fat (%E) <10 (9) 13.0 (0.11) 13.10 (0.10) 14.5 (0.07) 13.83 (0.07) 13.0 (0.10) 13.4 (0.10) 13.8 (0.14) 13.52 (0.14) 14.57 (0.16) 14.13 (0.14) MUFA (%E) 10–20 (20) 12.80 (0.09) 12.90 (0.09) 13.67 (0.06) 13.17 (0.06) 11.8 (0.10) 11.6 (0.10) 12.92 (0.13) 12.4 (0.12) 11.70 (0.09) 11.3 (0.10) PUFA (%E) 5–10 (10) 5.5 (0.07) 5.7 (0.06) 5.52 (0.03) 5.65 (0.03) 6.22 (0.07) 6.16 (0.08) 6.20 (0.08) 6.26 (0.08) 5.87 (0.1) 5.9 (0.1) Cholesterol (mg/day) 300 320 (5.15) 263 (3.9) NA NA 400.4 (0.76) 297 (5.58) 288.6 (6.16) 205.8 (3.84) 392 (8.10) 262 (4.83) Abbreviations: RI, Recommended Intake; %E, percentage of total energy; MUFA, Monounsaturated fatty acids; NA, Not Available; PUFA, Polyunsaturated fatty acids. Note: Standard error of mean are in the parentheses. Recommended intakes are based on Nordic Nutrition Recommendation 2012. Recommended intake for fruits and vegetables together is 500 g/day, excluding fruit juice. The amount was divided equally for fruits and vegetables. * Recommended intakes for ﬁber and fatty acids are provided as range in the Nordic Nutrition Recommendations (NNRs), and a single value (in the parentheses) is used in the model simulation. n dietary component intake versus recommended intake (RI) for men and women in Nordic Countries. Abbreviations: RI, Recommended Intake; %E, percentage of total energy; MUFA, Monounsaturated fatty acids; NA, Not Available; PUFA, Polyunsaturated fatty acids. Note: Standard error of mean are in the parentheses. 2.3. The Simulation Model A comparative risk assessment macrosimulation model, PRIME (Preventable Risk Integrated ModEl) [14] has been used for this study. This model simulates the eﬀect of changes in consumption of foods (fruits and vegetables) and nutrients (dietary ﬁber, salt and fatty acids) through risk factors, such as serum cholesterol, blood pressure, and overweight/obesity to diet-related mortality from CVDs and diet-related cancers. In a technical report, the details regarding the underlying assumptions of the model are available [14]. To be included in the model, food components have to be recognized as statistically associated with CVD outcomes and cancer, or biological risk factors for these diseases. Meta-analyses obtained from prospective cohort studies and randomized controlled trials are used to parameterize changes in nutritional risk factors and mortality as a result of the change in the population’s intake of food items and nutrients [14]. PRIME estimates the diﬀerences in mortality in one single year between the baseline scenario (actual dietary intake, in this case) and the counterfactual scenario (recommended dietary intake). The model is based on a number of key assumptions: 1. The counterfactuals are based on changing dietary variables that are continuous (e.g., fruit consumption (g/day)), rather than binary exposures (meet recommendations for fruit (yes/no)). Therefore, a distribution of each variable within the population is used as a baseline for the model. For the counterfactuals, a shift of distribution is made so that the new mean level of consumption matches the recommendation, but the variance in the population remains the same as the baseline. This is equivalent to everyone in the population making the same changes to their diet, implying that approximately 50% of the population will still not meet the recommendations in the counterfactual scenario, but this is appropriate since population-level targets (such as dietary recommendations) are monitored by tracking a population’s mean consumption levels. 2. 2. Combined changes in the risks for individuals are multiplicative. For example, if one extra serving of fruits reduces the risk of CVD by 11% and reducing salt intake by 1 g per day reduces the risk by 10%, then both of these behavior changes jointly reduce the risk of CVD death by 19.9% (1 −(1 −0.11) × (1 −0.10)). The PRIME model accounts for competing risks by combining relative risks multiplicatively. 2.2. Actual Intake Recommended intakes are based on Nordic Nutrition Recommendation 2012. Recommended intake for fruits and vegetables together is 500 g/day, excluding fruit juice. The amount was divided equally for fruits and vegetables. * Recommended intakes for ﬁber and fatty acids are provided as range in the Nordic Nutrition Recommendations (NNRs), and a single value (in the parentheses) is used in the model simulation. Nutrients 2019, 11, 1434 6 of 17 2.3. The Simulation Model However, the model is unable to account for interactions between risk factors (e.g., if increasing fruit and vegetable consumption provides more health beneﬁt for low-ﬁber consumers than high-ﬁber consumers). 3. Another assumption is that changes in risk follow a log-linear, dose-response relationship, except for obesity, which follows a J-shaped curve. For example, a change in the consumption of fruits and vegetables from 3 to 4 servings has the same effect on relative risk as a change in consumption from 6 to 7 servings. However, an upper threshold was included, above which there are no additional health benefits. The upper thresholds are based on the range of data collected in the meta-analyses used to parameterize the models. It is unlikely that the effects of different food components are independent and additive. By combining parameters multiplicatively, the PRIME model estimates the overlap in estimated changes in the risk of cause-specific mortality as they relate to changes in different dietary components (i.e., the outcome of changing several dietary components simultaneously is less than the sum of its parts and can never exceed 100% risk reduction). 3. We also assume that Nordic people are similar in all aspects other than food intake, for example in terms of other health-related behaviors, such as physical exercise, alcohol drinking, and smoking habits, although within-country diﬀerences do prevail in health behaviors [28]. PRIME has been previously used to answer similar research questions in and France [29], Canada [30], the UK [31], and Sweden [32]. Nutrients 2019, 11, 1434 7 of 17 7 of 17 2.5. Uncertainty Analysis A Monte Carlo simulation was conducted to estimate the Uncertainty Intervals (UI) around the results. Each of the estimates in the model were allowed to vary according to the distribution reported in the accompanying literature. The 95% UI estimates are based on the 2.5th and 97.5th percentiles of results obtained from 5000 iterations of the model. 2.4. Population Statistics The model requires age- and sex-speciﬁc population mortality for speciﬁc diseases for a given year. The mortality data for diet-related cancers (ICD-10: C00-14, C16, C23, and C33-34), coronary heart diseases (ICD-10: I20-25) and stroke (ICD-10: I60-69) were obtained from country speciﬁc national databases. This model also required the age- and sex-speciﬁc number of populations in that country for that speciﬁc year. The population statistics were obtained from the oﬃcial statistical websites of each country. The latest population and mortality data (from 2016) were used for all the countries except Norway, where the population and death statistics are from 2013. 3. Results The reported intake of fruits and vegetables was lowest for Finish men and women among all Nordic countries (Table 2). Women consumed more fruits than men consumed in all the countries. Women also consumed more vegetables than men consumed, except in Norway and Iceland. Danes had the highest intake of ﬁber, whereas Icelanders had the lowest intake of ﬁber. Men consumed more salt than women in all the countries and for both groups, the intake of salt was higher than the recommended intake level (6 g/day). The salt intake was highest among the Danish men and women compared to the other Nordic Countries. Saturated fat intake was higher than the recommendation in all the countries, and Icelanders had the highest intake of saturated fat. The MUFA and PUFA intake was within the range of recommendations but lower than the optimum value (Table 2). The model estimates that the highest number of deaths that could be prevented by following the recommendation intake is for Iceland, where 19.7% of the deaths can be prevented, followed by the Finish population (18.9%) (Table 3). In terms of food groups, the highest percentage of deaths can be saved by following the recommendations of fruit and vegetable intake for all the countries except Denmark (Figure 1). For Danes, following salt recommendations could prevent 42.74% of the deaths related to the dietary intake. For the Fins, 64.95% of the deaths could be prevented by following the dietary recommendations for fruit and vegetable intake. In terms of gender, more deaths could be prevented or delayed among men than women (Table 3). Modifying dietary intake to meet fruit and vegetable recommendations could save more lives for men than for women in all the Nordic countries. This is also true for salt intake; more lives of men than women could be saved by consuming the recommended salt intake. However, women would beneﬁt more from consuming more ﬁber, except in Iceland. Most of the deaths that could be prevented or delayed by improving dietary intake are related to coronary heart diseases, followed by stroke in all the Nordic countries (Supplementary Materials, Annex 2). In terms of cancer, only colorectal cancers and lung cancer were inﬂuenced by simulated dietary changes. The scenario is the same for both men and women in all the Nordic countries. 8 of 17 Nutrients 2019, 11, 1434 Table 3. Abbreviations: RI, Recommended Intake; note: 95% uncertainty intervals are provided in the parentheses. Due to the stochastic nature of the model, the total ﬁgure might not be the same for adding up male and female together. Actual death is the number of deaths for a year in the speciﬁc countries due to the diseases used in the simulation mode. Abbreviations: RI, Recommended Intake; note: 95% uncertainty intervals are provided in the parentheses. Due to the st for adding up male and female together. Actual death is the number of deaths for a year in the speciﬁc countries due t changes i 4. Discussion Most of the deaths that could be prevented or delayed by improving dietary intake are related to coronary heart diseases, followed by stroke in all the Nordic countries (Supplementary materials Annex 2). In terms of cancer, only colorectal cancers and lung cancer were influenced by simulated dietary changes. The scenario is the same for both men and women in all the Nordic countries. 4. Discussion In this model-based simulation study, we show that a considerable number of deaths could be prevented or delayed if the Nordic population adhere to the NNR. Among the Nordic Countries, Iceland would beneﬁt the most by adhering to the NNR. We also ﬁnd that the most lives could be saved by changes attributable to an increase in fruits and vegetable consumption except for Denmark, where most of the lives can be saved by reducing salt intake. Furthermore, it also revealed that more lives of men than women could be saved. In this model-based simulation study, we show that a considerable number of deaths could be prevented or delayed if the Nordic population adhere to the NNR. Among the Nordic Countries Iceland would benefit the most by adhering to the NNR. We also find that the most lives could be saved by changes attributable to an increase in fruits and vegetable consumption except for Denmark where most of the lives can be saved by reducing salt intake. Furthermore, it also revealed that more lives of men than women could be saved. The simulation model predicts that the highest benefit would be gained for Iceland. This resul is reasonable since Icelanders had the lowest intake of fiber and the highest intake of total fat The simulation model predicts that the highest beneﬁt would be gained for Iceland. This result is reasonable since Icelanders had the lowest intake of ﬁber and the highest intake of total fat, saturated fat, and a low intake of fruits and vegetables (Table 2). This result may be surprising since Iceland is one of the healthiest nations in the world according to the Bloomberg Healthiest Country index [33]. This index is based on several factors like health risks, availability of clean water, life expectancy, malnutrition, and causes of death where dietary habit is just one factor. Another reason might be that Icelanders have the lowest rate of physical inactivity among the Nordic countries [34], which is also a determinant of good health. 3. Results Percentage of deaths (together with a 95% Uncertainty Intervals) delayed or saved by dietary changes in a year in the Nordic Countries. 3. Results Estimated number of total deaths or delayed by speciﬁc dietary changes according to guidelines in a year in the Nordic countries. Country Food Groups All Dietary Guidelines Combined Actual Death % Averted by RI Fruits and Vegetables Fiber Fats Salt Sweden Men 1905 (1262–2152) 718 (512–1275) 623 (471–792) 666 (335–1175) 3626 (2994–4175) 21,638 16.75% Women 1073 (811–1420) 1285 (656–1577) 245 (224–487) 180 (63–237) 2553 (2030–2980) 22,816 11.18% Altogether 3013 (2080–3566) 2025 (1197–2792 969 (709–1274) 1057 (391–1423) 6405 (5086–7086) 44,454 14.41% Denmark Men 563 (406–725) 349 (196–502) 55 (11–99) 755 (326–1166) 1591 (1156–1997) 16,150 9.85% Women 212 (136–288) 380 (219–545) 12 (7–33) 282 (122–447) 846 (623–1072) 16,418 5.15% Altogether 773 (547–1002) 726 (413–1041) 67 (5–132) 1040 (453–1605) 2433 (1799–3053) 32,568 7.47% Norway Men 584 (389–773) 324 (180–475) 126 (82–173) 391 (159–638) 1312 (1020–1605) 11,162 11.75% Women 432 (265–591) 494 (296–688) 79 (46–120) 30 (5–76) 968 (739–1188) 12,271 7.89% Altogether 1016 (662–1378) 820 (464–1163) 204 (132–289) 422 (171–727) 2285 (1786–2770) 23,433 9.75% Finland Men 1985 (1357–2525) 845 (446–1248) 207 (119–297) 506 (212–800) 3141 (2517–3708) 14,549 21.59% Women 1529 (1043–1975) 903 (512–1293) 37 (−22 – 99) 16 (4–38) 2286 (1776–2764) 14,097 16.21% Altogether 3521 (2412–4503) 1747 (946–2541) 243 (101–396) 516 (207–850) 5421 (4280–6476) 28,646 18.92% Iceland Men 68 (48–87) 51 (28–71) 20 (17–23) 28 (12–45) 141 (117–163) 586 24.06% Women 46 (32–58) 37 (22–51) 7 (5–9) 2 (1–5) 81 (66–96) 543 14.9% Altogether 114 (82–145) 88 (51–121) 27 (22–32) 31 (12–50) 223 (185–257) 1129 Abbreviations: RI, Recommended Intake; note: 95% uncertainty intervals are provided in the parentheses. Due to the stochastic nature of the model, the total ﬁgure might not be the same for adding up male and female together. Actual death is the number of deaths for a year in the speciﬁc countries due to the diseases used in the simulation mode. or delayed by speciﬁc dietary changes according to guidelines in a year in the Nordic countries. Nutrients 2019, 11, 1434 than women coul benefit more from 9 of 17 n would 9 of 17 n would Figure 1. Percentage of deaths (together with a 95% Uncertainty Intervals) delayed or saved by dietary Figure 1. Percentage of deaths (together with a 95% Uncertainty Intervals) delayed or saved by dietary changes in a year in the Nordic Countries. Figure 1. Percentage of deaths (together with a 95% Uncertainty Intervals) delayed or saved by dietar Figure 1. changes i 4. Discussion In order to achieve the Nordic Ambition, i.e., by 2021, at least 70% of the population complies with the NNR of a daily intake of 500 g of fruits and vegetables, there is an urgent need, for policy implications in Nordic countries, to increase the fruits and vegetables intake of the population [39]. The beneﬁts from following the recommended salt intake are the third highest except for Denmark where it is the highest, according to the simulation model. For Denmark, the major portion of the salt comes from processed or semi-processed food, such as bread, meat, meat products, and cheese. Finland had the lowest salt intake among the Nordic countries. Since the 1970s, Finland has aimed to reduce salt intake in its National Nutrition Policy [40] by reformulation and raising public awareness of the harmful eﬀects of salt on health. This has led to a signiﬁcant reduction in salt intake of 3 g/day from 1979 to 2002 (12 to 9 g/day), as measured by urinary sodium [41], where the reduction was higher among women than men. The dietary survey (FINDIET’2012) [25] even provided a lower estimate of a 4.4 g/day reduction (from 12 to 7.6 g/day). This was accompanied by a fall in blood pressure and a decrease of 75%–80% in coronary heart disease and stroke mortality, with an increase of 5–6 years in life expectancy [42,43]. Finish action policy can be used as an example for other Nordic countries in terms of the reduction of salt intake. However, it is noteworthy that the sodium values are underestimated, as information on the addition of salt during cooking and at the dinner table was not systematically obtained during the diet interviews in Norway [26], as well as in the dietary surveys of Sweden [23] and Denmark [24]. Studies other than dietary surveys also indicate that salt consumption values might be underestimated in the dietary surveys [44–46]. This means that the eﬀect we estimated by simulation is probably also underestimated. Another interesting fact is that the salt intake of elderly women from Finland (65–74 years) and Norway (60–70 years) was lower than the recommended level, 5.70 g/day and 5.75 g/day, respectively (Supplementary Materials). This means that even an intake lower than the recommended intake of 6 g/day is possible. changes i 4. Discussion is reasonable since Icelanders had the lowest intake of fiber and the highest intake of total fat saturated fat, and a low intake of fruits and vegetables (Table 2). This result may be surprising since Iceland is one of the healthiest nations in the world according to the Bloomberg Healthiest Country index [33]. This index is based on several factors like health risks, availability of clean water, life expectancy, malnutrition, and causes of death where dietary habit is just one factor. Another reason might be that Icelanders have the lowest rate of physical inactivity among the Nordic countries [34] which is also a determinant of good health. The highest number of deaths could be prevented or delayed by increasing the intake of fruits and vegetables for most countries where Finland would gain the most benefit. These findings are The highest number of deaths could be prevented or delayed by increasing the intake of fruits and vegetables for most countries where Finland would gain the most beneﬁt. These ﬁndings are consistent with the Global Burden of Disease study, which revealed that health beneﬁts are higher for food categories that are consumed in an insuﬃcient amount, such as fruits and vegetables, than for foods and nutrients which are consumed in excess [35]. Findings for adolescent eating habits also indicated that fruit intake increased in Norway and Denmark, and the intake was the lowest for Finland [36]. In Denmark, a nation-wide 6-a-day initiative has been conducted since 2001 to increase the intake of fruits and vegetables in the population, which was eﬀective in increasing the fruit and vegetable intake of the country [37]. In Norway, a free program of fruit in school (without parental payment) was implemented nationwide in 2007, which showed an increase in the consumption of fruits not only in school children but also in their parents [38]. It is worth mentioning that a maximum of one portion of fruit juice is considered as fruit in all the countries, except Iceland where any portion of fruit juice is included as fruit [19]. Therefore, the inclusion of fruit juice may have contributed to the 10 of 17 Nutrients 2019, 11, 1434 mean intake of fruits and vegetables in the Nordic countries, which have an impact on our results. Nutrients 2019, 11, 1434 Nutrients 2019, 11, 1434 11 of 17 The results from this study can be compared with studies that use the earlier version of the PRIME model to estimate the health impact of achieving dietary recommendations in Canada [30] and the UK [31]. The UK study suggested that 46% of the deaths averted or delayed could be attributed to meeting fruit and vegetable recommendations [31], with a further 23% attributed to achieving the salt recommendation. For Canada, it was 72% and 10% for fruits and vegetables and salt, respectively [30]. Sweden, Norway, and Iceland are close to the UK study for the fruit and vegetable intake, whereas Finland is close to the Canadian study for the salt intake. The reason for this result might be the diﬀerence between recommended fruits and vegetables consumption in the Nordic countries, Canada and the UK. The recommendation for Canada is at least seven servings (depending on sex and age) [30] and in the UK, it is ﬁve servings per day [55] (equivalent to 400 g), whereas we used the NNR which is 500 g per day [1]. The Nordic food culture is diﬀerent from food cultures from the UK and Canada. Since the food culture, dietary practices, and recommendations are country-speciﬁc, a study on the health beneﬁts for each Nordic country is justiﬁed. Meier et al. showed that 12 dietary factors contributed to 22.4% of all deaths in 51 European countries, based on the Global Burden of Disease study [56]. Our model-based simulation ﬁndings for Nordic countries are lower than that. One reason for this result could be that the PRIME model does not consider deaths related to processed meat and sugar and sweetened beverages. The need for comparable data on nutrient intake across the Nordic countries is complicated due to diverse study methodologies. The methods used in the Nordic countries for the dietary surveys were diﬀerent (Table 1). The 4 or 7 day consecutive surveys, 24 or 48 h recall, and food frequency questionnaire (FFQ) each have their pros and cons [57]. While 24 h recall suﬀers from underreporting, it is less onerous for the respondents [58]. Collection over more days better reﬂects usual intake due to greater control over day-to-day variation but is associated with within-person errors and cannot capture the wide variations of intake within the population. changes i 4. Discussion The Nordic countries could consider lowering the recommended level, since the American Heart Association has suggested reducing salt intake to 3.75 g/day for the primary prevention of CVDs [47]. Furthermore, the European Food Safety Authority (EFSA) has recently positioned their assessment of sodium consumption for public consultation, where 3 g of sodium (equivalent to 5 g of salt) is suggested to reduce the risk of CVDs in the population [48]. The health beneﬁts from changing the intake of fats and fatty acids are fewer compared to other dietary components. The estimates from the simulation model report on both the strength of the association between dietary factors and its health outcomes. The actual intake of fat and fatty acids for the Nordic population are very close to the recommended intake. Moreover, the population of the Nordic countries consumes a considerable number of dairy products and ﬁsh. Fish consumption has been measured to be the highest in Norway, followed by Iceland and Finland [20]. Fatty ﬁshes are a good source of PUFA, and epidemiological studies suggested that diets rich in PUFA and MUFA are associated with low mortality [49,50]. It is noteworthy that women could gain the most from increased ﬁber intake, except in Iceland and Finland, whereas men could gain higher health beneﬁts from an increased intake of fruits and vegetables. The reason for this result may be that men consume fewer servings of fruits and vegetables than women, whereas women consume less ﬁber than men (Table 2). Since a signiﬁcant amount of ﬁber is are available from fruits and vegetables, this ﬁnding is questionable. One explanation is that fruit, vegetable, and ﬁber intake were separated in the simulation model. Moreover, the caloric intake of men was higher than women, and a signiﬁcant portion of the calories came from grains, which are a high source of ﬁber, especially whole grains [51]. The most commonly consumed whole grain cereals in the Nordic countries are wheat, rye, and oats, with a considerable inter-country variation in consumption patterns [52], as well as inter-age group variation within the country. For example, rye bread is an important feature in the Danish diet [53]. In Sweden, older adults consume more whole grain products than younger adults [54]. Nutrients 2019, 11, 1434 A detailed description of diﬀerent dietary surveys to estimate national dietary intake Nutrients 2019, 11, 1434 12 of 17 using diﬀerent methods, nutrient databases, and the problem with inter-country comparison can be found elsewhere [64]. These diﬀerences are a major drawback for inter-country comparison, and thus our ﬁndings need to be interpreted with caution. The strength of this study is that it uses the same simulation model, which facilitates cross-country comparison. The mortality statistics and population data were obtained from country-speciﬁc credible sources, and it is known that Nordic countries maintain good epidemiological data due to unique personal identiﬁcation numbers and validated national registers [65]. These registers are well maintained with a high coverage rate [66], which is also a strength of the study. However, it is worth mentioning that, from the Danish statistical website, some of the mortality data required for the model input were unavailable (e.g., Heart failure, Aortic aneurysm), so the results might be underestimated for Denmark. PRIME is a transparent model for which all the risk equations related to changes in diet and mortality come from high-quality meta-analyses [14]. Moreover, the model has been used several times in diﬀerent countries (for example, in the UK [55,67,68], Ireland [69], New Zealand [70], France [29], and Canada [30]). The estimates of relative risks that have been used to parameterize the model were taken from the results of published meta-analyses, which is an additional strength of this study. However, not all of the studies included in the meta-analyses adjusted their results for each of the dietary factors or biological risk factors that are included in the model. For example, the eﬀect of fruits and vegetables on CVDs is likely to be partially mediated by dietary ﬁber, which is not accounted for in the model [14]. The model-based ﬁndings may be aﬀected by double counting to some extent. On these grounds, an overestimation of deaths prevented or delayed is possible, which is a limitation. Another limitation is that the analysis does not account for the health risks of consuming red meat. Furthermore, that the health beneﬁts will be achieved in the same year if people follow NNR is an assumption of the model. It would, however, take years (e.g., the eﬀects of salt reduction on CVDs) [71] or even decades (e.g., eﬀects of dietary ﬁber on cancer) for the full health gain to be realized. Nutrients 2019, 11, 1434 The FFQ can capture the inﬂated energy and nutrient intake but is burdensome for the respondent [59]. Given that the Nordic countries perform dietary surveys regularly, standardizing the survey methodology would vastly improve data comparability across the Nordic Countries. For example, both Norway and Iceland used 2 × 24 h recall together with an FFQ, which is recommended by the EFSA [60]. This can be a way forward for the harmonization of dietary surveys in the Nordic countries and could thus facilitate comparison. Diﬀerences in dietary assessment methodologies present further limiting factors when making inter-country comparisons. For example, the mean energy intakes of Norwegian men aged 18–29 years were 3059 kcal per day [26], which is much higher than in the same age group in Sweden, (2246 kcal per day) [24], despite the fact that both national dietary were conducted in the same years (Supplementary Materials). These diﬀerences could thus result from either diﬀerent methodological approaches to calculate the energy or a disparity in the intake. The participation rates in the national dietary surveys vary to a large extent, and there is, in general, a low participation rate (Table 1). The highest participation rate was for Iceland (68.6%) [27]. Therefore, one cannot reject the notion of selection bias as only motivated people participated in the dietary surveys. Thus, the dietary surveys might not capture the true dietary intake of the population. Furthermore, underreporting is common and varies across methods and is aﬀected by multiple other factors, making it diﬃcult for comparison. For example, Norway excluded under-reporters, whereas Denmark included under-reporters in their analysis; other countries did not specify [61]. The lack of alignment and completeness of national nutrient databases and classiﬁcation systems present further limitations. Nutrient databases are required to calculate energy and nutrient intakes from food consumption data and are prone to random and systematic dietary measurement errors, which can aﬀect population means and the distribution of nutrient intakes [62]. An inter-country comparison is diﬃcult due to a lack of harmonization of nutrients, i.e., modes of expression, units, and chemical analytical methods of analysis. For example, the Englyst method provides lower estimates of dietary ﬁbers from certain cereals, fruits, white beans, and peanuts compared to the AOAC method [63]. Nutrients 2019, 11, 1434 Nonetheless, we provide a comparative scenario in the Nordic countries in regard to discrepancies in terms of actual dietary intake and recommended dietary intake by NNR with a simulation model. This simulation model study has the potential for future research. We found that diﬀerent countries require diﬀerent areas for the policy implications of changes in dietary habits. Danes need to reduce their salt intake while Finns need to increase their fruit and vegetable intake. The next question would be to investigate how decision makers can intervene to modify the consumption of dietary components (e.g., increase fruit and vegetable intake or decrease salt intake by using taxes or subsidies [72–74] or by interventions at the workplace, for example, free fruit or healthy meals in the canteen [75–77]). A subsidy on grain products can modify the dietary ﬁber intake to the recommended level in the Swedish population [78,79]. The subsidy also resulted in an increased intake of other food components, such as fat, salt, and sugar. This indicates that both subsidies and taxes need to be used in order to modify the dietary behavior of the population [78,79]. For Denmark, a tax on saturated fat (16 Danish Krona per kilogram of saturated fat) reduced the intake of saturated fat by 4%, and at the same time, increased the consumption of vegetables, fruits, and ﬁber [80] in the population. Furthermore, Nordic countries can learn from each other on successful interventions/policies. For example, Denmark could beneﬁt from the salt policy implemented in Finland, and the other Nordic counties may beneﬁt from the 6-a-day campaign in Denmark, to increase fruit and vegetable intake. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/11/6/1434/s1, Annex 1: Country-speciﬁc estimated number of deaths averted or delayed by adhering to Nordic Nutrition References 1. Secretary of the Nordic Council of Ministers, Nordic Council of Ministers. Nordic Nutrition Recommendations 2012: Integrating Nutrition and Physical Activity, 5th ed.; Nordisk Ministerråd: Copenhagen, Denmark, 2014; p. 627. 2. Organisation for Economic Co-operation and Development (OECD). Health at a Glance: Europe 2018; State of Health in the EU Cycle: Brussels, Belgium, 2018. 2. Organisation for Economic Co-operation and Development (OECD). Health at a Glance: Europe 2018; State of Health in the EU Cycle: Brussels, Belgium, 2018. 3. Becker, W. Dietary guidelines and patterns of food and nutrient intake in Sweden. Br. J. Nutr. 1999, 81 (Suppl. S1), S113–S117. [CrossRef] [PubMed] 3. Becker, W. Dietary guidelines and patterns of food and nutrient intake in Sweden. Br. J. Nutr. 1999, 81 (Suppl. S1), S113–S117. [CrossRef] [PubMed] 4. Jonsdottir, S.E.; Brader, L.; Gunnarsdottir, I.; Kally Magnusdottir, O.; Schwab, U.; Kolehmainen, M.; Risérus, U.; Herzig, K.-H.; Cloetens, L.; Helgegren, H.; et al. Adherence to the Nordic Nutrition Recommendations in a Nordic population with metabolic syndrome: High salt consumption and low dietary ﬁbre intake (The SYSDIET study). Food Nutr. Res. 2013, 57, 21391. [CrossRef] [PubMed] 4. Jonsdottir, S.E.; Brader, L.; Gunnarsdottir, I.; Kally Magnusdottir, O.; Schwab, U.; Kolehmainen, M.; Risérus, U.; Herzig, K.-H.; Cloetens, L.; Helgegren, H.; et al. Adherence to the Nordic Nutrition Recommendations in a Nordic population with metabolic syndrome: High salt consumption and low dietary ﬁbre intake (The SYSDIET study). Food Nutr. Res. 2013, 57, 21391. [CrossRef] [PubMed] 5. Handeland, K.; Kjellevold, M.; Wik Markhus, M.; Eide Graﬀ, I.; Frøyland, L.; Lie, Ø.; Skotheim, S.; Stormark, M.K.; Dahl, L.; Øyen, J. A Diet Score Assessing Norwegian Adolescents’ Adherence to Dietary Recommendations—Development and Test-Retest Reproducibility of the Score. Nutrients 2016, 8, 467. [CrossRef] [PubMed] 5. Handeland, K.; Kjellevold, M.; Wik Markhus, M.; Eide Graﬀ, I.; Frøyland, L.; Lie, Ø.; Skotheim, S.; Stormark, M.K.; Dahl, L.; Øyen, J. A Diet Score Assessing Norwegian Adolescents’ Adherence to Dietary Recommendations—Development and Test-Retest Reproducibility of the Score. Nutrients 2016, 8, 467. [CrossRef] [PubMed] 6. Ewers, B.; Trolle, E.; Jacobsen, S.S.; Vististen, D.; Almdal, T.P.; Vilsbøll, T.; Bruun, J.M. Dietary habits and adherence to dietary recommendations in patients with type 1 and type 2 diabetes compared with the general population in Denmark. Nutrition 2019, 61, 49–55. [CrossRef] [PubMed] 7. Stanaway, J.D.; Afshin, A.; Gakidou, E.; Lim, S.S.; Abate, D.; Abate, K.H.; Abbafati, C.; Abbasi, N.; Abbastabar, H.; Abd-Allah, F.; et al. 5. Conclusions In conclusion, this study shows that by modifying dietary intake, a considerable number of deaths could be prevented or delayed in the Nordic countries. Thus, policy makers should take the necessary steps to modify the dietary intake of the Nordic population. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/11/6/1434/s1, Annex 1: Country-speciﬁc estimated number of deaths averted or delayed by adhering to Nordic Nutrition 13 of 17 Nutrients 2019, 11, 1434 Recommendations and Annex 2: Country-speciﬁc age-group and sex-speciﬁc mean (standard deviation) dietary intake incorporated in the PRIME model Author Contributions: Conceptualization, S.S. and J.N.; methodology, S.S., J.N., I.M., U.-G.G.; Model, S.S.; formal analysis, S.S. and U.G.-G.; writing—original draft preparation, S.S. and J.N.; writing—review and editing S.S., J.N., I.M., P.M.N., U.G.-G.; funding acquisition, S.S., J.N., I.M., P.M.N., U.G.-G. Funding: This research was funded by Swedish Research Council for Health, Working Life and Welfare, grant number (dnr 2016-00312), Swedish Research Council, grant number (dnr 2014-646). The Health Economics Program at Lund University also receives core funding from Government Grant for Clinical Research, and Region Skåne (Gerdtham). Acknowledgments: We thank Peter Scarborough for allowing us to use the PRIME model and for his help in the analysis of the simulation model. Acknowledgments: We thank Peter Scarborough for allowing us to use the PRIME model and for his help in nalysis of the simulation model. 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. 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https://openalex.org/W2332842682	http://uu.diva-portal.org/smash/get/diva2:934299/FULLTEXT01	English	null	Topology based identification and comprehensive classification of four-transmembrane helix containing proteins (4TMs) in the human genome	BMC genomics	2,016	cc-by	15,038	* Correspondence: helgi.schioth@neuro.uu.se 1Department of Neuroscience, Functional Pharmacology, Uppsala University, BMC, Box 593, 751 24, Uppsala, Sweden 2Institutionen för neurovetenskap, BMC, Box 593, 751 24, Uppsala, Sweden © 2016 Attwood 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. Topology based identification and comprehensive classification of four- transmembrane helix containing proteins (4TMs) in the human genome Misty M. Attwood1, Arunkumar Krishnan1, Valentina Pivotti1, Samira Yazdi1, Markus Sällman Almén1 and Helgi B. Schiöth1,2* * Correspondence: helgi.schioth@neuro.uu.se 1Department of Neuroscience, Functional Pharmacology, Uppsala University, BMC, Box 593, 751 24, Uppsala, Sweden 2Institutionen för neurovetenskap, BMC, Box 593, 751 24, Uppsala, Sweden © 2016 Attwood 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. Attwood et al. BMC Genomics (2016) 17:268 DOI 10.1186/s12864-016-2592-7 Attwood et al. BMC Genomics (2016) 17:268 DOI 10.1186/s12864-016-2592-7 Background The preponderance of membrane proteins attach to the membrane with transmembrane α- helices, while the rest are characterized by transmembrane β-strands forming β-barrels. Previous topology based ana- lyses of membrane proteins in the human genome have shown that the largest category of α-helical membrane proteins is composed of only one transmembrane span- ning helix, while the second largest category includes the two transmembrane helix containing proteins [5] [7]. In particular, several studies point toward a strong correlation between membrane protein structure and function and that the number of transmembrane heli- ces substantially determines what function the protein carries out [5, 8–10]. Moreover, analyses of structural topologies of membrane proteomes have shown that the C-terminus is predominantly located in the inside of the membrane, as found across humans [5], Escherichia coli [11] and Saccharomyces cerevisiae [12], and can be en- gaged in activities such as stabilization, signaling, protein interactions, and channel gating amongst others [13, 14]. This C-terminus localization is ubiquitous as the majority of proteins containing odd numbers of helices, such as those in the large 1TM and 7TM groups, have intracellu- lar C-termini with N-termini in the extracellular environ- ment, and protein groups with even helical numbers have a greater amount of both termini located intracellularly. Our earlier complete curation of the membrane proteome in the human genome showed that certain membrane topologies are more common for specific functions such as enzymatic activity, receptors, and transporters [5]. For example, the majority of receptors fall into two major categories: those that contain either one transmem- brane helix (TM), or the well-studied 7TM G protein- coupled receptor (GPCR) group [15–17] which composes 67 % of human receptors [5]. This means that receptors have a greater number of their N-termini in the extracellu- lar environment, which functions in activities such as pro- tein interactions, protein targeting, and signaling [18]. Transporters, another well-researched group, tend to have six or more membrane-spanning helices, such as the solute carriers (SLC) that contain 10–14TM [19]. However, mem- brane bound enzymes generally contain fewer helices and they largely fall within 1TM or 2TM containing proteins y Within these larger protein families, selected proteins have been well characterized, however there still remain many 4TM proteins that have yet to be fully classified. Background Among these TM families that are categorized by the number of α-helices that span the membrane, the four- transmembrane helix containing proteins are characterized by an array of protein families, such as the neurotransmitter gated ion channels, claudins, connexins, and tetraspanins, that display large degrees of diversity in their functions. Neurotransmitter gated ion channels (NGIC) mediate a rapid transmission of signals at chemical synapses and are a heavily investigated group that act as both receptors and transporters [20]. They have four evolutionarily and struc- turally related 4TM families that include 45 members [21] plus one more family consisting of one gene, zinc activated ligand-gated ion channel (ZACN) [22]. Claudins are an- other major 4TM family and are one of the main structural components of tight junctions and mediate cell-cell adhe- sion. There are currently 27 mammalian claudin members that are found solely in epithelial cells. [23]. They contain the PMP22_Claudin domain (PF00822) which is a member of the Transporter superfamily clan, as well as two con- served cysteine residues in extracellular loop one, and most claudins contain a PDZ binding motif at the C-terminus position [24]. Connexins, which are also members of the Transporter superfamily clan, have been extensively studied as they are the structural components of gap junctions, which are involved in cell-cell communication. There are 21 different connexin proteins described by several conserved features: a four membrane-spanning domain (PF00029) and a conserved three-cysteine residue domain (PF10582) found on each of the two extracellular loops. And finally, the Tetraspanins include 33 members in which some members have a wide and abundant distribu- tion throughout tissues and others are more selectively expressed [25]. These ‘true’ tetraspanins (to differentiate from proteins with 4TMs that can also be called tetra- spanins) contain the tetraspanin functional domain (PF00335), which is a member of the Tetraspanin-like clan, as well as a conserved CCG motif with 4–6 conserved extracellular cysteine residues [26] Membrane proteins are essential in several cell processes and participate in a wide variety of functions, including playing pivotal roles in signaling pathways, acting as regulatory elements, functioning as receptors, and also facilitating the transport of ions and molecules across the impermeable lipid bilayer [1]. Due to their diverse and important functional activities, membrane proteins serve as major targets for pharmaceutical industries [2, 3]. Approximately 20–30 % of most animal proteomes are transmembrane proteins, which in humans amounts to ~5500 proteins [4–6]. Abstract Background: Membrane proteins are key components in a large spectrum of diverse functions and thus account for the major proportion of the drug-targeted portion of the genome. From a structural perspective, the α-helical transmembrane proteins can be categorized into major groups based on the number of transmembrane helices and these groups are often associated with specific functions. When compared to the well-characterized seven-transmembrane containing proteins (7TM), other TM groups are less explored and in particular the 4TM group. In this study, we identify the complete 4TM complement from the latest release of the human genome and assess the 4TM structure group as a whole. We functionally characterize this dataset and evaluate the resulting groups and ubiquitous functions, and furthermore describe disease and drug target involvement. Results: We classified 373 proteins, which represents ~7 % of the human membrane proteome, and includes 69 more proteins than our previous estimate. We have characterized the 4TM dataset based on functional, structural, and/or evolutionary similarities. Proteins that are involved in transport activity constitute 37 % of the dataset, 23 % are receptor-related, and 13 % have enzymatic functions. Intriguingly, proteins involved in transport are more than double the 15 % of transporters in the entire human membrane proteome, which might suggest that the 4TM topological architecture is more favored for transporting molecules over other functions. Moreover, we found an interesting exception to the ubiquitous intracellular N- and C-termini localization that is found throughout the entire membrane proteome and 4TM dataset in the neurotransmitter gated ion channel families. Overall, we estimate that 58 % of the dataset has a known association to disease conditions with 19 % of the genes possibly involved in different types of cancer. Conclusions: We provide here the most robust and updated classification of the 4TM complement of the human genome as a platform to further understand the characteristics of 4TM functions and to explore pharmacological opportunities. Keywords: Human proteome, Four transmembrane, 4TM, Function, Topology prediction, Structure function, Cancer, Drug targets Keywords: Human proteome, Four transmembrane, 4TM, Function, Topology prediction, Structure function, Cancer, Drug targets Attwood et al. BMC Genomics (2016) 17:268 Attwood et al. BMC Genomics (2016) 17:268 Page 2 of 16 Background To the best of our knowledge, the complete complement of four-transmembrane helical containing proteins has not been characterized before in any vertebrate genome, and curation of the 4TM component of the human gen- ome can provide an important basis to understand the diversity of the important 4TM proteome. First, we aim to provide a qualitative functional classification of the complete 4TM dataset screened from the latest release of the human genome (GRCh38.p3). Second, we sought to analyze whether there is any correlation between the topological orientations (N-terminus inside or outside of the membrane) and their functional activities, that is if certain topologies are favored for certain functional Attwood et al. BMC Genomics (2016) 17:268 Page 3 of 16 Page 3 of 16 Page 3 of 16 Additionally, the proteins need to be evaluated for an- notation quality and protein validity, i.e. that they have recognized protein-coding descriptions with acceptable transcriptional support. classes. Third, we aim to identify how many of these 4TM proteins are associated with diseases and how many of them are potential drug targets. Here, we present the first complete gene repertoire and detailed functional classification of the human four membrane-spanning protein dataset. The predicted proteins were evaluated for validity and reliability through comparison with the CCDS dataset and then further manually refined (Fig. 1B). The 4TM dataset includes 101 sequences identified as fragments. There are 106 proteins established as isoforms that in- clude 88 proteins that contain Pfam functional domains that are cited through literature as containing more than four helices, indicating the protein has an incomplete domain and consequently functionality cannot be in- ferred. Additionally, 15 proteins have been identified through literature and database resources as suspected false-positive hits from TOPCONS-single. None of these proteins are included in the 4TM unambiguous dataset. Forty additional proteins were manually evaluated and added to the dataset to include members of known 4TM protein families (for example, claudins and tetraspanins). The final dataset includes 373 unambiguous 4TM pro- teins, representing a single protein product from each gene. These 373 proteins are categorized into five major groups based on functional, structural, and/or evolution- ary similarities: Transporters (66), Enzymes (45), Dual Identification and curation of the 4TM dataset Identification and curation of the 4TM dataset de t cat o a d cu at o o t e dataset A combination of in silico automatic classification followed by individual sequence curation was used to analyze proteins with four membrane-spanning regions (see Fig. 1: Parts A and B, and Methods section for details). The initial human proteome contained 93,129 protein coding sequences, and after excising signal peptides 1,296 4TM sequences were initially predicted using TOPCONS-single. The predicted dataset was reduced by selecting the longest sequence length per unique gene (using ENSEMBL gene identification), re-evaluated by TOPCONS-single, and the non-redundant dataset includes 555 proteins. This dataset does include proteins that may have incomplete sequences that do not contain initial and/ or terminal residues; that are possibly false-positive hits from TOPCONS-single; as well as proteins that are isoforms and contain incomplete functional domains. Fig. 1 (Parts A and B). Automatic and manual classification process. Part 1A: Schematic diagram of the automatic classification process. The first step in the automatic characterization process included downloading the Homo sapiens proteome from the Genome Reference Consortium human genome 38 (GRCh38) release with the GenCode annotation information. SignalP standalone was used to assess and excise any signal peptides. The four membrane-spanning regions were predicted using TOPCONS-single, which comprises five different prediction tools and returns a consensus decision of the number of transmembrane areas. The longest sequence for each genomic location, or gene, was then selected and then those sequences were re-evaluated with TOPCONS-single. Uniprot, which is a large comprehensive repository of protein sequences with both manually curated and automatically generated annotations, was used to download associated annotations for each sequence in the predicted dataset. Part 1B: Schematic overview of the manual curation process for each individual sequence. The purpose of the individual sequence inspections was to ensure the 4TM dataset was composed of valid proteins with four membrane-spanning regions so that the function could be inferred and described. The predicted dataset was evaluated for validity and reliability using the CCDS dataset. The predicted dataset includes proteins that may be fragments, possible false-positive hits from TOPCONS-single, and protein isoforms that contain incomplete functional domains. The Uniprot annotation data included information such as whether the sequence was a fragment or not, any associated Pfam functional domains or families, the Transportation Classification (TC) number, the Enzyme Commission (EC) number, as well as Gene Ontology annotation terms Fig. Identification and curation of the 4TM dataset 1 (Parts A and B). Automatic and manual classification process. Part 1A: Schematic diagram of the automatic classification process. The first step in the automatic characterization process included downloading the Homo sapiens proteome from the Genome Reference Consortium human genome 38 (GRCh38) release with the GenCode annotation information. SignalP standalone was used to assess and excise any signal peptides. The four membrane-spanning regions were predicted using TOPCONS-single, which comprises five different prediction tools and returns a consensus decision of the number of transmembrane areas. The longest sequence for each genomic location, or gene, was then selected and then those sequences were re-evaluated with TOPCONS-single. Uniprot, which is a large comprehensive repository of protein sequences with both manually curated and automatically generated annotations, was used to download associated annotations for each sequence in the predicted dataset. Part 1B: Schematic overview of the manual curation process for each individual sequence. The purpose of the individual sequence inspections was to ensure the 4TM dataset was composed of valid proteins with four membrane-spanning regions so that the function could be inferred and described. The predicted dataset was evaluated for validity and reliability using the CCDS dataset. The predicted dataset includes proteins that may be fragments, possible false-positive hits from TOPCONS-single, and protein isoforms that contain incomplete functional domains. The Uniprot annotation data included information such as whether the sequence was a fragment or not, any associated Pfam functional domains or families, the Transportation Classification (TC) number, the Enzyme Commission (EC) number, as well as Gene Ontology annotation terms Attwood et al. BMC Genomics (2016) 17:268 Page 4 of 16 Function (47), Receptors (2), and Miscellaneous (213). The proteins identified as Miscellaneous are further clas- sified into subgroups with similar functions based on GO terms, Pfam domain descriptions, and protein family descriptions. are identified as drug targets: B-lymphocyte antigen CD20 [SwissProt: P11836] is involved in the regulation of B-cell activation and proliferation; and Calcium release-activated calcium channel protein 1 [SwissProt: Q96D31] which mediates calcium influx following depletion of calcium stores [28]. Sixteen genes in the alpha-type channel proteins are identified in various disease conditions and several of the most common disorders are displayed in Fig. 2. For a complete overview of the dataset including gene-disease associations, see Additional file 1. Functional classifications Transporters The Transporter category is the largest functional group with 66 proteins that are further divided into seven different classes (Fig. 2). Proteins identified with a Transport Classification number (TC) are included in this functional class. The alpha-type channel (TCDB ID: 1.A.-.-.) is the largest group and contains 26 proteins. Alpha-type channels transport solutes such as potassium, calcium, sodium, and chloride ions through transmem- brane pores or channels via an energy-independent process [27]. Eight of these proteins function as subunits in gap junctions, where six are connexins and two are pannexins. There are two proteins within this group that Eighteen proteins are identified as auxiliary transport proteins (8.A.-.-.-) that facilitate transport across mem- branes but do not directly participate in the transport process [27]. Eleven of these proteins belong to the Transporter superfamily clan and six proteins are true tetraspanins. All eight proteins that belong to the paracellu- lar channel: claudin tight junction class (1.H.-.-.-) contain the PMP22_Claudin domain (Pfam family: PF00822) and are members of the previously described claudin proteins. Paracellular transport transpires outside of cells and Fig. 2 The human 4TM Transporter class. The figure shows the 66 proteins of the Transporter class that are further categorized into seven different subclasses. The Transporter Classification number (TC) is determined by the Transporter Classification Database, which has been cross-referenced through Uniprot to obtain the TC number associated with the protein. The TC system is specific for membrane transport proteins and includes both functional and phylogenetic information in the numbering information. The transporter proteins identified as drug targets and the drug indications are also presented. An updated dataset of all current targeted and potential proteins and genes involved in drug studies or experimentation was used to identify proteins that are drug targets as well as the drug indications. In addition, the top common gene-disease associations are displayed for each subgroup, with the number of proteins involved in parenthesis. Three different resources were used to identify gene-disease associations: the Online Mendelian Inheritance in Man (OMIM) database; the Functional Disease Ontology (FunDO) resource; and the Jensen Lab Diseases database (see Methods for details). Proteins that participate in transport activity but do not have an associated TC number are not included in this class, but rather in a subgroup of the Miscellaneous proteins. The number in parenthesis represents the number of proteins that have been identified in that group Fig. Functional classifications Transporters 2 The human 4TM Transporter class. The figure shows the 66 proteins of the Transporter class that are further categorized into seven different subclasses. The Transporter Classification number (TC) is determined by the Transporter Classification Database, which has been cross-referenced through Uniprot to obtain the TC number associated with the protein. The TC system is specific for membrane transport proteins and includes both functional and phylogenetic information in the numbering information. The transporter proteins identified as drug targets and the drug indications are also presented. An updated dataset of all current targeted and potential proteins and genes involved in drug studies or experimentation was used to identify proteins that are drug targets as well as the drug indications. In addition, the top common gene-disease associations are displayed for each subgroup, with the number of proteins involved in parenthesis. Three different resources were used to identify gene-disease associations: the Online Mendelian Inheritance in Man (OMIM) database; the Functional Disease Ontology (FunDO) resource; and the Jensen Lab Diseases database (see Methods for details). Proteins that participate in transport activity but do not have an associated TC number are not included in this class, but rather in a subgroup of the Miscellaneous proteins. The number in parenthesis represents the number of proteins that have been identified in that group Fig. 2 The human 4TM Transporter class. The figure shows the 66 proteins of the Transporter class that are further categorized into seven different subclasses. The Transporter Classification number (TC) is determined by the Transporter Classification Database, which has been cross-referenced through Uniprot to obtain the TC number associated with the protein. The TC system is specific for membrane transport proteins and includes both functional and phylogenetic information in the numbering information. The transporter proteins identified as drug targets and the drug indications are also presented. An updated dataset of all current targeted and potential proteins and genes involved in drug studies or experimentation was used to identify proteins that are drug targets as well as the drug indications. In addition, the top common gene-disease associations are displayed for each subgroup, with the number of proteins involved in parenthesis. Three different resources were used to identify gene-disease associations: the Online Mendelian Inheritance in Man (OMIM) database; the Functional Disease Ontology (FunDO) resource; and the Jensen Lab Diseases database (see Methods for details). Functional classifications Transporters Proteins that participate in transport activity but do not have an associated TC number are not included in this class, but rather in a subgroup of the Miscellaneous proteins. The number in parenthesis represents the number of proteins that have been identified in that group Attwood et al. BMC Genomics (2016) 17:268 Page 5 of 16 solutes passively follow concentration gradients or transcel- lular electrical potentials [27]. Four proteins are identified as putative transport proteins (9.B.-.-.-) where transport function has been suggested for them, but evidence is not yet complete and these proteins will eventually be classified elsewhere or eliminated [27]. Two of these putative trans- porters contain the MARVEL domain (PF01284): Occludin [SwissProt: Q16625] is a member of the tight junction asso- ciated MARVEL proteins (TAMP) [29] and is involved in several diseases including cancer and neurologic disorders; and Synaptophysin [SwissProt: P08247] is involved with structural functions and in targeting vesicles to the plasma membrane [30] and is also involved in cancer, Alzheimer’s disease, schizophrenia, and mental retardation. Four proteins are porters (2.A.-.-.-) that use a carrier-mediated process to catalyze solutes through the membrane [27]. P- P-bond hydrolysis-driven transporters (3.A.-.-.-), which hydrolyze ATP to drive the active transport of solutes, in- cludes just one protein [SwissProt: P33897]. Recognized transporters of unknown biochemical mechanism (9.A.-.-.-) comprises five proteins, of which three are members of the Transporter superfamily clan. reduction reactions in which H and O atoms or electrons are transferred from one substance to another include 12 enzymes. There are varied specific functions within this group, including iron ion binding and lipid metabolic processes [28]. Transferases (EC 2.-.-.-) are the largest enzymatic group with 25 proteins, and 18 of these contain the zf-DHHC palmitoyltransferase functional domain (PF01529) which is involved in zinc as well as other ion binding [31]. While there are 23 mammalian DHHC pro- teins identified in the membrane proteome [32], only 18 of them are predicted to have 4TMs. There are two proteins described as hydrolases (EC 3.-.-.-), which use hydrolysis to form two products. Three proteins are lyases (EC 4.-.-.-) and two of these contain the Protein tyrosine phosphatase- like protein domain (PF04387) which functions in very long chain fatty acid biosynthesis [31]. All three lyases are in- volved in a variety of disease conditions. And three proteins are classified as ligases (EC 6.-.-.-) in which all are involved as E3 ubiquitin ligases. Enzymes The Enzyme class includes 45 proteins with a corre- sponding Enzyme Commission (EC) number (Fig. 3). Oxidoreductases (EC 1.-.-.-) that catalyze oxidation/ Fig. 3 The human 4TM Enzyme class. The figure displays the 45 proteins identified with an EC number that belong to the Enzyme class. The enzymes are divided into five groups which correspond to the type of chemical reaction that they catalyze. Uniprot cross-references the ENZYME nomenclature database and IntEnz (Integrated relational Enzyme database) to obtain the EC number associated with a protein. The single protein classified as a drug target as well as the drug indications are displayed. The drug target and drug indications were identified through an updated dataset of all current targeted and potential proteins and genes involved in drug studies or experimentation. Some of the most common gene- disease associations are also shown for each of the subgroups in the Enzyme class. Three different resources were used to identify gene-disease associations: the Online Mendelian Inheritance in Man (OMIM) database; the Functional Disease Ontology (FunDO) resource; and the Jensen Lab Diseases database (see Methods for details). Proteins that are involved in enzymatic activity but do not have an associated EC number are not included in this class, but rather in a subgroup of the Miscellaneous proteins. The number in parenthesis represents the number of proteins that have been identified Fig. 3 The human 4TM Enzyme class. The figure displays the 45 proteins identified with an EC number that belong to the Enzyme class. The enzymes are divided into five groups which correspond to the type of chemical reaction that they catalyze. Uniprot cross-references the ENZYME nomenclature database and IntEnz (Integrated relational Enzyme database) to obtain the EC number associated with a protein. The single protein classified as a drug target as well as the drug indications are displayed. The drug target and drug indications were identified through an updated dataset of all current targeted and potential proteins and genes involved in drug studies or experimentation. Some of the most common gene- disease associations are also shown for each of the subgroups in the Enzyme class. Three different resources were used to identify gene-disease associations: the Online Mendelian Inheritance in Man (OMIM) database; the Functional Disease Ontology (FunDO) resource; and the Jensen Lab Diseases database (see Methods for details). Dual functions Forty-seven proteins are distinguished as having dual functions as either receptor/transporter (46 proteins) or enzyme/transporter (1 protein) (Fig. 4). The complete repertoire of the 4TM neurotransmitter gated ion chan- nel family, also known as the anionic and cationic cys- Enzymes Proteins that are involved in enzymatic activity but do not have an associated EC number are not included in this class, but rather in a subgroup of the Miscellaneous proteins. The number in parenthesis represents the number of proteins that have been identified Attwood et al. BMC Genomics (2016) 17:268 Page 6 of 16 Fig. 4 Proteins identified in the Dual functions class. The figure shows the 47 proteins identified as having specific dual functions. All of the neurotransmitter gated ion channel (NGIC) proteins are presented here, which include 46 members that are characterized by being a transporter and having a TC number (1.A.-.-.-; alpha-type channel) as well as being identified as a receptor. The NGIC are divided into their five different protein families, and the number of proteins identified as drug targets and the drug indications are presented. In addition, one protein is determined to be both an enzyme and transporter and the information is presented in the bottom row of the figure. The drug target and drug indications were identified through an updated dataset of all current targeted and potential proteins and genes involved in drug studies or experimentation. The gene-disease associations for all NGIC proteins are also displayed. Three different resources were used to identify gene-disease associations: the Online Mendelian Inheritance in Man (OMIM) database; the Functional Disease Ontology (FunDO) resource; and the Jensen Lab Diseases database (see Methods for details). The number in parenthesis represents the number of proteins that have been identified Fig. 4 Proteins identified in the Dual functions class. The figure shows the 47 proteins identified as having specific dual functions. All of the neurotransmitter gated ion channel (NGIC) proteins are presented here, which include 46 members that are characterized by being a transporter and having a TC number (1.A.-.-.-; alpha-type channel) as well as being identified as a receptor. The NGIC are divided into their five different protein families, and the number of proteins identified as drug targets and the drug indications are presented. In addition, one protein is determined to be both an enzyme and transporter and the information is presented in the bottom row of the figure. The drug target and drug indications were identified through an updated dataset of all current targeted and potential proteins and genes involved in drug studies or experimentation. The gene-disease associations for all NGIC proteins are also displayed. Enzymes Three different resources were used to identify gene-disease associations: the Online Mendelian Inheritance in Man (OMIM) database; the Functional Disease Ontology (FunDO) resource; and the Jensen Lab Diseases database (see Methods for details). The number in parenthesis represents the number of proteins that have been identified loop receptor group, includes all 45 proteins that belong to four different families as well as the single zinc activated ligand-gated ion channel protein [SwissProt: Q401N2]. The four families include gamma-aminobutryic- acid (GABAA), glycine, 5-hydroxytryptamine-3 (5HT3), and acetylcholine (nAChR) receptors and each protein contains the conserved neurotransmitter ligand binding domain (PF02931) as well as the ion channel domain (PF02932). All 46 NGIC proteins are described with a TC number as well as being identified as a receptor. In addition, these proteins are characterized by extracellular N- and C-termini. Thirty-five of the proteins are identified as drug targets which include a range of indications, as shown in Fig. 4. The common disease conditions include neurological diseases such as epilepsy, autistic disorder, schizophrenia, Alzheimer’s disease, various dependencies, as well as different cancers. loop receptor group, includes all 45 proteins that belong to four different families as well as the single zinc activated ligand-gated ion channel protein [SwissProt: Q401N2]. The four families include gamma-aminobutryic- acid (GABAA), glycine, 5-hydroxytryptamine-3 (5HT3), and acetylcholine (nAChR) receptors and each protein contains the conserved neurotransmitter ligand binding domain (PF02931) as well as the ion channel domain (PF02932). All 46 NGIC proteins are described with a TC number as well as being identified as a receptor. In addition, these proteins are characterized by extracellular N- and C-termini. Thirty-five of the proteins are identified as drug targets which include a range of indications, as shown in Fig. 4. The common disease conditions include neurological diseases such as epilepsy, autistic disorder, schizophrenia, Alzheimer’s disease, various dependencies, as well as different cancers. Uncharacterized proteins Thirty-two of the Miscellaneous proteins have an uncharacterized function and twenty-four of these pro- teins have a recognized Pfam domain. Within the uncharacterized proteins, there are six interesting clus- ters of sequences: there are two sets that each have three members with the same conserved domain (PF04103 and PF05805); three sets of proteins with two members each that have the same domain (PF05255, PF14967, and PF10269); and a sixth group with two proteins that do not contain a conserved domain, but share ~38 % se- quence similarity and belong to the same protein family. (See Fig. 6 for details). In addition, there are three pro- teins that have possible homologue hits through BLASTP searches and two proteins that have conserved domains that were identified through the Conserved do- main database; the Reticulon domain and the Claudin 2 superfamily, which contains the claudins. Several of the clusters and homologue hits are discussed below, be- cause while the uncharacterized proteins do not have any associated function ascribed to them through GO terms, valuable information was gleaned through litera- ture searches. Two of the Miscellaneous proteins are identified as drug targets. One is Gap junction alpha 1 protein [Swis- sProt: P17302], which is a connexin subunit and plays a possibly critical role in hearing and in bladder functional capacity [28]. It is being investigated for use as a wound healing accelerant [35]. The other identified drug target is Leukocyte antigen CD37 [SwissProt: P11049] – a tet- raspanin that is involved in negative regulation of cell proliferation and positive regulation of immunoglobulin production, amongst other functions [28]. This protein Fig. 5 The functional subgroups of the Miscellaneous class. The graph displays the breakdown of the larger functional subgroups that contain 158 of the 213 proteins that belong to the Miscellaneous class, as well as the remaining 55 proteins under Various functions. The subgroups were determined by individually examining GO (general) and GO (molecular) terms as well as Pfam domain and protein family descriptions to categorize proteins with similar functions. Many proteins participate in multiple activities, and the subgroups are meant to highlight some of the main functional tasks within the dataset and how many proteins are involved with them. The Transport activity column includes the four subgroups transport activity, vesicle mediated transport, chemotaxis, and channel activity and totals 30 proteins altogether. Receptors The two proteins identified solely as receptors each be- long to different receptor types. The first receptor is the high affinity immunoglobin epsilon receptor subunit beta [SwissProt: Q01362] and is a member of the IgE receptors, which combine with specific molecular sites on the surface of B- and T-lymphocytes. This protein is also identified as a drug target and is used as an anti- asthmatic agent. The second receptor is the type-1 angiotensin II receptor-associated protein [SwissProt: Q6RW13] and is a cell surface protein that binds angio- tensins, which cause vasoconstriction that increases blood pressure, and activates intracellular changes that influence cell behavior. The Receptor class was identified using manual screening with two resources: the Medical Subject Headings (MeSH) [33] database and IUPHAR/ BPS: Guide to Pharmacy [34]. Attwood et al. BMC Genomics (2016) 17:268 Attwood et al. BMC Genomics (2016) 17:268 Page 7 of 16 Miscellaneous is being targeted as a possible anti-neoplastic agent in cancer treatment. Many of the 213 proteins classified as Miscellaneous are multi-functional and involved in a variety of activities. Fourteen of the Miscellaneous proteins have signal pep- tides predicted and five proteins have extracellular ter- minal locations predicted. Fig. 5 shows several of the larger subgroups of proteins with similar functions that have been categorized using GO terms. The subgroups are meant to show the broad activities within the dataset and present some of the main functional tasks and how many proteins are involved in them. These functional subgroups can be composed of a single protein family, for example all 15 proteins associated with gap junctions are connexins, or be characterized by several different families, such as the 25 proteins involved in cell adhe- sion (in which 22 are claudins and 3 are tetraspanins). Uncharacterized proteins The Receptor-like activity column includes both the signaling and regulation subgroups and totals 36 proteins as well Fig. 5 The functional subgroups of the Miscellaneous class. The graph displays the breakdown of the larger functional subgroups that contain 158 of the 213 proteins that belong to the Miscellaneous class, as well as the remaining 55 proteins under Various functions. The subgroups were determined by individually examining GO (general) and GO (molecular) terms as well as Pfam domain and protein family descriptions to categorize proteins with similar functions. Many proteins participate in multiple activities, and the subgroups are meant to highlight some of the main functional tasks within the dataset and how many proteins are involved with them. The Transport activity column includes the four subgroups transport activity, vesicle mediated transport, chemotaxis, and channel activity and totals 30 proteins altogether. The Receptor-like activity column includes both the signaling and regulation subgroups and totals 36 proteins as well Attwood et al. BMC Genomics (2016) 17:268 Page 8 of 16 Fig. 6 Uncharacterized proteins. This figure presents the six sets of uncharacterized proteins that have multiple members with the same conserved Pfam domain or high sequence similarity. In addition, five uncharacterized proteins that have either conserved domains or hits through BLASTP searches are also shown Fig. 6 Uncharacterized proteins. This figure presents the six sets of uncharacterized proteins that have multiple members with the same conserved Pfam domain or high sequence similarity. In addition, five uncharacterized proteins that have either conserved domains or hits through BLASTP searches are also shown TMEM50A and TMEM50B [SwissProt: O95807 and P56557] both contain the UPF0220 Pfam domain (PF05255) and are the sole members of the TMEM50 protein family. TMEM50A is located on chromosome 1p36.11 in the RH gene locus, between the RHD and RHCE genes, where its position may be linked to RH haplotypes and contribute to selective pressures regarding certain RH haplotypes [36, 37]. According to the Gene Expression Omnibus (GEO) [38], TMEM50A appears to be highly upregulated in late stage cervical cancer in comparison to normal cells. In a study that assesses biomarkers to investigate the etiology of Down syndrome, TMEM50B was found to be upregulated two fold in Down syndrome samples compared to normal [39]. located on chromosome 3. Only one gene, TMEM14C, has any associated function – heme biosynthetic process activity; all others have yet to be characterized. Uncharacterized proteins However, in yeast TMEM14A stabilizes the mitochondrial membrane potential and inhibits retinamide-induced apoptosis [41]. Neither TMEM179 nor TMEM179B [SwissProt: Q6ZVK1 and Q7Z7N9] contain a conserved Pfam domain but they share ~38 % sequence identity and belong to the same protein family. No function could be found asso- ciated with these proteins, however an article from 2009 [42] identifies TMEM179, which is located on chromosome 14q32.33, as an ‘evolutionary breakpoint’ region. This region is repeat rich and ‘reused’ during karyotypic evolution in that breakpoint features that are retained may have predisposed these genomic re- gions to large scale chromosomal instability. TMEM14B [SwissProt: Q9NUH8] is a member of the TMEM14 protein family which includes TMEM14A, TMEM14B, TMEM14C, TMEM14D (which is a pseudo- gene), and TMEM14E, of which TMEM14B and TMEM14C are predicted as 4TMs. According to a recent 2015 article [40], NMR shows that 4TMs are predicted in TMEM14A and TMEM14C, but only 3 TMs span the en- tire membrane. Perhaps this contributes to TMEM14C as one of the few proteins with the N-terminus predicted to be located in the extracellular environment. TMEM14A/B and C are located on chromosome 6 and TMEM14E is TMEM203 and TMEM60 [SwissProt: Q969S6 and Q9H2L4] both contain the Pfam domain TMEM185A (PF10269), which is also conserved in the proteins TMEM185A and TMEM185B (neither of these are pre- dicted as 4TM). This domain has been identified to be in- volved with the Fragile-X syndrome through the protein TMEM185A [43], however TMEM203 and TMEM60 share only ~10 % identity to the TMEM185A/B proteins and ~21 % identity between each other. In a recent Attwood et al. BMC Genomics (2016) 17:268 Page 9 of 16 belong to any clan and thus do not have any identified homologous sister families. publication, the previously uncharacterized TMEM203 protein was described as an evolutionarily conserved regulator of intracellular calcium levels that is required for spermatogenesis [44]. The largest clan, the Transport superfamily clan, comprises 78 proteins from six different conserved Pfam domain families. The six families within this clan include: the aforementioned connexin domain (PF00029, 21 proteins); PMP22_claudin (PF00822, 37 proteins); Claudin_2 (PF13903, 10 proteins); L_HGMIC_fpl (PF10242, 5 proteins); GSG-1 (PF07803, 2 proteins); and innexin (PF00876, 2 proteins). The predominant functions include cell adhesion, transporter activity, regulation, and cell communication via gap junctions. Major Pfam families and clans within 4TM dataset Major Pfam families and clans within 4TM dataset There are eight major Pfam domain families and clans within the dataset that contain between 5–81 members (see Fig. 7): Transport superfamily clan (78 proteins), Tetraspanin-like clan (53 proteins), Marvel-like clan (24 proteins), Zinc beta ribbon clan (18 proteins), NGIC family (46 proteins), Reticulon family (7 proteins), L6- membrane family (6 proteins), and Got1/Sft2-like family (5 proteins). The clans are composed of homologous do- main families and include the unique four-transmembrane protein families such as claudins, connexins and tetraspa- nins. The domain families are formed from the collection of proteins with the same conserved Pfam domain, such as the 46 members of the NGIC family, and which do not The 24 proteins that contain the MARVEL domain belong to the Marvel-like clan. Five of the MARVEL proteins are classified as transporters while others have shown association with specialized membrane microdo- mains (rafts) that could be involved in cholesterol-rich membrane apposition events in cellular processes such as biogenesis of vesicular transport carriers or tight junction regulation [47]. The eighteen proteins in the Zinc beta ribbon clan contain the zf-DHHC domain Fig. 7 Functional classification breakdown within the 4TM major clans and families. The figure displays the breakdown of how many proteins within each major clan and family are identified for each functional class, i.e. Transporter, Enzyme, Dual function, Receptor, and Miscellaneous classes. Approximately 61 % of the dataset is described by these eight clans and families. The number in parenthesis corresponds to the total number of proteins within that clan or family Fig. 7 Functional classification breakdown within the 4TM major clans and families. The figure displays the breakdown of how many proteins within each major clan and family are identified for each functional class, i.e. Transporter, Enzyme, Dual function, Receptor, and Miscellaneous classes. Approximately 61 % of the dataset is described by these eight clans and families. The number in parenthesis corresponds to the total number of proteins within that clan or family Fig. 7 Functional classification breakdown within the 4TM major clans and families. The figure displays the breakdown of how many proteins within each major clan and family are identified for each functional class, i.e. Transporter, Enzyme, Dual function, Receptor, and Miscellaneous classes. Approximately 61 % of the dataset is described by these eight clans and families. Uncharacterized proteins FAM189A1, FAM189B, and TMEM212 [SwissProt: O60320, P81408, and A6NML5] each contain the CD20 domain (PF04103), which is a member of the Tetraspanin- like clan. All three are uncharacterized, however FAM189B has been shown to possibly interact with the WW domain binding and be involved in protein binding [45]. Addition- ally, the three proteins TM4SF18, TM4SF19, and TM4SF20 [SwissProt: Q96CE8; Q96DZ7; Q53R12] all contain the L6_membrane domain (PF05805), which has unknown functions. However, there are three other proteins in the TM4SF protein family with the L6 domain and two are involved in regulation and signaling while the third is a transporter. The Tetraspanin-like clan is the second largest clan with 53 proteins and is composed of two families: the ‘true’ tetraspanin domain family (PF00335, 33 proteins) and the CD20 domain family (PF04103, 20 proteins). The ‘true’ tetraspanins are involved in transport activity and have also been identified as building tetraspanin enriched domains (TEMs) that facilitate protein scaffolding and as- sembly of specialized complexes [26]. The other domain family, CD20, appears to be cell-surface proteins that are involved in important cell regulation and differentiation activities, as well as possibly facilitate intracellular protein- protein interactions [46]. Major Pfam families and clans within 4TM dataset those that completely span the membrane, they do not take into account anomalies such as reentrant loops, short breaks in helices, and helices that lie along the surface of the membrane [8]. As mentioned previously with TMEM14C, this method limitation could possibly affect the topology prediction. For example, those 11 proteins that have predicted extracellular orientation (excluding the NGIC family) might be interesting proteins to study for trans- membrane structural purposes. Examples of common structures and topologies for each functional class are represented in Fig. 8, which highlights conserved fea- tures and N- and C-terminus orientations found in the 4TM dataset. For example, claudins, tetraspanins, and connexins categorized in the Miscellaneous class are shown with the four conserved membrane regions, the intracellular location of the N- and C-termini, and also conserved cysteine residues that are often found in the extracellular loops and are involved in forming disul- phide bonds. Some members of these homologous families have been well-studied; however there are others that are still not fully described. For example, of the 27 members of the claudin family, only limited functional knowledge is available on at least 13 of the proteins: Claudins 6, 9, 12, 13, 18, and 20–27 [48]. Additionally, meaningful GO functional terms are lacking for at least five proteins of the ‘true’ tetraspanins (Tspan11, Tspan13, Tspan16, Tspan18, and Tspan19), as well as at least eleven pro- teins that contain the CD20 domain and are members of the MS4A protein family which is involved in important cell regulation and differentiation activities. Disease-gene and cancer associations and drug targets There are 215 genes identified with disease conditions. Various types of cancers, such as lung, colon, breast, and liver, are the most common diseases identified with 72 genes recognized in association with them. Many of the genes are described in multiple disease conditions, as can be expected from the critical functional activities that membrane proteins participate in. There are 14 genes associated with epilepsy, 13 genes with schizo- phrenia, and 13 with autism. Conditions involving deaf- ness are associated with 12 genes; 10 genes are involved with nicotine dependence; diabetes, Alzheimer’s disease and alcohol dependence each have 7 genes identified with them. As NGIC receptors are involved in a plethora of neurological disease conditions, it is not surprising that disease conditions such as schizophrenia, autism, Alzheimer’s and dependence/addiction are heavily repre- sented. Major Pfam families and clans within 4TM dataset The number in parenthesis corresponds to the total number of proteins within that clan or family Attwood et al. BMC Genomics (2016) 17:268 Page 10 of 16 Page 10 of 16 Page 10 of 16 however the presence of a signal peptide can influence dif- ferent orientations. Signal peptides are short sequences of amino acid residues attached to the N-terminus domain that target the protein to the membrane and are then subsequently removed by proteolysis post membrane insertion. In addition, when using transmembrane pro- tein prediction methods, it is important to assess and excise signal peptides from sequences as otherwise they can be mistaken as transmembrane helices due to the hydrophobicity of the peptide sequence [49]. The re- sults of the TOPCONS-single topology predictions for this dataset include 316 proteins with the N- and C- termini located within the lumen of the membrane and 57 proteins with the terminals in the outside environment. The complete NGIC group, which compose virtually all of the Dual function class with 46 proteins, have the N- and C-termini located in the extracellular environment. The extracellular N-terminus is consistent with the other large receptor groups, i.e., the 1TMs and 7TMs, however the C- terminal is usually located in the intracellular environment due to the important activities it is typically involved in, particularly signaling transduction. Additionally, 40 of the NGIC proteins are predicted from the SignalP signal pep- tide prediction software to have signal peptides. In total there are 60 proteins that are predicted to have signal pep- tides. As current transmembrane protein prediction methods are based on classical helical structures, i.e. those that completely span the membrane, they do not take into account anomalies such as reentrant loops, short breaks in helices, and helices that lie along the surface of the membrane [8]. As mentioned previously with TMEM14C, this method limitation could possibly affect the topology prediction. For example, those 11 proteins that have predicted extracellular orientation (excluding the NGIC family) might be interesting proteins to study for trans- membrane structural purposes. Examples of common structures and topologies for each functional class are represented in Fig. 8, which highlights conserved fea- tures and N- and C-terminus orientations found in the 4TM dataset. Discussion This consensus topology based screening of 4TMs followed by manual curation presents the most robust and updated dataset of the 4TM complement of the hu- man proteome. Our initial screening of the human proteome predicts 555 sequences with four membrane- spanning helices and this estimate is comparable with Major Pfam families and clans within 4TM dataset Additional file 1 includes the complete gene- disease association descriptions. There are 44 proteins identified as a current targeted or potential protein that is involved in drug studies or experimentation, with 35 of them identified in the NGIC family. Major Pfam families and clans within 4TM dataset For example, claudins, tetraspanins, and connexins categorized in the Miscellaneous class are shown with the four conserved membrane regions, the intracellular location of the N- and C-termini, and also conserved cysteine residues that are often found in the extracellular loops and are involved in forming disul- phide bonds. and act as transferases. The 46 members of the NGIC family has been discussed previously and the seven pro- teins in the Reticulon family are involved in activities such as transport and regulation as well as the apop- totic process, but one of them is also completely uncharacterized. The last two domain families include the six proteins of the L6-membrane family in which several are involved in transport and regulation, how- ever three of them are also completely uncharacterized, and the five proteins of the Got/Sft2-like proteins that function in vesicle mediated transport. however the presence of a signal peptide can influence dif- ferent orientations. Signal peptides are short sequences of amino acid residues attached to the N-terminus domain that target the protein to the membrane and are then subsequently removed by proteolysis post membrane insertion. In addition, when using transmembrane pro- tein prediction methods, it is important to assess and excise signal peptides from sequences as otherwise they can be mistaken as transmembrane helices due to the hydrophobicity of the peptide sequence [49]. The re- sults of the TOPCONS-single topology predictions for this dataset include 316 proteins with the N- and C- termini located within the lumen of the membrane and 57 proteins with the terminals in the outside environment. The complete NGIC group, which compose virtually all of the Dual function class with 46 proteins, have the N- and C-termini located in the extracellular environment. The extracellular N-terminus is consistent with the other large receptor groups, i.e., the 1TMs and 7TMs, however the C- terminal is usually located in the intracellular environment due to the important activities it is typically involved in, particularly signaling transduction. Additionally, 40 of the NGIC proteins are predicted from the SignalP signal pep- tide prediction software to have signal peptides. In total there are 60 proteins that are predicted to have signal pep- tides. As current transmembrane protein prediction methods are based on classical helical structures, i.e. Topology dd In addition to the number of membrane spanning helices, the orientations of the N- and C-termini are important factors in determining the functional activity of the pro- tein. The terminal orientations are usually determined by the initial insertion of the peptide into the membrane, Attwood et al. BMC Genomics (2016) 17:268 Page 11 of 16 Fig. 8 (Parts a, b, c, d, and e). Common topologies and conserved features within the 4TM dataset. Part a The Miscellaneous class includes proteins that have been characterized into subgroups through similar functional activities. Common features include intracellular termini and conserved cysteine residues (yellow outlined in red ovals) in the extracellular loops that either engage in forming disulphide bonds (e.g. claudins and tetraspanins) or interact and form bonds with other proteins (i.e. connexins). Tetraspanins have 4-6 conserved cysteine residues as well as a conserved CCG (cysteine-cysteine-glycine) motif in the large extracellular loop 2. Part b The example here, MS4A2, is one of the two identified receptors and a member of the MS4A protein family, in which 16 members are characterized by 4TMs, a CD20 domain, and an in N-terminus. Part c The Transporter class includes 66 proteins, of which 65 have an in N-terminus and conserved cysteine residues in the extracellular loops are common. TMEM205 is the sole transporter with the opposite topology, and is interesting due to its use of novel mechanisms in cisplatin (chemotherapy) resistance [82]. Part d Of the 45 Enzyme class proteins, all except five maintain an N-terminus intracellular location. ZDHHC-3 is a typical protein of the ZDHHC protein family, characterized by 4TMs, a conserved DHHC domain, and a conserved DPG (aspartate-proline-glycine) motif as well as a TTxE (threonine-threonine-asparagine-glutamate) motif. Part e The Dual function class contains 47 proteins and all 46 proteins of the neurotransmitter gated ion channel family (NGIC) are included here. The NGIC family has a long extracellular N-terminus that contains several important binding sites as well as two conserved cysteine residues that participate in disulphide bonds. The NGIC family is unique in that it has extracellular N- and C-termini and also has signal peptides predicted in 40 of the proteins Fig. 8 (Parts a, b, c, d, and e). Common topologies and conserved features within the 4TM dataset. Part a The Miscellaneous class includes proteins that have been characterized into subgroups through similar functional activities. Topology dd Part c The Transporter class includes 66 proteins, of which 65 have an in N-terminus and conserved cysteine residues in the extracellular loops are common. TMEM205 is the sole transporter with the opposite topology, and is interesting due to its use of novel mechanisms in cisplatin (chemotherapy) resistance [82]. Part d Of the 45 Enzyme class proteins, all except five maintain an N-terminus intracellular location. ZDHHC-3 is a typical protein of the ZDHHC protein family, characterized by 4TMs, a conserved DHHC domain, and a conserved DPG (aspartate-proline-glycine) motif as well as a TTxE (threonine-threonine-asparagine-glutamate) motif. Part e The Dual function class contains 47 proteins and all 46 proteins of the neurotransmitter gated ion channel family (NGIC) are included here. The NGIC family has a long extracellular N-terminus that contains several important binding sites as well as two conserved cysteine residues that participate in disulphide bonds. The NGIC family is unique in that it has extracellular N- and C-termini and also has signal peptides predicted in 40 of the proteins Transporters make up the largest category of 4TMs and consist of 37 % of the dataset, with 17 % identified solely in the Transporter class, an additional 12 % in the Dual function category, and 8 % more in the Miscellan- eous class that are members of subgroups such as trans- port activity, vesicle mediated transport, channel activity, and chemotaxis (see Fig. 5). In the entire human mem- brane proteome [5], transporters compose approximately 15 % of the proteins, which shows that the 4TM dataset contains more than double that amount proportionally. And when comparing against the 6TM protein set, an- other membrane group known to be involved in transport activity, roughly 30 % of that group of proteins function in transport. Comparatively, 23 % in total of 4TM proteins are involved in receptor activity and 13 % function in en- zymatic activity, which is commensurate to the roughly 25 % of receptors and 10 % of enzymes found in the entire membrane proteome. This might suggest that the four- transmembrane topological architecture is more favored for transporting molecules than over other functions. the human tissue-based Protein Atlas database that cur- rently predicts 554 sequences with four-transmembrane regions [50]. Topology dd Common features include intracellular termini and conserved cysteine residues (yellow outlined in red ovals) in the extracellular loops that either engage in forming disulphide bonds (e.g. claudins and tetraspanins) or interact and form bonds with other proteins (i.e. connexins). Tetraspanins have 4-6 conserved cysteine residues as well as a conserved CCG (cysteine-cysteine-glycine) motif in the large extracellular loop 2. Part b The example here, MS4A2, is one of the two identified receptors and a member of the MS4A protein family, in which 16 members are characterized by 4TMs, a CD20 domain, and an in N-terminus. Part c The Transporter class includes 66 proteins, of which 65 have an in N-terminus and conserved cysteine residues in the extracellular loops are common. TMEM205 is the sole transporter with the opposite topology, and is interesting due to its use of novel mechanisms in cisplatin (chemotherapy) resistance [82]. Part d Of the 45 Enzyme class proteins, all except five maintain an N-terminus intracellular location. ZDHHC-3 is a typical protein of the ZDHHC protein family, characterized by 4TMs, a conserved DHHC domain, and a conserved DPG (aspartate-proline-glycine) motif as well as a TTxE (threonine-threonine-asparagine-glutamate) motif. Part e The Dual function class contains 47 proteins and all 46 proteins of the neurotransmitter gated ion channel family (NGIC) are included here. The NGIC family has a long extracellular N-terminus that contains several important binding sites as well as two conserved cysteine residues that participate in disulphide bonds. The NGIC family is unique in that it has extracellular N- and C-termini and also has signal peptides predicted in 40 of the proteins Fig. 8 (Parts a, b, c, d, and e). Common topologies and conserved features within the 4TM dataset. Part a The Miscellaneous class includes proteins that have been characterized into subgroups through similar functional activities. Common features include intracellular termini and conserved cysteine residues (yellow outlined in red ovals) in the extracellular loops that either engage in forming disulphide bonds (e.g. claudins and tetraspanins) or interact and form bonds with other proteins (i.e. connexins). Tetraspanins have 4-6 conserved cysteine residues as well as a conserved CCG (cysteine-cysteine-glycine) motif in the large extracellular loop 2. Part b The example here, MS4A2, is one of the two identified receptors and a member of the MS4A protein family, in which 16 members are characterized by 4TMs, a CD20 domain, and an in N-terminus. Topology dd Both of these 4TM prediction datasets include all protein products (such as isoforms), and the Protein Atlas database also uses a majority decision method but with seven different prediction methods based on the Ensembl gene annotation. Our protein dataset con- sists of 373 members after manual curation and also appending 40 selected proteins, which represents 7 % of the ~5550 human membrane proteome. This dataset con- tains 69 more predicted proteins that what was previously estimated in our human membrane proteome, which also accounted for a single protein product from each gene, obtained from the IPI human version 3.39 in 2009. The different number of predicted proteins might be attributed to our manual curation process, as well as different num- ber and types of prediction methods and different gene annotation sources. There are 341 proteins characterized in the dataset, which amounts to approximately 91 % of the proteins having an associated description. the human tissue-based Protein Atlas database that cur- rently predicts 554 sequences with four-transmembrane regions [50]. Both of these 4TM prediction datasets include all protein products (such as isoforms), and the Protein Atlas database also uses a majority decision method but with seven different prediction methods based on the Ensembl gene annotation. Our protein dataset con- sists of 373 members after manual curation and also appending 40 selected proteins, which represents 7 % of the ~5550 human membrane proteome. This dataset con- tains 69 more predicted proteins that what was previously estimated in our human membrane proteome, which also accounted for a single protein product from each gene, obtained from the IPI human version 3.39 in 2009. The different number of predicted proteins might be attributed to our manual curation process, as well as different num- ber and types of prediction methods and different gene annotation sources. There are 341 proteins characterized in the dataset, which amounts to approximately 91 % of the proteins having an associated description. Page 12 of 16 Attwood et al. BMC Genomics (2016) 17:268 Page 12 of 16 Page 12 of 16 Attwood et al. BMC Genomics (2016) 17:268 Intriguingly, in our topology analysis we found an im- portant exception to the ubiquitous membrane prote- ome intracellular locale of the C-terminus in the NGIC group that performs dual functions; all 46 members have extracellular N- and C-termini. And concomitantly, 40 of the 46 NGIC proteins are predicted to have signal peptides. Conclusions In conclusion, we have functionally classified and manually curated the 4TM complement of the human proteome, which is characterized by four-transmembrane helices and the majority of proteins containing conserved N- and C- termini intracellular location. This examination of the 4TM structural group and related functions shows the ubiquitous transport activity that is unique in a membrane group with such few transmembrane helices. While 4TM proteins are not necessarily identified as classical receptors and trans- porters, we show they are still heavily involved in these ac- tivities as well as cell communication, cell adhesion, and working as scaffolding and structural elements. This has led to perhaps an oversight in pharmacological research efforts. As this detailed characterization of the 4TM dataset and the associated gene-disease information exhibit, these proteins participate in a host of important activities and it is becoming more apparent that they are also involved in various disease conditions including cancer and neuro- logical conditions. Further, this dataset highlights particu- lar 4TM proteins that are waiting to be studied in connection to diseases and as possible drug targets. An interesting aspect of the functional classification of the 4TM dataset is that 61 % of the proteins can be de- scribed by 8 large Pfam families or clans, containing a range from 5 to 81 members. Fig. 7 depicts the eight clans and domain families and displays the number of proteins within each functional class for each clan or family. Furthermore, there are only 12 proteins that do not contain any type of conserved functional domain, so up to 97 % of the 4TM dataset contains conserved features that are found in homologous domains within other pro- teins. As described in the results, some members of these homologous families have been well-studied, however many others have still not been investigated and function is inferred through homology. Additionally, this evaluation elucidates six groups (14 proteins) that have uncharacter- ized functional activity and that share not only conserved sequence features but also a similar 4TM structure (see Fig. 6). These groups of proteins indicate interesting avenues of future investigation. Topology dd It has been shown [8] that the prokaryotic 2TM glutamate receptor is an example of where the addition of a cleavable signal peptide can influence the terminal orientations of proteins that originally had a cytoplasmic N-terminus, particularly with proteins with a fewer number of transmembrane helices. The addition of a signal peptide can cause reorientation of the termini ends through causing the translocation of the N- terminus to the outside environment. When the signal peptide is subsequently cleaved, the location of the ter- minal is in the extracellular region [51]. Additionally, the NGIC family possesses a long (~200 amino acids) N- terminus tail which contributes to a stably folded ligand binding domain [52, 53]. As shown in the 7TM GPCR families, signal peptides are more common in those pro- teins that contain those two factors, i.e. a long N tail and an N-terminus that engages in stabilizing the ligand binding domain [54, 55]. This set of factors regarding the signal peptides and terminal orientations are inter- esting components that could possibly contribute to the evolution of the NGIC topology orientations. had 17 % of their proteins identified with cancer. Part of this might be accounted for by more research on proteins involved in transport activities. The involvement of 4TM proteins in different types of cancer and other disease con- ditions point toward the importance of this less explored class of transmembrane proteins and it may be possible to further utilize this class of transmembrane proteins as drug targets. Generating the initial 4TM dataset The predicted four-transmembrane sequences were re- trieved and sorted by gene identification (Ensembl gene identification) and sequence length. The dataset was re- duced by retaining the longest sequence per gene from the predicted sequences. At this juncture, the dataset possibly includes isoforms of proteins as the predicted 4TM sequence may not necessarily be the canonical sequence of that gene. The dataset was then re-evaluated with TOPCONS-single again to attempt to mitigate false- positive proteins and obtain as accurate dataset as possible, and the final non-redundant predicted 4TM dataset was produced. transmembrane prediction web server to assess membrane topology including the number of membrane-spanning helices and orientation of the terminal ends [57]. As pre- diction methods use different algorithms to discriminate transmembrane helices, the number of alpha helices pre- dicted by each method varies. One manner to improve the accuracy in membrane prediction and is to use several prediction methods and make a consensus or majority decision regarding topology. TOPCONS-single uses a consensus decision method that comprises five different prediction tools: SCAMPI-single [58]; S-TMHMM [59]; MEMSAT 1.0 [60]; HMMTOP [61]; and Phobius [62]. The Benchmark of membrane helix predictions from se- quence website was used to assess the accuracy of the methods used in TOPCONS-single, and the percentage accuracy for which all membrane helices were correctly predicted for the methods ranged from 56–72 % [63], which is comparable to the TOPCONS-single published benchmark results (51–73 %) [57]. The initial membrane topology was performed using all five prediction methods. The predicted four-transmembrane sequences were re- trieved and sorted by gene identification (Ensembl gene identification) and sequence length. The dataset was re- duced by retaining the longest sequence per gene from the predicted sequences. At this juncture, the dataset possibly includes isoforms of proteins as the predicted 4TM sequence may not necessarily be the canonical sequence of that gene. The dataset was then re-evaluated with TOPCONS-single again to attempt to mitigate false- positive proteins and obtain as accurate dataset as possible, and the final non-redundant predicted 4TM dataset was produced. the most prevalent, the most similar to orthologous se- quences, the properties of the amino acid composition, or in lieu of nothing else, then the longest sequence. The se- quence status is defined as either complete or fragment, in which the canonical sequence is missing amino acid resi- dues, often in the initial or terminal ends. Generating the initial 4TM dataset A two-step analysis process was used: in silico transmem- brane prediction (Fig. 1a) followed by manual curation and classification. The Homo sapiens genome assembly GRCh38 with genome annotation GenCode v21 transla- tions was downloaded from GenCode [56] on February 10, 2015. The genome annotation file contained 93,139 gene products from 19,881 protein-coding genes. As transmembrane protein prediction methods have difficulty differentiating between N-terminal alpha helices and cleavable signal peptides, the genome annotation file was evaluated with a standalone version of SignalP 4.1 software [49]. SignalP uses a neural network based method to distinguish between N-terminal helices and signal peptides. The parameters used were: eukaryotic organism, default minimum signal peptide length of 10aa, the best method was chosen which designated TM regions might be present, and the default cutoff value of 0.45. The mature sequences with the signal peptides excised were then evaluated with TOPCONS-single Overall, we estimate that 58 % of the genes in our 4TM dataset are identified as being involved in various disease conditions and roughly 19 % of the genes are possibly involved in different types of cancer. It is interest- ing to note that proportionally the Transporter class has a higher percentage of genes that are possibly involved in cancer, with 29 % of the proteins in that class identified. In comparison, the Miscellaneous and Enzyme classes both Page 13 of 16 Page 13 of 16 Page 13 of 16 Attwood et al. BMC Genomics (2016) 17:268 transmembrane prediction web server to assess membrane topology including the number of membrane-spanning helices and orientation of the terminal ends [57]. As pre- diction methods use different algorithms to discriminate transmembrane helices, the number of alpha helices pre- dicted by each method varies. One manner to improve the accuracy in membrane prediction and is to use several prediction methods and make a consensus or majority decision regarding topology. TOPCONS-single uses a consensus decision method that comprises five different prediction tools: SCAMPI-single [58]; S-TMHMM [59]; MEMSAT 1.0 [60]; HMMTOP [61]; and Phobius [62]. The Benchmark of membrane helix predictions from se- quence website was used to assess the accuracy of the methods used in TOPCONS-single, and the percentage accuracy for which all membrane helices were correctly predicted for the methods ranged from 56–72 % [63], which is comparable to the TOPCONS-single published benchmark results (51–73 %) [57]. The initial membrane topology was performed using all five prediction methods. Generating the initial 4TM dataset Those protein sequences identified as fragments were considered invalid proteins and culled from the dataset. To reduce false- positive predictions from TOPCONS-single, proteins that were identified in literature or database sources as having less than or greater than four-transmembrane segments were also removed from the dataset. The initial gene annotation source, GenCode, has ~1050 more protein-coding gene entries than the most conserva- tive annotation resource, the Consensus Coding Sequence dataset (CCDS) [64]. Therefore the CCDS identifier was used to assess the validity of each protein, i.e. that each has acceptable transcriptional support and recognized protein-coding annotation. Additionally, the sequence sta- tus, CCDS identifier, and sequence length were used to ensure that the predicted protein was the main (or canon- ical) protein product of the gene and not an isoform. There are eight predicted proteins in the dataset that do not have a CCDS identifier associated with them. Five of these have annotation support through RefSeq [65], Vega [66], UCSC, and Ensembl [67] and the authors have chosen to retain them in the dataset. Two genes are iden- tified as pseudogenes (Gje1 and Cyp2d7) however they ap- pear to be capable of some functional activity and were retained in the dataset: while Gje1 does not form func- tional gap junction channels, it causes enhanced ATP re- lease from HeLa cells, and with two SNPs Cyp2d7 may result in an open reading frame with a protein-coding product [65]. Unc93b1 was discussed in our previous examination of the membrane proteome [5] and has been evaluated in literature as having multiple functions [68] with truncated isoforms and thus could be an important protein to include. Manual curation Determining the unambiguous 4TM proteins The manual classification process included four steps, which are highlighted in Fig. 1B. Universal protein re- source (Uniprot) [28] was used to access annotation in- formation on the dataset by using the unique Ensembl protein identification associated with each protein. Uni- prot is a large comprehensive repository of protein se- quences with manually curated as well as automatically generated associated annotations. For each protein, the associated UniProt annotations were used in the cur- ation process: gene name, sequence status, review status, Consensus Coding Sequence identifier, Transporter Clas- sification number, Enzyme Commission number, Pfam domain information, Gene Ontology annotation terms, and protein family information. The functional domains and families for each sequence were obtained through cross-referencing the Pfam data- base. The Pfam [69], InterPro [31], and ProSite [70] data- bases as well as appropriate literature were used to assess the characteristics and number of transmembrane helices of each Pfam domain associated with each protein. Pro- teins that contained a domain that had evidence of more than four-transmembrane helices, and thus the predicted 4TM protein contained an incomplete functional domain, were also discarded from the dataset. The remaining pro- teins compose the 4TM unambiguous dataset. The Uniprot sequence information is derived from translated sequences that have been submitted to the International Nucleotide Sequence Database Collabor- ation (INSDC), which is EMBL-bank, GenBank, and DDBJ. The canonical sequence is determined from either Determining gene-disease associations and identifying drug targets g Three different resources were utilized to investigate gene-disease associations within the dataset. The Online Mendelian Inheritance in Man (OMIM) [76] database was cross-referenced through UniProt and the associated annotations for each protein were downloaded. OMIM contains a catalogue of genetic traits and disorders with referenced overviews on all known Mendelian disorders. The Functional Disease Ontology (FunDO) [77] resource incorporates Disease Ontology terms, which identifies gene-disease associations, but has a simplified vocabulary list called DOLite to enable more interpretable results [78]. The DOLite gene-disease mapping file was down- loaded and gene names within the dataset were searched. The third resource was the Diseases database [79] which incorporates disease-gene associations from automatic text mining, manually curated literature, cancer mutation data, and genome-wide association studies with evidence confidence scores for each association [80]. The filtered (non-redundant) text mining file was downloaded and searched to identify gene-disease associations within the dataset. To help mitigate possible false-negative hits from TOPCONS-single that missed true 4TM proteins and to provide as complete a 4TM protein repertoire as possible, missing proteins from known protein families (such as claudins and tetraspanins) were manually explored and added to ensure the families were fully represented in the dataset. In total, forty additional proteins were added to the final dataset that had been identified in literature or database resources as containing 4TM regions. Proteins that were not classified as Enzymes, Trans- porters, Receptors, or Dual Functions were grouped into the Miscellaneous class. To further describe the miscel- laneous proteins, GO (general) and GO (molecular) terms were used to categorize proteins with similar func- tions into subgroups such as cell adhesion, chemotaxis, gap junction, enzymatic activity, regulation, etc. The GO terms are cross-referenced from the GO project, which aims for a consistent and comprehensive functional an- notation for gene products across databases [73]. There were several proteins that did not have any associated GO terms, but there were Pfam domains that described the function of the protein. There were also proteins where even with a conserved Pfam domain the function of the protein was unknown or uncharacterized. Those miscellaneous proteins that did not have any described function were categorized as uncharacterized. An updated dataset of all current targeted as well as potential proteins and genes involved in drug studies or experimentation was obtained from Rask-Anderson et al. [35]. Determining gene-disease associations and identifying drug targets This dataset is based on data from DrugBank [81], which provides extensive drug data and target informa- tion, and then has been manually curated to create a com- prehensive non-redundant dataset. Both the proteins and genes within the unambiguous 4TM dataset were investi- gated to identify drug targets. Investigating the uncharacterized proteins The uncharacterized proteins were further researched using the National Center for Biotechnology Information (NCBI) BLASTP [74] resource. BLASTP was used to de- termine if there were any possible homologues with the uncharacterized protein. The Non-redundant protein dataset (nr) was the search dataset and the default pa- rameters were used including the Blosum 62 substitution matrix; Word size: 3; and Expectation threshold: 10. Resulting hits with acceptable scores for investigating Abbreviations 4TM: four-transmembrane helix containing protein; NGIC: neurotransmitter gated ion channel; TC: Transporter classification number; EC: Enzyme commission number; GO: Gene ontology. Additional file Additional file 1: Contains the UniProt identifications, gene symbols, and Ensembl protein identifications for the classification of the 494 valid 4TM dataset. Also included within the table is the following information: signal peptides, topology, Pfam domains, functional classifications, review status, CCDS identifier, and gene-disease associations. (XLSX 176 kb) Classifying the unambiguous 4TM proteins Classifying the unambiguous 4TM proteins The Transporter Classification number (TC), Enzyme Commission number (EC), Gene Ontology (GO) terms, Pfam domain characteristics, and protein family information Attwood et al. BMC Genomics (2016) 17:268 Page 14 of 16 distant homologues were retained: greater than ~25 % sequence similarity; an E-value between 0 –1e-6; and a bit score value >50 [75]. In addition, literature searches through NCBI PubMed were performed on selected proteins. were used to describe the functions of the unambigu- ous 4TM dataset and categorize them into appropriate classes. The Transporter Classification Database [27] is cross-referenced by Uniprot to provide all TC numbers. The Transporter Classification system is an IUBMB ap- proved classification system for membrane transport proteins that includes both functional and phylogenetic information. Proteins that had an associated TC number were classified as Transporters. The Enzyme Commission number is produced by cross-referencing the ENZYME nomenclature database [71] and IntEnz (Integrated rela- tional Enzyme database [72]. If a protein had an associated EC number, they were categorized as Enzymes. 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https://openalex.org/W1529966314	https://scientiamarina.revistas.csic.es/index.php/scientiamarina/article/download/253/251/251	English	null	Age and growth rate of juvenile bluefin tuna <i>Thunnus thynnus</i> from the Mediterranean sea (Sicily, Italy)	Scientia marina	2,005	cc-by	6,719	Age and growth rate of juvenile bluefin tuna Thunnus thynnus from the Mediterranean Sea (Sicily, Italy)* MARIO LA MESA1, MAURO SINOPOLI 2 and FRANCO ANDALORO 2 MARIO LA MESA1, MAURO SINOPOLI 2 and FRANCO ANDALORO 2 1 CNR, Istituto di Scienze Marine (ISMAR), Sezione Pesca Marittima di Ancona, Largo Fiera della Pesca, 60125 Ancona, Italy. E-mail: m.lamesa@ismar.cnr.it 2 ICRAM - Istituto Centrale per la Ricerca scientifica e tecnologica Applicata al Mare, STS Palermo, via Emerico Amari 124, 90139, Palermo, Italy. stituto di Scienze Marine (ISMAR), Sezione Pesca Marittima di Ancona, Largo Fiera della Pesca, 60125 Ancona, Italy. E-mail: m.lamesa@ismar.cnr.it 2 ICRAM - Istituto Centrale per la Ricerca scientifica e tecnologica Applicata al Mare, STS Palermo, via Emerico Amari 124, 90139, Palermo, Italy. SUMMARY: The microstructural analysis of the sagittal otoliths of juvenile Atlantic bluefin tuna, Thunnus thynnus, can be used to estimate the age and growth rate of young-of-the-year individuals born in the Mediterranean. Samples of juvenile bluefin tuna, obtained as by-catch of the local small-scale pelagic fishery for Atlantic bonito and dolphinfish, were collect- ed off the northern coast of Sicily between late August and early November 2002. Otolith age readings were carried out on 56 specimens ranging between 195 and 400 mm fork length and between 112 and 1266 g total weight. Based on microin- crement analysis along a counting path from the otolith core to the antirostrum, juvenile fishes were found to be between 77 days and 153 days old. The estimated growth rate was practically linear over the whole size range, accounting for about 2.0- 2.37 mm/day. Derived from the length-weight relationship and the estimated length-at-age data, the mean weight-at-age was 168, 429 and 813 g for 2, 3 and 4 month-old juveniles respectively. Furthermore, the hatching date distribution of bluefin tuna, obtained by means of the back-calculation of ageing data, indicated a spawning period of at least two months, name- ly from mid-May to mid-July, with a peak in mid-June. Our data indicate that juvenile bluefin tuna have a very high growth rate in the first part of their life, reaching a weight of more than 1 kg in four months. Keywords: age and growth, bluefin tuna, otoliths, Mediterranean Sea. RESUMEN: EDAD Y TASA DE CRECIMIENTO DE LOS JUVENILES DE ATÚN THUNNUS THYNNUS DEL MAR MEDITERRÁNEO (SICILIA, ITALIA). SCIENTIA MARINA SCIENTIA MARINA SCI. MAR., 69 (2): 241-249 2005 Received May 24, 2004. Accepted September 16, 2004. Palabras clave: edad y crecimiento, atún, otolitos, Mar Mediterráneo. Age and growth rate of juvenile bluefin tuna Thunnus thynnus from the Mediterranean Sea (Sicily, Italy) – El análisis microestructural de los otolitos sagitta de juveniles del atún Thunnus thynnus, ha permitido estimar la edad y tasa de crecimiento de individuos del primer año de vida, nacidos en el Mediterráneo. Los juveniles de atún, obteni- dos como especie acompañante de la pesquería artesanal pelágica del bonito y lampuga, se recolectaron en las costas de Sici- lia desde finales de agosto a principios de noviembre de 2002. Las lecturas de edad de los otolitos se realizaron en 56 ejem- plares entre 195 mm y 400 mm de longitud de furca y entre 112 g y 1266 g de peso total. En base a los análisis de microin- crementos a lo largo del eje de lectura, desde el núcleo del otolito hasta el antirostrum, se determinó que los juveniles tení- an entre 77 y 153 días de edad. La tasa de crecimiento estimada era prácticamente lineal a lo largo de todo el rango de tallas, representando alrededor de 2.0-2.37 mm/dia. A partir de las relaciones talla-peso y talla estimada-edad se determinó el peso medio-edad en 168 g, 429 g y 813 g para juveniles de 2, 3 y 4 meses de edad, respectivamente. Además, la fecha de eclo- sión del atún, estimada a partir del retrocálculo de la edad, indica que el período reproductor se extiende al menos durante dos meses, desde mediados de mayo a mediados de julio con un pico a mediados de junio. Nuestros datos indican que los juveniles de atún presentan una elevada tasa de crecimiento en las fases tempranas de vida, alcanzando un peso de mas de 1 kg en cuatro meses. Palabras clave: edad y crecimiento, atún, otolitos, Mar Mediterráneo. AGE AND GROWTH OF BLUEFIN TUNA 241 INTRODUCTION FIG. 1. – Sampling area off the northern coast of Sicily, showing the three main landing locations. The northern bluefin tuna, Thunnus thynnus, is a highly migratory species occurring in temperate and subtropical waters of the Atlantic and Pacific Oceans (Collette and Nauen, 1983). Two sub- species have been recognised: T. thynnus thynnus (Linnaeus) in the north Atlantic and T. thynnus ori- entalis (Temminck and Schlegel) in the north Pacific. Atlantic bluefin tuna are distributed throughout the Atlantic Ocean, as well as in the Mediterranean Sea and the Gulf of Mexico (Gibbs and Collette, 1967), which represent the most important spawning areas of this subspecies (Math- er et al., 1995; Magnuson et al., 1994). Age and growth rate of juvenile bluefin tuna Thunnus thynnus from the Mediterranean Sea (Sicily, Italy)* In May- June, adult bluefin tuna migrate through the Strait of Gibraltar into the Mediterranean for feeding and spawning activities. Soon after spawning, which occurs from the beginning of June to the end of August (Arena, 1979), most individuals leave the Mediterranean to overwinter in the Atlantic off Morocco (Sella, 1929; Rodriguez-Roda, 1964; Sara, 1973; Compeán-Jimenez and Bard, 1983). FIG. 1. – Sampling area off the northern coast of Sicily, showing the three main landing locations. of juveniles come from tag-and-recapture studies (Cort and Rey, 1985; Cort, 1990), and rearing exper- iments (Le Gall and Bard, 1979; Katavic et al., 2002). Very few data on age estimation of juvenile Atlantic bluefin tuna have been reported to date. Larvae and young-of-the-year bluefin tuna caught in the western Atlantic and the Gulf of Mexico were aged by means of microincrement counts in sagittal otoliths (Brothers et al., 1983). In the Mediterranean Sea, age estimation of juvenile bluefin tuna was determined by otolith microstructure analysis (Radtke and Morales-Nin 1989; Santamaria et al., 2003) and by annulus-formation in dorsal spines (Megalofonou and De Metrio, 2000). Besides its importance as a spawning area for Atlantic bluefin tuna, the Mediterranean is one of the most important fishing grounds, accounting for 53% of total catch of this species (ICCAT, 2000). In addi- tion, being a large pelagic species, it is considered to be the most economically valuable fish in both the Atlantic and the Mediterranean (CEC, 1995). g In order to provide new data on age and growth of young-of-the-year bluefin tuna, samples were collected off the northern coasts of Sicily on a monthly basis, from just after the spawning season (spring-summer) until the beginning of winter. The main aim was to determine the growth rate of the early life of juveniles until their first winter of life, probably a period of fundamental importance for the subsequent year class strength and survival. Sagittal otoliths were thus collected and aged by counting the daily growth increments. In addition, the hatch date distribution obtained from the back-calculation of ageing data allowed us to gain new insight into the time and duration of the spawning season of the Atlantic bluefin tuna in this area As for the western Atlantic and Gulf of Mexico population of bluefin tuna (see in Brothers et al., 1983), most studies concerning the spawning period and the age and growth of juveniles of T. Age and growth rate of juvenile bluefin tuna Thunnus thynnus from the Mediterranean Sea (Sicily, Italy)* thynnus in Mediterranean waters were undertaken using indirect methods. Indeed, the spawning time was frequently estimated according to the seasonal occurrence of eggs and larvae or younger juveniles (Scaccini et al., 1975; Richards, 1976; Piccinetti and Piccinetti-Man- frin, 1979; Piccinetti and Manfrin, 1993), as well as through the study of gonadal maturation in adult fish (Serna and Alot, 1992; Susca et al., 2000; Medina et al., 2002; Hattour and Macías, 2002). MATERIAL AND METHODS Agata palamitara drift net 15-10 S. Agata ferrettara drift net 16-10 S. Agata ferrettara drift net 17-10 S. Agata ferrettara drift net 24-10 C. d’Orlando ferrettara drift net 28-10 S. Agata palamitara drift net 13-11 C. d’Orlando palamitara drift net and by means of drift and purse seine nets. Sampling details are summarised in Table 1. Each specimen was measured as total length (TL) and fork length (FL) to the lowest mm and weighed as total weight and eviscerated weight (g). Fish were dissected and the heads removed and frozen. To study age and growth of juvenile bluefin tuna, we decided to use otoliths, as they are not susceptible to resorp- tion or alterations once formed (Campana and Neil- son 1985, Campana et al., 1995). In addition, otoliths are particularly useful for age determination of species with minute scales, such as bluefin tuna. Otolith removal was carried out following the “open the hatch method” (Secor et al., 1992). A cranio-cau- dal frontal cut was made through the head to expose the brain, which was removed allowing the localisa- tion of the sagittae. The sagittae were extracted by means of forceps, carefully cleaned from adhering vestibular tissue and stored dry in plastic vials. We preferred this method to the more common procedure (Nichy and Berry, 1976), as we were unable to prop- erly locate the right position of the vertical cut through the head without breaking or loosing the sagittae into the cranial cavity. Owing to the thickness of the antirostrum, there was a considerable individual variation in otolith readability. However, the particular three-dimen- sional morphology of the otolith, namely the pro- jecting surfaces and the deep sulcus acusticus, did not allow the otolith surface to be ground without loss of increments in the core or on the margin of the antirostrum. By means of ordinary least squares linear regres- sion analysis, a straight line was fitted to the age- length data pairs obtained by the increment counts of otoliths. Furthermore, from the ageing data and the date of capture, the monthly distribution of hatching dates of our specimens was back-calculated. The relationship between fish length (FL) and maximum otolith diameter was investigated by means of linear regression analysis. Finally, the length-weight relationship was calculated according to the exponential equation in the commonly used form: Following Brothers et al. MATERIAL AND METHODS Available data on growth rate of juvenile Atlantic bluefin tuna are mainly based on length frequency analysis (Furnestin and Dardignac, 1962; Le Gall and Bard, 1979; Cort, 1990; Liorzou and Bigot, 1995; Orsi Relini et al., 1997), as size distributions are well-defined and correspond to early age class- es, as a consequence of fast growth and a single and relatively short spawning period (Compeán-Jimenez and Bard, 1983). Additional data on the growth rate The study area was the northern coast of Sicily between Milazzo and S. Agata di Militello (Fig. 1). Samples were collected from commercial landings of the local small scale fishery, in the framework of the ICRAM Project “Eolide”. Juvenile bluefin tuna were caught offshore (about 10 miles) as by-catch, close to floating fish aggregating devices (FADs) 242 M. LA MESA et al. 242 M. LA MESA et al. TABLE 1. – Sampling data of juvenile bluefin tuna collected off the northern coast of Sicily in 2002. TABLE 1. – Sampling data of juvenile bluefin tuna collected off the northern coast of Sicily in 2002. side and increments are almost exposed on the otolith surface. According to Brothers et al. (1983), the increment counts were made from the core to the margin of the antirostrum (Secor et al., 1992), an area with a relatively unambiguous and continuous pattern of increments. Each increment was a bipartite concentric ring comprised of a dis- continuous and an incremental zone (defined D- zone and L-zone respectively, Secor et al., 1995). The number of D-zones were counted as the num- ber of increments, and assumed to be the age of the fish (see below). Two counts were made from the core to the margin and vice-versa, and the mean value was considered. When the counts dif- fered by more than ten increments they were dis- carded. Out of 72 otoliths examined, about 22% were discarded as they were difficult to read. The index of average percent error (APE) (Beamish and Fournier, 1981), as well as the mean coeffi- cient of variation (CV) (Chang, 1982), were calcu- lated to estimate the relative precision between the counts. TABLE 1. – Sampling data of juvenile bluefin tuna collected off the northern coast of Sicily in 2002. Date Area Gear Type 18-08 S. Agata palamitara drift net 29-08 Milazzo cianciolo purse-seine 04-09 Milazzo cianciolo purse-seine 05-09 S. Agata cianciolo purse-seine 10-09 S. Agata cianciolo purse-seine 12-09 S. MATERIAL AND METHODS (1983) and Foreman (1996), sagittal otoliths were placed in a drop of immersion oil on a depression slide to improve res- olution and examined under a light microscope at 100-400x magnification. The light microscope was equipped with a television camera connected to a video-analysis image program (OPTIMAS 6.5). The software was also used to measure the maximum diameter (OD), i.e. the maximum distance between the rostrum and the postrostrum (see Secor et al., 1992), of each otolith to an accuracy of 0.01 mm. In addition, the sagittae were weighed to mg. W = a FLb where W is the total wet weight (g), FL the fork length of fish (mm) and a and b are regression para- meters. By linearisation of the above equation, the log10-transformed length-weight data were comput- ed to determine the regression parameters. All sta- tistical inferences were based on a significance level of P = 0.05. Increment counts were made on the distal side, as the overburden deposition is very thin on this AGE AND GROWTH OF BLUEFIN TUNA 243 FIG. 4. – Sagittal otolith of juvenile bluefin tuna, showing the mar- gin of antirostrum with more regular and distinct increments. Scale bar = 100 µm. FIG. 2. – Sagittal otolith of juvenile bluefin tuna, showing the count- ing path of micro increments from the core to the margin of the antirostrum. FIG. 2. – Sagittal otolith of juvenile bluefin tuna, showing the count- ing path of micro increments from the core to the margin of the antirostrum. FIG. 4. – Sagittal otolith of juvenile bluefin tuna, showing the mar- gin of antirostrum with more regular and distinct increments. Scale bar = 100 µm. Otolith morphology Off the core area, the first four-five increments are quite regular, spherical and of similar thickness (1-2 µm). Increments then become progressively wider and elongated along the posterior-anterior axis. Similarly, within such bipartite rings, the L- zone (or light subunit) becomes progressively wider than the D-zone (dark subunit). In correspondence of the thicker zone of antirostrum, approximately after the fifteenth-twentieth increment, growth increments are optically very dense and their thick- ness increases up to 20 µm. Close to the margin of the antirostrum, the increment thickness decreases and they tend to progressively become more regular and distinct (Fig. 4). As observed in previous stud- ies (Brothers et al., 1983), subdaily growth incre- ments are quite common along the counting path, but a proper reading method consisting in the use of a high focal point of the light microscope largely prevented their enumeration. The sagittae of juvenile bluefin tuna are elongated along the anterior-posterior axis, flattened and with a pronounced thin rostrum. The external (distal) side of the otoliths is slightly concave and has an evident pro- trusion in the dorsal-posterior area. The internal (medial) side has a very deep sulcus acusticus. The antirostrum, i.e. an otolith protrusion opposite the ros- trum, is well developed and its thickness is highly variable (Fig. 2). The primordium is an optically dense and irregular round area in the centre of the otolith. It is surrounded by two diffuse layers, or non-incremen- tal rings, forming together the core (Fig. 3). Following some authors (Brothers et al., 1983; Itoh et al., 2000), these rings were not included in increment counts due to their faintness and anomalous width. FIG. 3. – Sagittal otolith of juvenile bluefin tuna, showing the core region. Arrows indicate the two diffuse layers surrounding the pri- mordium; spots indicate the first six daily rings. Scale bar = 10 µm. The maximum otolith diameter, recorded in fish- es between 195 mm and 400 mm FL, ranged from 3.3 mm to 6.26 mm. The relationship between max- imum otolith diameter (OD) and fork length of fish (FL) was linear (Fig. 5) and is summarised in the following equation: OD = 0.898 + 0.013 FL (n = 35, r2 = 0.94) OD = 0.898 + 0.013 FL (n = 35, r2 = 0.94) Age and growth Diel periodicity of growth increment formation has been largely documented in several scombrids, such as skipjack tuna, Katsuwonus pelamis, black skipjack tuna, Euthynnus lineatus, yellowfin tuna, Thunnus albacares, and albacore, Thunnus alalunga FIG. 3. – Sagittal otolith of juvenile bluefin tuna, showing the core region. Arrows indicate the two diffuse layers surrounding the pri- mordium; spots indicate the first six daily rings. Scale bar = 10 µm. 244 M. LA MESA et al. 0 1 2 3 4 5 6 7 8 100 200 300 400 500 Fork length (mm) Otolith diameter (mm) FIG. 5. – Relationship between maximum otolith diameter (OD) and fork length (FL) of juvenile bluefin tuna. 0 1 2 3 4 5 6 7 8 100 200 300 400 500 Fork length (mm) Otolith diameter (mm) FIG. 5. – Relationship between maximum otolith diameter (OD) and fork length (FL) of juvenile bluefin tuna. northern coast of Sicily. FL Age classes (months) (mm) II III IV V 190 2 1 200 2 210 1 220 1 230 1 240 250 2 260 1 3 270 1 3 280 290 3 300 2 310 3 2 320 5 2 330 4 1 340 2 350 2 360 6 1 370 1 380 3 390 400 1 N 8 27 20 1 Mean 220.2 290.4 350.6 360 Std 3.0 3.5 2.5 0 100 200 300 400 500 0 50 100 150 200 Estimated age (days) Fork length (mm) FIG. 6. – Simple linear regression fitted to age-length data of juvenile bluefin tuna from the northern coast of Sicily. Otolith diameter (mm) Fork length (mm) FIG. 5. – Relationship between maximum otolith diameter (OD) and fork length (FL) of juvenile bluefin tuna. FIG. 5. – Relationship between maximum otolith diameter (OD) and fork length (FL) of juvenile bluefin tuna. (Wild and Foreman, 1980; Uchiyama and Struhsak- er, 1981; Laurs et al., 1985; Wexler, 1993). Similar- ly, age estimation by means of otolith increment counts has been validated in larval and juvenile bluefin tuna, Thunnus thynnus, from fertilisation to fish of 68 cm in fork length (Foreman, 1996; Itoh et al., 2000), as well as in adult specimens (Radtke, 1984). Following these authors, we assume that the growth increments we describe for young-of-the- year bluefin tuna are laid down daily, providing the true age (in days) of aged specimens. Age and growth length-at-age data revealed the mean weight-at-age of two, three and four month old juveniles to be 168, 429 and 813 g respectively. Age and growth Furthermore, as the first increment is formed on approximately the fourth day after hatching, i.e. at the onset of feeding (Brothers et al., 1983; Itoh et al., 2000), age estimates were corrected by adding four days to total increment counts. 0 100 200 300 400 500 0 50 100 150 200 Estimated age (days) Fork length (mm) Overall, 56 specimens between 195 and 400 mm FL and between 112 and 1266 g total weight were aged. Age estimates ranged from 77 days to 153 days and they are summarised in an age-length key (Table 2). As far as the precision of readings is con- cerned, the low value of imprecision indices observed (0.020 and 0.029 for APE and CV, respec- tively) indicates a successful consistency between subsequent readings, supporting the suitability of the preparation techniques adopted. Based on the mean value of fork length at age (see Table 2), the growth rate was about 70 mm between the second and third month of life, then it decreased to 60 mm between the third and fourth month of life. Unfortu- nately we had only one fish older than four months, so we were unable to give the growth rate between the fourth and fifth month of life. FIG. 6. – Simple linear regression fitted to age-length data of juvenile bluefin tuna from the northern coast of Sicily. FL = 41.20 + 2.37 age (days) (n = 56, r2 = 0.71) FL = 41.20 + 2.37 age (days) (n = 56, r2 = 0.71) The slope of the linear fit thus indicates a growth rate of 2.37 mm/day. Fork lengths of fish were plot- ted against catch dates to provide an estimate of growth rate and to compare such results with those obtained from the age-length data. A least square regression was then fitted to the data, giving a growth rate of 2.02 mm/day. This value was not sig- nificantly different from that obtained from the age- length relationship (F = 1.61, P> 0.1). The growth model which best fits age estimates- length data was a simple linear regression (Fig. 6), providing the following relationship: AGE AND GROWTH OF BLUEFIN TUNA 245 0 5 10 15 20 25 30 mid-may end-may mid-june end-june mid-july N° of specimens FIG. 7. – Monthly pattern of hatching dates of juvenile bluefin tuna, back-calculated from the ageing data and date of catch. DISCUSSION The bluefin tuna fishery can be considered one of the most traditional and ancient activities in the Mediterranean Sea, with evidence dating back to 7000 BC (Doumenge, 1998). As a consequence of the considerable exploitation of this marine resource, studies concerning the bluefin tuna biolo- gy and its fisheries began in the early 20th century, especially in the Mediterranean Sea (Sanzo, 1910, Roule, 1924; Buen, 1925; Heldt, 1926, 1932; Sella, 1929, 1930; Scordia, 1933). FIG. 7. – Monthly pattern of hatching dates of juvenile bluefin tuna, back-calculated from the ageing data and date of catch. Starting from the age estimates and date of cap- ture, hatching date distribution was back-calculated (Fig. 7). Estimated hatching period was quite long, lasting from mid-May to mid-July with a peak on mid-June. Similarly to other Atlantic tunas, the bluefin tuna fishery is currently managed by the International Commission for the Conservation of the Atlantic Tuna (ICCAT), through a complete stock assessment performed every two years since 1970 and based on an age-structured population model. However, the stock management of this species recently encoun- tered some difficulties, because of increasing uncer- tainties concerning catch and effort data and lack of information on some biological and ecological fea- tures, such as age, growth rate and natural mortality (Fromentin, 2003). REFERENCES Arena, P. – 1979. Aspectes biologiques et de comportement des concentrations genetiques du thon en Mediterranee. In: F.X. Bard and J.Y. Le Gall (eds.), Le thon rouge en Méditerranée: biologie, pêche et aquaculture. Actes Colloq. CNEXO, 8: 100-106. Beamish, R.J. and D.A. Fournier. – 1981. A method of comparing the precision of a set of age determinations. Can. J. Fish. Aquat. Sci., 38: 982-983. Brothers, E.B., E.D. Prince and D.W. Lee. – 1983. Age and growth of young-of-the-year bluefin tuna, Thunnus thynnus, from otolith microstructure. NOAA Tech. Rep.NMFS, 8: 49-59. Buen, F. de. – 1925. Biologia del Atún Orcynus thynnus (L.). Resul- tado de las campañas realizadas por acuerdos internacionales. Inst. Esp. Oceanogr., 1: 1-118. p g Campana, S.E. and J.D. Neilson. – 1985. Microstructure of fish otoliths. Can. J. Fish. Aquat. Sci., 42: 1014-1032. Campana, S.E. J.A. Gagne and J.W. McLaren. – 1995. Elemental fingerprinting of fish otoliths using ID-ICPMS. Mar. Ecol. Progr. Ser., 122(1-3): 115-120. g Cavallaro, G., A. Cefali and A. Potoschi. – 1998. Alcuni aspetti bio- logici e pesca di pescespada, tonno ed alalunga in studi esegui- ti tra il 1984 ed il 1996 nel Tirreno meridionale e nello Ionio. Biol. Mar. Medit., 5(3): 241-251. A further evidence to test the reliability of age estimates is the comparison of the back-calculated hatching dates with the spawning period of adults and the seasonal appearance of early larvae. Our data show a hatching period of bluefin tuna extend- ing from mid-May to mid-July, with a peak in mid- June. The present results are fully consistent with previous studies on reproduction of bluefin tuna in Mediterranean, which report a spawning period between May and July indicated by the presence of hydrated oocytes (Rodriguez-Roda, 1967; Susca et Chang, W.Y.B. – 1982. A statistical method for evaluating the reproducibility of age determination. Can. J. Fish. Aquat. Sci., 39: 1208-1210. Collette, B.B., and C.E. Nauen. – 1983. FAO Species catalogue, Vol. 2: scombrids of the world, an annotated and illustrated cat- alogue of tunas, mackerels, bonitos and related species known to date. FAO Fish. Synop., 125: 1-137. y p Commission of European Community (CEC). – 1995. Characteri- zation of large pelagic stocks (Thunnus thynnus L., Thunnus alalunga Born., Sarda sarda Bloch., Xiphias gladius L.) in the Mediterranean. Final report. p Compean-Jiménez, G. and F.X. Bard. – 1983. Length-weight relationship At this stage, the first otolith increment is laid down (Sanzo, 1932; Broth- ers et al., 1983). We thus added four days to incre- ment counts to obtain the age of fish from fertilisa- tion to catch. al., 2001; Medina et al., 2002; Corriero et al., 2003). Similarly, the backcalculated hatching dates are in agreement with data on the occurrence of eggs and larvae of bluefin tuna in the Mediterranean from mid-June to early August (Piccinetti and Manfrin, 1993; Piccinetti et al., 1996; Cavallaro et al., 1998). In conclusion, the analysis of otolith microstruc- ture and chemical composition in juvenile bluefin tuna can be considered of great importance in stock management, providing information on age and growth rate and on putative nursery areas. Further- more, as previously tested in other studies, the microincrements or growth units found in the otolith microstructure of juvenile bluefin tuna are laid down daily, allowing individuals to be aged with a high degree of accuracy. Although we were unable to validate our age estimates, the daily formation of otolith increment has been recently validated both in larvae and juve- nile bluefin tuna, i.e. from larvae 5 days after fertil- isation to fish 68 cm in fork length ((Foreman, 1996; Itoh et al., 2000). However, we attempted to indi- rectly evaluate the accuracy of age estimates by comparing the results obtained from increment counts with other independent measures of age and growth. The growth rates, i.e. the slopes of the least squares regression lines fitted to age-length and date-of-catch-length pair data, were very similar to each other. The estimated growth rate of our sam- ples was between 2.0 mm/day and 2.4 mm/day, which is similar to that reported for juvenile bluefin tuna of similar size sampled in the Ionian Sea and obtained by means of otolith increment counts (2.9 mm/day, Santamaria et al., 2003). On the other hand, such values are slightly higher than those reported for juveniles bluefin tuna caught between October-November in the Ligurian Sea and obtained by means of length-frequency analysis and mark- recapture (0.71-2.0 mm/day). It should be men- tioned, however, that such discrepancies are proba- bly due to the different sampling period and tech- niques used. Length-weight relationship The relationship between fish length (FL) and weight was determined from 72 specimens (Fig. 8), ranging from 195 mm to 400 mm FL and from 110 g to 1266 g. The values of parameters a and b of the allometric power function, derived from linear regression of the log10-transformed length-weight data pairs, were the following: A further problem is the stock delimitation and the degree of intermixing between different stocks, as the bluefin tuna is a highly migratory species that is widely distributed throughout the north Atlantic and the Mediterranean Sea (ICCAT, 2002). On the basis of elemental analysis of otoliths, used as nat- ural tags which reflect differences in the chemical and physical environment of a fish, some studies have recently attempted to identify Atlantic bluefin tuna stocks from putative nurseries, i.e. the Gulf of Mexico and the Mediterranean Sea (Secor et al., 2002; Rooker et al., 2003). Similarly, chemical char- acteristics of otolith and mercury body burden have been used to discriminate the two postulated popu- lations found in the Mediterranean, one that spends longer periods of time in the Mediterranean and one that migrates from the Atlantic to the Mediterranean just for spawning, returning to the Atlantic after breeding (Renzoni et al., 1978; Morales-Nin and Fortuño, 1990). W = 1.9210-6 FL 3.39 (n = 72, r2 = 0.98) W = 1.9210-6 FL 3.39 (n = 72, r2 = 0.98) A positive allometric growth (i.e. b>3) was thus observed in the early life of bluefin tuna. Coupling of the length-weight relationship and the estimated 0 200 400 600 800 1000 1200 1400 0 100 200 300 400 500 Fork length (mm) Total weight (g) FIG. 8. – Fork length-weight relationship of juvenile bluefin tuna from the northern coast of Sicily. In the present paper we analysed the inner struc- ture of the otoliths, using the microincrement pat- terns to estimate the age and growth of juvenile FIG. 8. – Fork length-weight relationship of juvenile bluefin tuna from the northern coast of Sicily. 246 M. LA MESA et al. bluefin tuna. In this species, otoliths (sagittae and probably lapilli) are known to be present at hatching (Sanzo, 1932). Bluefin tuna eggs hatch in just two days, and the early larvae resorb their yolk two or three days after hatching when the onset of exoge- nous feeding probably occurs. ACKNOWLEDGEMENTS We are much indebted to the whole staff of Icram-Palermo involved in the field activities for providing fish samples. Length-weight relationship Similarly, the growth rates for juvenile bluefin tuna reported from the Atlantic stocks are very different among each other, again as a conse- quence of different approaches (see Rivas, 1954; Mather and Schuck, 1960; Furnestin and Dardignac, 1962; Brothers et al., 1983). REFERENCES Growth increments on dorsal spines of eastern Atlantic bluefin tuna, Thunnus thyn- nus and their possible relation to migration patterns. NOAA Tech. Rep. NMFS, 8: 145-150. AGE AND GROWTH OF BLUEFIN TUNA 247 AGE AND GROWTH OF BLUEFIN TUNA 247 bluefin tuna. Collect. Vol. Sci. Pap. ICCAT, 5: 302-306. Corriero, A., S. Desantis, M. Deflorio, F. Acone, C.R. Bridges, J.M. de la Serna, P. Megalofonou and G. De Metrio. – 2003. Histolog- ical investigation on the ovarian cycle of the bluefin tuna in the western and central Mediterranean. J. Fish Biol., 63: 108-119. Orsi Relini, L., G. Palandri, F. Garibaldi, M. Relini, C. Cima and G. Torchia. – 1997. 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https://openalex.org/W2944605535	https://www.frontiersin.org/articles/10.3389/fphar.2019.00493/pdf	English	null	Corrigendum: Ion Channel Targeted Mechanisms of Anti-arrhythmic Chinese Herbal Medicine Xin Su Ning	Frontiers in pharmacology	2,019	cc-by	475	Copyright © 2019 Wang, Xie, Yu, Ellory, Wilkins, Zhu and Ma. 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. CORRECTION published: 09 May 2019 doi: 10.3389/fphar.2019.00493 CORRECTION published: 09 May 2019 doi: 10.3389/fphar.2019.00493 Approved by: Frontiers in Pharmacology Editorial Ofﬁce, A Corrigendum on Ion Channel Targeted Mechanisms of Anti-arrhythmic Chinese Herbal Medicine Xin Su Ning by Wang, T., Xie, W., Yu, J., Ellory, C., Wilkins, R., Zhu, Y., et al. (2019). Front. Pharmacol. 10:70. doi: 10.3389/fphar.2019.00070 In the original article, we incorrectly used the university internal grant code which should be replaced by the Chinese Medicine Research Fund in University of Oxford. The authors apologize for this error and state that this does not change the scientiﬁc conclusions of the article in any way. The original article has been updated. Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Received: 01 April 2019 Accepted: 18 April 2019 Published: 09 May 2019 Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology g p The funding statement should read: The funding statement should read: The funding statement should read: This work was supported by the Chinese Medicine Research Fund in University of Oxford. The grant was sponsored by Shaanxi Momentum Pharmaceutical Co., Ltd. Shaanxi Momentum Pharmaceutical Co., Ltd. had no involvement in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. All authors declare no conﬂict of interest. Taiyi Wang 1, Weiwei Xie 2, Jiahui Yu 2, Clive Ellory 1, Robert Wilkins 1, Yan Zhu 2 and Yu-ling Ma 1* Taiyi Wang 1, Weiwei Xie 2, Jiahui Yu 2, Clive Ellory 1, Robert Wilkins 1, Yan Zhu 2 and Yu-ling Ma 1* 1 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom, 2 Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China Keywords: anti-arrhythmic drugs, premature ventricular contractions, Xin Su Ning, Chinese Herbal Medicine, electrophysiology Approved by: Frontiers in Pharmacology Editorial Ofﬁce, Frontiers Media SA, Switzerland Correspondence: Yu-ling Ma yu-ling.ma@dpag.ox.ac.uk Approved by: Frontiers in Pharmacology Editorial Ofﬁce, Frontiers Media SA, Switzerland Correspondence: Yu-ling Ma yu-ling.ma@dpag.ox.ac.uk Frontiers in Pharmacology \| www.frontiersin.org Citation: Wang T, Xie W, Yu J, Ellory C, Wilkins R, Zhu Y and Ma Y (2019) Corrigendum: Ion Channel Targeted Mechanisms of Anti-arrhythmic Chinese Herbal Medicine Xin Su Ning. Front. Pharmacol. 10:493. doi: 10.3389/fphar.2019.00493 May 2019 \| Volume 10 \| Article 493 Frontiers in Pharmacology \| www.frontiersin.org
https://openalex.org/W4214903323	https://pubs.rsc.org/en/content/articlepdf/2022/ma/d2ma00093h	English	null	The significance of nanoparticle shape in chirality transfer to a surrounding nematic liquid crystal reporter medium	Materials advances	2,022	cc-by	9,387	Showcasing research from the laboratories of Professors Torsten Hegmann and Claudio Zannoni, Advanced Materials and Liquid Crystal Institute, Kent State University, Kent. (Ohio), USA and Department of Industrial Chemistry "Toso Montanari", University of Bologna, Bologna, Italy. As featured in: See Claudio Zannoni, Torsten Hegmann et al ., Mater . Adv ., 2022, 3 , 3346. PAPER Katsuyoshi Hoshino et al . Edge-on lamellar crystallization of oligo(3-methoxythiophene) in polyester matrix films and a gold tone development thereof Materials Advances rsc.li/materials-advances ISSN 2633-5409 Volume 3 Number 8 21 April 2022 Pages 3335–3664 As featured in: Materials Advances The significance of nanoparticle shape in chirality transfer to a surrounding nematic liquid crystal reporter medium As it turns out, the efficacy of chirality transfer may be understood by surprisingly simple geometric considerations. A combination of theory and experiment helped elucidate a mechanism based on the product of an established pseudoscalar chirality indicator and a scalar geometric shape compatibility factor. The experimental system is based on precisely engineered gold nanoshapes in a nematic liquid crystal solvent reporting medium. The predictive power of this approach may find use in macromolecular and biological systems, from chiral metamaterials to understanding nature’s innate ability to transfer homochirality. a Advanced Materials and Liquid Crystal Institute (AMLCI), Kent State University, Kent (OH) 44242-0001, USA. E-mail: thegmann@kent.edu b Materials Science Graduate Program, Kent State University, Kent (OH) 44242-0001, USA c Institute for Solid State Physics, The University of Tokyo, Tokyo, Japan d Department of Chemistry and Biochemistry, Kent State University, Kent (OH) 44242-0001, USA e Dipartimento di Chimica Industriale ‘‘Toso Montanari’’ and INSTM, Universita` di Bologna, Viale Risorgimento 4, IT-40136 Bologna, Italy. E-mail: claudio.zannoni@unibo.it f Brain Health Research Institute (BHRI), Kent State University, Kent (OH) 44242- 0001, USA PERSPECTIVE Open Access Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. rsc.li/materials-advances Registered charity number: 207890 The significance of nanoparticle shape in chirality transfer to a surrounding nematic liquid crystal reporter medium Anshul Sharma,ab Taizo Mori, c Ahlam Nemati, ab Diana P. N. Gonçalves, bd Lara Querciagrossa, e Claudio Zannoni e and Torsten Hegmann abdf Cite this: Mater. Adv., 2022, 3, 3346 Anshul Sharma,ab Taizo Mori, c Ahlam Nemati, ab Diana P. N. Gonçalves, bd Lara Querciagrossa, e Claudio Zannoni e and Torsten Hegmann abdf Defined based on geometric concepts, the origin of biological homochirality including the single handedness of key building blocks, D-sugars and L-amino acids, is still heavily debated in many ongoing research endeavors. Origin aside, transmission and amplification of chirality across length scales are likely essential for the predominance of one handedness over the other in chiral systems and are attracting an unabated interest not only in biology but also in material science. To oﬀer a measure for chirality and through-space chirality transfer, we here provide a report on recent progress toward the development of a suitable approach for an a priori prediction of chirality ‘‘strength’’ and eﬃcacy of chirality transfer from a chiral solute to an achiral nematic solvent. We achieve this by combining an independently calculated, suitable pseudoscalar chirality indicator for the solute with another, independently calculated scalar solute–solvent shape compatibility factor. In our ongoing pursuit to put this approach to the test, we are advancing and refining a versatile experimental platform based on achiral gold nanoparticle cores varying in size, shape, and aspect ratio capped with monolayers of chiral molecules or on intrinsically chiral cellulose nanocrystals that serve as chiral solutes in an achiral nematic liquid crystal phase acting as a reporter medium. The pitch of the ensuing induced chiral nematic liquid crystal phase ultimately serves as a reporter medium that allows us to experimentally quantify and com- pare chirality and efficacy of chirality transfer. Received 27th January 2022, Accepted 3rd March 2022 DOI: 10.1039/d2ma00093h rsc.li/materials-advances rsc.li/materials-advances Materials Advances View Article Online View Journal \| View Issue 1 Introduction call any geometrical figure, or group of points, chiral, and say it has chirality, if its image in a plane mirror, ideally realized, cannot be brought to coincide with itself.’’4 Considering its unquestion- able significance in many areas of science ranging from cell–cell interactions,5 drug discovery,6 and catalysis7 to chiral optics8 and chiral nanomaterials,9–12 some key questions asked and addressed by many scientists are: Does chirality scale, can it be quantified even for abstract geometrical objects, and how does it vary solely as a function of shape? Kurt Mislow, a pioneer in this field of research, published one of the most detailed reports on purely geometric chirality metrics to quantify chirality.13 Now almost three decades ago, that report primarily focused on geometric figures in 2- and 3-D space representing molecules, rather than nanomaterials, but many of the concepts have since been applied to chiral nanostructures. Within the boundaries of each model or chirality measure, objects including molecules can be defined as ‘‘more’’ or ‘‘less’’ chiral. In due course, Mislow and colleagues concluded: ‘‘there remains the daunting challenge of bridging the gap between the results of chiral shape analysis and the world of experimental observables.’’13 Chirality, defined as a geometric property of a group of points or atoms in space, or of any object that is not superimposable onto its mirror image, is ubiquitous at all scale levels1—from subatomic particles2 to galaxies in space.3 For any system to be chiral, irrespective of any physical or chemical manifestation of said chirality, all that is necessary is the complete absence of improper rotations in the symmetry group of the system. Lord Kelvin defined chirality by the absence of mirror symmetry: ‘‘I In this perspective, we summarize recent attempts by our teams to bridge this gap by showing that an appropriate 3346 \| Mater. Adv., 2022, 3, 3346–3354 © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online View Article Online Fig. 1 Visual summary depicting the concept of using an induced chiral nematic liquid crystal (N-LC phase) as a reporter medium to study the chirality transfer eﬃcacy (i.e., amplification across length scales) depend- ing on the size, shape, and aspect ratio (AR) of gold nanocarriers capped with an identical chiral ligand shell; p/2 refers to the helical half-pitch of the induced N-LC phase formed by doping the achiral N-LC phase. 2.1 Basic concepts The intrinsic chirality of any given chiral shape (object or molecule) can be calculated using an absolute, as opposed to being relative to a reference, pseudoscalar chirality indicator15 —termed the average chirality index hGa oai.†,16,17 For chiral nanoshapes, such as those of interest here, this index can be derived from coarse grained (CG) representations of the chiral ligand molecules suitably distributed on the nanoshape surface as we will see later. Experimental datasets correlated to the chirality of these nanoshapes are acquired by investigating dispersions of said chiral nanoshapes in an achiral nematic liquid crystal (N-LC)18 serving as a reporter medium.19 The induced chiral nematic LC (N-LC) phase allows a ranking of the eﬃcacy of chirality transfer depending on the dimensions of the nanoshape via microscopically (visually)-observable and easily measurable helical pitch, p, values (Fig. 1). For any chiral nanoshape solute X in an achiral N-LC phase N, p of the induced N-LC phase is inversely proportional to the concen- tration of the nanoshape, c X, and to the helical twisting power bXN (eqn (1)):20 Early experiments by our teams focused on size eﬀects of quasi-spherical (polyhedral) nanoparticles. Within each of the studied series of quasi-spherical gold nanoparticles (GNP), a particular GNP core diameter showed maximum values for both bXN and/or hGa oai.19 With the chirality emanating exclusively from a combination of spatial arrangement and chiral nature of the ligand shell molecules (considering an achiral metal core based on experimental i.e., spectroscopic evidence), the molar helical twisting power bmol defined in eqn (3): bmol = 1/(pxLigandr) (3) (3) in which p is the measured helical pitch, xLigand the mole fraction of the chiral ligand in the N-LC mixture, and r the enantiomeric purity of the chiral species), was used as the most accurate measure for the eﬃcacy of chirality transfer to the N-LC reporter medium. 1 Introduction The right-side of the figure depicts diﬀerent sizes and aspect ratios of the gold nanoshapes among N-LC molecules in the induced N-LC phase. Materials Advances View Article Online ss Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1 Visual summary depicting the concept of using an induced chiral nematic liquid crystal (N-LC phase) as a reporter medium to study the chirality transfer eﬃcacy (i.e., amplification across length scales) depend- ing on the size, shape, and aspect ratio (AR) of gold nanocarriers capped with an identical chiral ligand shell; p/2 refers to the helical half-pitch of the induced N-LC phase formed by doping the achiral N-LC phase. The right-side of the figure depicts diﬀerent sizes and aspect ratios of the gold nanoshapes among N-LC molecules in the induced N-LC phase. Mater. Adv., 2022, 3, 3346–3354 \| 3347 Perspective Materials Advances pseudoscalar chirality index can fairly accurately predict the chirality transfer eﬃcacy in an otherwise unrelated experi- mental system largely by assessing shape and shape con- gruency. While introducing the scope of and a classification for chirality measures, Mislow and colleagues noted that any ‘‘degree of chirality’’ scales with shape and should be invariant with the size of the object.13 Starting with this assumption, metal nanoparticles, particu- larly those with a gold core, continue to be fertile ground for testing shape-chirality correlations since gold nanoparticles can now be reproducibly synthesized in a very wide range of sizes and shapes with suitably narrow size and shape polydispersity.14 For the purpose of this discussion here, we will refer to these gold nanoparticles varying in size as well as shape, capped with a monolayer of chiral molecules as chiral nanoshapes. 2.3 Desymmetrization from GNPs to GNRs (gold nanorods) Our initial hypothesis was that the observed enhancement of this through-space chirality transfer was caused by long-range, through-space interactions between the chiral molecules and the plasmonic nanostructures exemplified by spectroscopically observed enhanced anisotropy or Kuhn’s dissymmetry factors g (eqn (4)):22 textures commonly seen for achiral N-LC phases, thus indicating p-N and, as a consequence, bmol - 0. GNP2 showed a value of \|bmol\| = 16 mm1; about one order of magnitude lower than the value detected for GNP5 but compared reasonably well to some commercially available organic chiral additives developed to induce N-LC phases with \|bmol\| values ranging from about 10 to 40 mm1.19 g = De/e (4) (4) where De and e are the molar circular dichroism and molar extinction coeﬃcient, respectively) here for chiral molecules in the vicinity of plasmonic nanostructures. Even higher g values were reported when such quasi-spherical GNPs were replaced by elongated GNRs,23 which, based on our hypothesis, should lead to increased \|bmol\| values when anisometric GNRs capped with the same chiral molecules are dispersed as a chiral solute in the identical N-LC host. Another GNP series investigated the role of axially chiral ligands. GNPs capped with three sets of (R)- and (S)-enantiomers based on axially chiral 1,10-binaphthyl-thiol derivatives that diﬀered only in the length of a non-tethered aliphatic chain were investigated as chiral solutes in the same N-LC host (Fig. 3).21 The variation in length of the non-tethered aliphatic chain in the 20-position, potentially due to reasons of varying dihedral angles in solution and the resulting changes in steric demand (or due to diﬀerences in solubility) gave rise to some variation in GNP core diameter during synthesis (overall ranging from about 1.1–2.5 nm in core diameter). The binaphthyl-thiol-capped GNP with the Experimental data indeed confirmed our hypothesis,24 and each of the two GNRs diﬀering in their aspect ratio (AR) and capped with a siloxane networked cholesterol-thiol ligand shell led to significantly higher \|bmol\| values (Fig. 4). MAR-GNR1 (MAR here standing for the medium aspect ratio of AR = 4.3 with the transversal cross-sectional diameter of this GNR of d = 10 nm closely matching the diameter of GNP10) produced a value of \|bmol\| = 1064 mm1. Similarly, MAR-GNR2 with AR = 2.2 induced an N-LC phase with a value of \|bmol\| = 895 mm1. 2.3 Desymmetrization from GNPs to GNRs (gold nanorods) Remarkably, the trends of independently calculated or acquired hGa oai and \|bmol\| values, respectively, for this GNP2–10/MAR-GNR series matched almost perfectly (Fig. 5). Our, for the most part, naı¨ve conclusions from these first couple of datasets were that desymmetrizing the shape of the nanocarrier and increasing its AR will increase hGa oai and \|bmol\|.24 Fig. 3 Series of GNPs capped with 1,10-binaphthyl-thiol ligands diﬀering in the length of the non-tethered aliphatic chain in the 20-position ((R)- and (S)-enantiomers) tested as chiral solutes in the N-LC phase formed by 5CB (40-pentyl-4-biphenylcarbonitrile). While cisoid-conformations were observed in organic solvents, transoid-conformations were confirmed in the induced N-LC phase. Reproduced from and adapted with permission from ref. 21 – Copyright (2016), American Chemical Society. This was further supported by the considerable amplification of the chirality transfer by MAR-GNR1 (the more eﬀective chiral inducer of the GNRs tested thus far) capped with the identical series of axially chiral binaphthyl-thiol ligands described earlier. This was further supported by the considerable amplification of the chirality transfer by MAR-GNR1 (the more eﬀective chiral inducer of the GNRs tested thus far) capped with the identical series of axially chiral binaphthyl-thiol ligands described earlier. For this particular MAR-GNR1BN series,25 the \|bmol\| values, now strongly depending on the length of the non-tethered alipha- tic chain in the 20-position on the 1,10-binaphthyl unit, ranged from \|bmol\| = 192 mm1 to a record \|bmol\| = 1312 mm1. Here again, independent calculations of hGa oai predicted these experimental trends both with respect to magnitude of \|bmol\| as well as the sign (aka handedness) of the induced N-LC phase (Fig. 6).25 For this particular MAR-GNR1BN series,25 the \|bmol\| values, now strongly depending on the length of the non-tethered alipha- tic chain in the 20-position on the 1,10-binaphthyl unit, ranged from \|bmol\| = 192 mm1 to a record \|bmol\| = 1312 mm1. Here again, independent calculations of hGa oai predicted these experimental trends both with respect to magnitude of \|bmol\| as well as the sign (aka handedness) of the induced N-LC phase (Fig. 6).25 Fig. 3 Series of GNPs capped with 1,10-binaphthyl-thiol ligands diﬀering in the length of the non-tethered aliphatic chain in the 20-position ((R)- and (S)-enantiomers) tested as chiral solutes in the N-LC phase formed by 5CB (40-pentyl-4-biphenylcarbonitrile). While cisoid-conformations were observed in organic solvents, transoid-conformations were confirmed in the induced N-LC phase. Reproduced from and adapted with permission from ref. 2.2 Early experimental nanoparticle systems 2 Studies focusing on the eﬀects of core diameter and presence (absence) of a chiral bias during GNP formation. GNP2 and GNP5 (the subscript number indicating the average core diameter) are capped with cholesterol-thiol ligands; GNP10(silox) is capped with a siloxane shell using 3-mercaptopropyltrimethoxysilane (MPS) that is further end- functionalized with cholesterol. Reproduced from and adapted with per- mission from ref. 19 – Copyright (2014), American Chemical Society. largest core diameter of about 2.5 nm (GNPBN(R)-1) turned out to be just slightly the strongest chiral inducer of this series with \|bmol\| = 66 mm1. However, in light of comparatively smaller differences in core diameter vis-a`-vis the earlier series19 overall, all six GNPBN essentially showed identical \|bmol\| values, ranging from \|bmol\| = 61 mm1 to \|bmol\| = 66 mm1. Most noteworthy, all GNPs discussed thus far (with the exception of GNP10) well outperformed the free cholesterol-thiol and 1,10-binaphthyl-thiol ligands with respect to their ability to helically distort the initially achiral N-LC, i.e., induce a signifi- cantly lower pitch (higher \|bmol\|) at any identical concentration of the chiral molecules in the N-LC matrix.19,21 Open Access Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 Studies focusing on the eﬀects of core diameter and presence (absence) of a chiral bias during GNP formation. GNP2 and GNP5 (the subscript number indicating the average core diameter) are capped with cholesterol-thiol ligands; GNP10(silox) is capped with a siloxane shell using 3-mercaptopropyltrimethoxysilane (MPS) that is further end- functionalized with cholesterol. Reproduced from and adapted with per- mission from ref. 19 – Copyright (2014), American Chemical Society. 2.2 Early experimental nanoparticle systems p / 1 bXNc X (1) (1) For the first set of GNPs with core diameters of about 2, 5 and 10 nm, capped with cholesterol-thiol or siloxane-networked cholesterol-thiol derivatives (Fig. 2), \|bmol\| was the largest (i.e., p was the tightest) for GNP5 characterized by an average core diameter of 5.5 nm (\|bmol\| = 178 mm1 (we here report values for the absolute molar helical twisting power, \|bmol\|, since the sign of bmol indicates the handedness and not the magnitude; ‘‘+’’ for a right-handed and ‘‘’’ for a left-handed helical distortion of the induced N-LC phase).19 Thus, bXN serves as a figure of merit or measure of chirality in these systems and is assumed to be proportional to the intrinsic chirality of the nanoshape (eqn (2)): bXN p hGa oai (2) (2) † This index depends only on geometric information, i.e., in this case the position and orientation of the chiral ligands with respect to the nanoparticle frame, and thus indirectly on the size, shape, and AR of the nanomaterial:16,17,24 † This index depends only on geometric information, i.e., in this case the position and orientation of the chiral ligands with respect to the nanoparticle frame, and thus indirectly on the size, shape, and AR of the nanomaterial:16,17,24 In contrast, GNP10 with the largest core diameter did not induce an apparent N-LC phase at any concentration judging from textural analysis data obtained by cross-polarized optical microscopy (cPOM) with the LC samples confined between substrates with varying surface boundary conditions (including homeotropic anchoring or a top surface formed by air). The observed Schlieren textures were indistinguishable from Ga oa ¼ P P rij rkl ril rij rjk rjk rkl n rijrjkrkl 2ril if iojokol 2 1; . . . ; n ½ 0 otherwise 8 < : , where n is P P n rijrjkrkl ril 0 otherwise < : otherwise the total number of atoms (or beads), rij the vector between two atoms i and j, with modulus corresponding to their distance rij, while P indicates all permutations of i, j, k, l atoms or beads. © 2022 The Author(s). Published by the Royal Society of Chemistry Mater. Adv., 2022, 3, 3346–3354 \| 3347 3347 Perspective View Article Online View Article Online Perspective Materials Advances Fig. © 2022 The Author(s). Published by the Royal Society of Chemistry 2.4 Variations in AR, other shapes, and shape matching Equipped with these pieces of the puzzle, the next question in our quest was: How do shape and AR of the nanocarrier of chiral information impact chirality transfer? Moreover, reflecting on our appreciation that an independently calculated hGa oai can predict experimental trends of \|bmol\|, we put this system to the test with a significant expansion of the scope of our research by focusing on more pronounced shape and AR variations.26 For all the nanoshapes described so far, the working principle for any amplification of the eﬃcacy of chirality transfer always appeared to be that the chiral ligand shell molecules act as a joined force or network, thereby amplifying chirality transfer throughout the N-LC medium both locally (to N-LC molecules in direct vicinity of the suspended GNRs) and throughout the N- LC bulk. Freeze-fracture transmission electron microscopy (TEM) experiments, showing arrays of appropriately spaced (based on simple volume fraction calculations) and seemingly twisted with respect to each other MAR-GNR2, lend support for an additional contribution to this eﬃcacious chirality transfer (Fig. 7).24 It appeared reasonable to assume that cooperative eﬀects among the now twisted GNRs would further amplify chirality transfer, and such cooperative eﬀects were experimen- tally seen in the plots of 1/p vs. the concentration of the GNRs c X in the N-LC. At a distinct threshold value of c X, the slope of the curve would abruptly rise with increasing c X rather than plateau as normally observed for chiral additives in N-LCs.24 One may think of this contribution as a feedback loop at and above the threshold concentration: a spatially massive (when compared to the N-LC building blocks, i.e., N-LC molecules) and strong chiral inducer amplifies its own twist and thus shortens p in the surrounding matrix, which, in turn leads to an This was ultimately accomplished by comparing calculations of a shape-corrected hGa oai, described next, with the experi- mental values gathered for \|bmol\| for a larger set of gold nanoshapes now including nanoprisms (GNPR), nanodisks (GND), nanostars (GNS) as well as GNRs diﬀering in AR. All gold nanoshapes were monolayer-capped with the identical cholesterol-thiol ligand shell shown in Fig. 2. The dimensions as well as \|bmol\| values for these and all other nanoshapes discussed in this perspective are summarized in Table 1. 2.3 Desymmetrization from GNPs to GNRs (gold nanorods) 5 (a) Atomistic and coarse-grained (CG) representation of the ligand molecule, (b) MAR-GNR1 spherocylindrical and GNP10 models each deco- rated with 20 CG ligands randomly distributed. (c) Trend of averaged chirality indicator \|Ga oa\| in comparison to the trend experimentally deter- mined for \|bmol\| for GNPs2–10 as well as MAR-GNR1 and MAR-GNR2. Values of chirality have been obtained for 20 000 random configurations on the GNPs and 1800 selected configurations on the GNRs. Reproduced from and adapted with permission from ref. 24 – Copyright (2018), Springer-Nature. increased torque to the GNRs that shortens p and so on and so forth. 2.3 Desymmetrization from GNPs to GNRs (gold nanorods) (a) GNRs with a cholesterol end-functionalized siloxane ligand shells show significantly elevated values of \|bmol\| in comparison to GNPs as shown in plots (b) and (c). ZLI-4572, COC, R-811, and CB15 are commercially available organic chiral dopants; the Chol-silane and Chol-disulfides are the neat organic ligand molecules tested as chiral additives, +Core is the core diameter of the GNP. \|bmol\| values here used the molar concentration of the entire ligand-capped GNR and thus the SI unit [mm1 mol1] (\|bmol\| values described in the text as well as in Table 1 use the more correct and dimensionless mole fraction of the chiral ligand shell molecules only since the GNR core is achiral; eqn (3)). (d) Cooperative eﬀects among the admixed GNRs are supported by analyzing plots of the inverse pitch (1/p) vs. the MAR-GNR concentration; the slope increases at a threshold concen- tration rather than plateauing as commonly observed for chiral additives in induced N-LC phases (green dotted curve) until a concentration when the GNRs begin the aggregate (dotted circle at cMARGNR1silox = 0.7 wt%). Reproduced from and adapted with permission from ref. 24 – Copyright (2018), Springer-Nature. Fig. 4 Amplification of the chirality transfer eﬃcacy by desymmetrization. (a) GNRs with a cholesterol end-functionalized siloxane ligand shells show significantly elevated values of \|bmol\| in comparison to GNPs as shown in plots (b) and (c). ZLI-4572, COC, R-811, and CB15 are commercially available organic chiral dopants; the Chol-silane and Chol-disulfides are the neat organic ligand molecules tested as chiral additives, +Core is the core diameter of the GNP. \|bmol\| values here used the molar concentration of the entire ligand-capped GNR and thus the SI unit [mm1 mol1] (\|bmol\| values described in the text as well as in Table 1 use the more correct and dimensionless mole fraction of the chiral ligand shell molecules only since the GNR core is achiral; eqn (3)). (d) Cooperative eﬀects among the admixed GNRs are supported by analyzing plots of the inverse pitch (1/p) vs. the MAR-GNR concentration; the slope increases at a threshold concen- tration rather than plateauing as commonly observed for chiral additives in induced N-LC phases (green dotted curve) until a concentration when the GNRs begin the aggregate (dotted circle at cMARGNR1silox = 0.7 wt%). Reproduced from and adapted with permission from ref. 24 – Copyright (2018), Springer-Nature. Fig. © 2022 The Author(s). Published by the Royal Society of Chemistry 2.3 Desymmetrization from GNPs to GNRs (gold nanorods) 21 – Copyright (2016), American Chemical Society. © 2022 The Author(s). Published by the Royal Society of Chemistry 3348 \| Mater. Adv., 2022, 3, 3346–3354 View Article Online View Article Online Fig. 4 Amplification of the chirality transfer eﬃcacy by desymmetrization. (a) GNRs with a cholesterol end-functionalized siloxane ligand shells show significantly elevated values of \|bmol\| in comparison to GNPs as shown in plots (b) and (c). ZLI-4572, COC, R-811, and CB15 are commercially available organic chiral dopants; the Chol-silane and Chol-disulfides are the neat organic ligand molecules tested as chiral additives, +Core is the core diameter of the GNP. \|bmol\| values here used the molar concentration of the entire ligand-capped GNR and thus the SI unit [mm1 mol1] (\|bmol\| values described in the text as well as in Table 1 use the more correct and dimensionless mole fraction of the chiral ligand shell molecules only since the GNR core is achiral; eqn (3)). (d) Cooperative eﬀects among the admixed GNRs are supported by analyzing plots of the inverse pitch (1/p) vs. the MAR-GNR concentration; the slope increases at a threshold concen- tration rather than plateauing as commonly observed for chiral additives in induced N-LC phases (green dotted curve) until a concentration when the GNRs begin the aggregate (dotted circle at cMARGNR1silox = 0.7 wt%). Reproduced from and adapted with permission from ref. 24 – Copyright (2018), Springer-Nature. Perspective This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Perspective Perspective p Fig. 5 (a) Atomistic and coarse-grained (CG) representation of the ligand molecule, (b) MAR-GNR1 spherocylindrical and GNP10 models each deco- rated with 20 CG ligands randomly distributed. (c) Trend of averaged chirality indicator \|Ga oa\| in comparison to the trend experimentally deter- mined for \|bmol\| for GNPs2–10 as well as MAR-GNR1 and MAR-GNR2. Values of chirality have been obtained for 20 000 random configurations on the GNPs and 1800 selected configurations on the GNRs. Reproduced from and adapted with permission from ref. 24 – Copyright (2018), Springer-Nature. Open Access Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. ess Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 Amplification of the chirality transfer eﬃcacy by desymmetrization. 2.4 Variations in AR, other shapes, and shape matching To account for a shape compatibility dependence of the chirality transfer, our new model assumed that there is a combi- nation of the chiral solute’s intrinsic chirality and its ability to twist the N-LC that contributes to the transfer chirality across length scales. For a chiral solute (nanoshape) X, we assumed that bXN can be written as: (5) Mater. Adv., 2022, 3, 3346–3354 \| 3349 Fig. 7 (a) Schematic depiction of the freeze-fracture (FF) method to prepare the TEM specimen. (b) A representative voxel of the N-LC droplet showing the multi-domain structure via the embedded GNRs. (c) and (d) FF-TEM images of the induced N-LC phase of Felix-2900-03 containing 0.5 wt% MAR-GNR2silox (scale bars: (c) 200 nm and (d) 1 mm, apparent circles are from the TEM grids). To obtain these images, it was important that the replica captured most of the MAR-GNRs on the fractured surface, thereby providing a direct visualization of the GNRs in the bulk material. Areas highlighted by a yellow box and (e–h) select areas from many of the obtained FF-TEM images show GNR arrays (often twisted) with an average separation closely matching the theoretically calculated particle–particle distances, DP–P, of ca. 90–100 nm by assuming well-dispersed GNRs in the induced N-LC matrix (scale bars in (e–h): 100 nm). Reproduced from and adapted with permission from ref. 24 – Copyright (2018), Springer- Nature. Perspective Materials Advances Fig. 6 (a) Schematic depiction of the MAR-GNR1BN series with the 2,2 substituted 1,10-binaphthyl-thiol ligands in the transoid conformati formed in the induced N-LC phase. (b) Chirality eﬃciency plot: calculat particle–particle distances, DP–P, vs. \|bmol\|. The most efficient chiral GN solutes occupy the top right corner of this plot, the bottom lower corn the least efficient ones. Inset (red dashed box) shows the section hig lighted in the lower left part of the plot (data points are either direc identified with the abbreviation of the nanoshape or highlighted in bl boxes. (c) Values of the chirality index, hGa oai, for 20 binaphthyl thiol liga molecules bound to the MAR-GNR1 compared to the values of t experimentally determined HTP data, \|bw\| (helical twisting power bas on the weight fraction of the chiral BN ligands in the N-LC), and \|bmol\| (w Materials Advances Open Access Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. © 2022 The Author(s). Published by the Royal Society of Chemistry 2.4 Variations in AR, other shapes, and shape matching 7 (a) Schematic depiction of the freeze-fracture (FF) method to prepare the TEM specimen. (b) A representative voxel of the N-LC droplet showing the multi-domain structure via the embedded GNRs. (c) and (d) FF-TEM images of the induced N-LC phase of Felix-2900-03 containing 0.5 wt% MAR-GNR2silox (scale bars: (c) 200 nm and (d) 1 mm, apparent circles are from the TEM grids). To obtain these images, it was important that the replica captured most of the MAR-GNRs on the fractured surface, thereby providing a direct visualization of the GNRs in the bulk material. Areas highlighted by a yellow box and (e–h) select areas from many of the obtained FF-TEM images show GNR arrays (often twisted) with an average separation closely matching the theoretically calculated particle–particle distances, DP–P, of ca. 90–100 nm by assuming well-dispersed GNRs in the induced N-LC matrix (scale bars in (e–h): 100 nm). Reproduced from and adapted with permission from ref. 24 – Copyright (2018), Springer- Nature. solvent transmission coefficient of the chiral nanoshape solute X to the achiral nematic mesogen N. Here, we employed for the first term the maximum chirality indicator (Fig. 8a); for SXN we now introduced a shape-complementarity model to quantify the shape resemblance (or shape incongruity) between the N-LC molecule serving as host and the chiral ligand surface-modified nanoshape.26 The use of \|Ga oa,max\| was justified by the fact that the HTP data were calculated from experimental pitch measurements, which are particularly sensitive to the highest rather than average chirality values (particularly for a system containing various contributions of diﬀerent chirality). Fig. 6 (a) Schematic depiction of the MAR-GNR1BN series with the 2,20- substituted 1,10-binaphthyl-thiol ligands in the transoid conformation formed in the induced N-LC phase. (b) Chirality eﬃciency plot: calculated particle–particle distances, DP–P, vs. \|bmol\|. The most efficient chiral GNR solutes occupy the top right corner of this plot, the bottom lower corner the least efficient ones. Inset (red dashed box) shows the section high- lighted in the lower left part of the plot (data points are either directly identified with the abbreviation of the nanoshape or highlighted in blue boxes. 2.4 Variations in AR, other shapes, and shape matching (c) Values of the chirality index, hGa oai, for 20 binaphthyl thiol ligand molecules bound to the MAR-GNR1 compared to the values of the experimentally determined HTP data, \|bw\| (helical twisting power based on the weight fraction of the chiral BN ligands in the N-LC), and \|bmol\| (with the largest values were set to 100%). Reproduced from and adapted with permission from ref. 25 – Copyright (2019), American Chemical Society. Hereby we further assumed that SXN is related to the inverse diﬀerence between the 2-D isoperimetric ratios (IPR2D; defined as IPR2D = c2/A, with c being the length of the perimeter and A the 2D shape surface area) of solute and solvent, 1/IPR2D XN = 1/(IPR2D X IPR2D N). The degree of correlation between SXN and bXN (i.e., \|bmol\|) will then provide direct insights into the importance of shape complementarity in chirality transfer. SXN, of course, is a scalar quantity such that the orientation of one nanoshape with respect to another is non-zero and scales invariantly under spatial transformation, even in the achiral case. We defined SXN as the absolute value of the inverse in which GX is the chirality indicator hGa oai of the nanoshape and SXN a compatibility factor representing a chiral solute – achiral in which GX is the chirality indicator hGa oai of the nanoshape and SXN a compatibility factor representing a chiral solute – achiral 3350 \| Mater. Adv., 2022, 3, 3346–3354 © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Perspective Materials Advances g. 8 Plots showing the trends of: (a) the maximum chirality indicato a oa,max\| in comparison to the experimentally derived values for \|bw\| an mol\| (with the largest value set to 100% and the lowest to 0%—here GNP th bmol = bw = 0, and every other value—and every other value weighte cordingly) for the calculated values of \|bmol\| see Table 1, (b) 1/(IPR2D) an 1/(IPR3D) each in comparison to \|bmol\| (yellow shaded areas in (c) signi me correlation in trends); values for IPR2D and IPR3D use the adjusted A lues considering the thickness of the ligand shell. (d) Plot showing the tren the shape compatibility-corrected maximum chirality indicator \|Ga oa,maxSXN r a subset of nanoshapes in comparison to \|bw\| and \|bmol\|. LAR, MAR, and HA ere refer to low, medium and high aspect ratio, respectively. Reproduce om and adapted with permission from ref. 2.4 Variations in AR, other shapes, and shape matching d The AR cannot be easily calculated for GNS; thus far the only concave shape investigated. Open Access Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Nanoshapea Nanoshape Core Dimensionsb (nm) (d = diameter, w = width, h = height, l = length, AR = aspect ratio) \|bmol\| (mm1) GNP2 d = 1.8 AR B1c 16 GNP5 d = 5.5 178 GNP10 d = 10.0 0 GNP10(silox) d = 10.0 0 GNPBN(R)-1 d = 2.5 61–66 GNPBN(S)-1 d = 1.7 GNPBN(R)-2 d = 2.1 GNPBN(S)-2 d = 1.1 GNPBN(R)-3 d = 1.2 GNPBN(S)-3 d = 1.1 MAR-GNR1 d = 10.0; l = 43.0 AR = 4.3 1064 MAR-GNR1silox 1303 MAR-GNR1BN(R)-1 196 MAR-GNR1BN(S)-1 192 MAR-GNR1BN(R)-2 410 MAR-GNR1BN(S)-2 393 MAR-GNR1BN(R)-3 1166 MAR-GNR1BN(S)-3 1312 MAR-GNR1BN(S)-3silox 210 MAR-GNR2silox d = 23.5; l = 51.0 AR = 2.2 896 LAR-GNR d =15.2; l = 25.0 AR = 1.7 53 HAR-GNR d = 12.5; l = 87.0 AR = 8.5 18 LAR-GND d = 79.0; h = 21.6 AR = 3.7 37 HAR-GND d = 45.0; h = 7.4 AR = 6.1 43 GNS dcore = 60.0; lspikes = 69.0 —d 105 GNPR ledge = 50.0; h = 12.5 AR = 5.3 216 5CB (N-LC) l = 1.8; w = 0.45 AR = 4.0 — licensed under a Creative Commons Attribution 3.0 Unported Licence. a All nanoshapes are capped with a thiolate ligand shell unless other- wise noted, e.g., the subscript ‘‘silox’’ refers to nanoshapes capped with a siloxane conjugated 3-mercaptopropylsilyloxy-ligand shell end- functionalized with aliphatic cholesteryl ester groups via a siloxane condensation reaction; BN = 1,10-binaphthyl; all other nanoshapes are capped with a cholesterol-thiol; LAR, MAR and LAR refer to large, medium or low aspect ratio, respectively. b Calculated AR values here are not considering the thickness of the ligand monolayer. Those referred to in the text used for the calculation of the 2-D isoperimetric ratios, IPR2D, consider the ligand shell of the chiral ligand-capped nanoshapes and thus diﬀer from those listed here for the nanoshape cores. c Assuming quasi-spherical overall shapes for polyhedral GNPs. d The AR cannot be easily calculated for GNS; thus far the only concave shape investigated. 2.4 Variations in AR, other shapes, and shape matching diﬀerence between the IPR2D of each nanoshape and the IPR2D of the N-LC host molecule 5CB (eqn (6)): ( ( )) SXN 1 D IPR2D ð Þ (6) (6) i.e., the closest match between their respective 2-D shapes was set to 100%. The 2-D approach proved to be significantly more sensitive than the corresponding 3-D IPR approach considering a comparison of the trends of IPR2D and IPR3D (defined as IPR3D = A3/V2, where V is the volume of the nanoshape) vs. \|bmol\| shown in Fig. 8b and c. IPR2D clearly shows a very close match to the experimentally derived \|bmol\| values (Fig. 8b). Hence, the chirality transfer is experienced by the 5CB host molecules from the 2-D projection of the suspended nanoshapes, i.e., from the distribution of chiral molecules on the nanoshape surface they are in direct contact with. Plotting i.e., the closest match between their respective 2-D shapes was set to 100%. The 2-D approach proved to be significantly more sensitive than the corresponding 3-D IPR approach considering a comparison of the trends of IPR2D and IPR3D (defined as IPR3D = A3/V2, where V is the volume of the nanoshape) vs. \|bmol\| shown in Fig. 8b and c. IPR2D clearly shows a very close match to the experimentally derived \|bmol\| values (Fig. 8b). Hence, the chirality transfer is experienced by the 5CB host molecules from the 2-D projection of the suspended nanoshapes, i.e., from the distribution of chiral molecules on the nanoshape surface they are in direct contact with. Plotting Fig. 8 Plots showing the trends of: (a) the maximum chirality indicator \|Ga oa,max\| in comparison to the experimentally derived values for \|bw\| and \|bmol\| (with the largest value set to 100% and the lowest to 0%—here GNP10 with bmol = bw = 0, and every other value—and every other value weighted accordingly) for the calculated values of \|bmol\| see Table 1, (b) 1/(IPR2D) and (c) 1/(IPR3D) each in comparison to \|bmol\| (yellow shaded areas in (c) signify some correlation in trends); values for IPR2D and IPR3D use the adjusted AR values considering the thickness of the ligand shell. (d) Plot showing the trend of the shape compatibility-corrected maximum chirality indicator \|Ga oa,maxSXN\| for a subset of nanoshapes in comparison to \|bw\| and \|bmol\|. LAR, MAR, and HAR here refer to low, medium and high aspect ratio, respectively. Reproduced from and adapted with permission from ref. 2.4 Variations in AR, other shapes, and shape matching 26 – Copyright (2022), AAAS. Materials Advance Table 1 Comparison of experimentally determined molar helical twisting power, \|bmol\|, values of the chiral ligand-capped gold nanoshapes as solutes in the N-LC 5CB. Data from ref. 19, 21 and 24–26 Table 1 Comparison of experimentally determined molar helical twisting power, \|bmol\|, values of the chiral ligand-capped gold nanoshapes as solutes in the N-LC 5CB. Data from ref. 19, 21 and 24–26 power, \|bmol\|, values of the chiral ligand capped gold nanoshapes as solutes in the N-LC 5CB. Data from ref. 19, 21 and 24–26 Nanoshapea Nanoshape Core Dimensionsb (nm) (d = diameter, w = width, h = height, l = length, AR = aspect ratio) \|bmol\| (mm1) GNP2 d = 1.8 AR B1c 16 GNP5 d = 5.5 178 GNP10 d = 10.0 0 GNP10(silox) d = 10.0 0 GNPBN(R)-1 d = 2.5 61–66 GNPBN(S)-1 d = 1.7 GNPBN(R)-2 d = 2.1 GNPBN(S)-2 d = 1.1 GNPBN(R)-3 d = 1.2 GNPBN(S)-3 d = 1.1 MAR-GNR1 d = 10.0; l = 43.0 AR = 4.3 1064 MAR-GNR1silox 1303 MAR-GNR1BN(R)-1 196 MAR-GNR1BN(S)-1 192 MAR-GNR1BN(R)-2 410 MAR-GNR1BN(S)-2 393 MAR-GNR1BN(R)-3 1166 MAR-GNR1BN(S)-3 1312 MAR-GNR1BN(S)-3silox 210 MAR-GNR2silox d = 23.5; l = 51.0 AR = 2.2 896 LAR-GNR d =15.2; l = 25.0 AR = 1.7 53 HAR-GNR d = 12.5; l = 87.0 AR = 8.5 18 LAR-GND d = 79.0; h = 21.6 AR = 3.7 37 HAR-GND d = 45.0; h = 7.4 AR = 6.1 43 GNS dcore = 60.0; lspikes = 69.0 —d 105 GNPR ledge = 50.0; h = 12.5 AR = 5.3 216 5CB (N-LC) l = 1.8; w = 0.45 AR = 4.0 — a All nanoshapes are capped with a thiolate ligand shell unless other- wise noted, e.g., the subscript ‘‘silox’’ refers to nanoshapes capped with a siloxane conjugated 3-mercaptopropylsilyloxy-ligand shell end- functionalized with aliphatic cholesteryl ester groups via a siloxane condensation reaction; BN = 1,10-binaphthyl; all other nanoshapes are capped with a cholesterol-thiol; LAR, MAR and LAR refer to large, medium or low aspect ratio, respectively. b Calculated AR values here are not considering the thickness of the ligand monolayer. Those referred to in the text used for the calculation of the 2-D isoperimetric ratios, IPR2D, consider the ligand shell of the chiral ligand-capped nanoshapes and thus diﬀer from those listed here for the nanoshape cores. c Assuming quasi-spherical overall shapes for polyhedral GNPs. Open Access Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40 This article is licensed under a Creative Commons Attribution 3.0 Unp Fig. 9 (a) Plot showing trends of the shape compatibility-corrected maximum chirality indicator \|Ga oa,maxSXN\| for the full set of nanoshapes, now including the powerful chiral inducer MAR-GNR1, in comparison to the experimentally derived values for \|bw\| and \|bmol\|. Models of the induced N-LC phase show a complete 3601 rotation of the local director leading to a tighter pitch from left to right depending on the size, shape, and AR of the suspended nanoshape (for GNP10: p - N). (b) Visualization showing the varying degree of shape compatibility among GNRs featuring diﬀerent AR values with the 2-D projection of 5CB scaled in size to match each GNR width. Reproduced from and adapted with permission from ref. 26 – Copyright (2022), AAAS. Fig. 10 (a–c) Chemical structures of the CNC surface modifications. The CNC core displays both morphological (twist of the CNC rods) and inherent core chirality (via the glucose building blocks). (d) Schematic representation of the N-LC phase induced by the addition of only minute amounts of the functionalized CNCs to the LC host, Felix-2900-03, in the nematic phase. Reproduced from and adapted with permission from ref. 27 – Copyright (2021), Wiley-VCH. Fig. 10 (a–c) Chemical structures of the CNC surface modifications. The CNC core displays both morphological (twist of the CNC rods) and inherent core chirality (via the glucose building blocks). (d) Schematic representation of the N-LC phase induced by the addition of only minute amounts of the functionalized CNCs to the LC host, Felix-2900-03, in the nematic phase. Reproduced from and adapted with permission from ref. 27 – Copyright (2021), Wiley-VCH. much need to alter the chemical nature of the chiral molecules decorating the nanoshape surface. the shape compatibility-corrected \|Ga oa,maxSXN\| against the experimentally obtained values shows extraordinary close correlation across almost all nanoshapes with respect to \|bw\| and \|bmol\| (Fig. 8d and 9a), most noteworthy all in the absence of any fitting parameter. But what if the nanoshape core was chiral? To elucidate the role of core chirality, we needed a system with an intrinsically chiral core. The perhaps closest match to the most potent MAR-GNR1, at least for now, appeared to be cellulose nanocrystals (CNCs). Accordingly, in another recently published study, we further studied such innately chiral CNCs, both neat and decorated with chiral or achiral molecules (Fig. Open Access Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40 This article is licensed under a Creative Commons Attribution 3.0 Unp 10).27 Here, a comparison between neat CNCs, CNCs-functionalized with cholesterol units (CNCs-Chol), and MAR-GNR1 revealed that the contribution to chirality transfer from a molecular and morphologically chiral core is likely considerable. The CNCs-Chol, lacking a dense chiral ligand shell present on MAR-GNR1 (translating to a 75% lower overall cholesterol content for the CNCs-Chol at a set constant weight fraction in an N-LC) showed an about eightfold decrease in \|bw\| in comparison to MAR-GNR1. However, replacing the chiral cholesterol units (Fig. 10b) with achiral pro-mesogenic moieties characterized by a close structural match to the N-LC host molecules (Fig. 10c) did not lead to a reduction of \|bw\|. The importance of the shape coupling or shape compatibil- ity factor SXN is most apparent when the AR of X is changing within a series of equal-shaped nanoshape solutes in the same N. This concept is best visualized by sketches shown in Fig. 9b, where the shape compatibility among GNRs, HAR-GNR, MAR- GNR1 and LAR-GNR proves the utility of introducing SXN. 2.4 Variations in AR, other shapes, and shape matching 26 – Copyright (2022), AAAS. Fig. 8 Plots showing the trends of: (a) the maximum chirality indicator \|Ga oa,max\| in comparison to the experimentally derived values for \|bw\| and \|bmol\| (with the largest value set to 100% and the lowest to 0%—here GNP10 with bmol = bw = 0, and every other value—and every other value weighted accordingly) for the calculated values of \|bmol\| see Table 1, (b) 1/(IPR2D) and (c) 1/(IPR3D) each in comparison to \|bmol\| (yellow shaded areas in (c) signify some correlation in trends); values for IPR2D and IPR3D use the adjusted AR values considering the thickness of the ligand shell. (d) Plot showing the trend of the shape compatibility-corrected maximum chirality indicator \|Ga oa,maxSXN\| for a subset of nanoshapes in comparison to \|bw\| and \|bmol\|. LAR, MAR, and HAR here refer to low, medium and high aspect ratio, respectively. Reproduced from and adapted with permission from ref. 26 – Copyright (2022), AAAS. Mater. Adv., 2022, 3, 3346–3354 \| 3351 © 2022 The Author(s). Published by the Royal Society of Chemistry Fig. 10 (a–c) Chemical structures of the CNC surface modifications. The CNC core displays both morphological (twist of the CNC rods) and inherent core chirality (via the glucose building blocks). (d) Schematic representation of the N-LC phase induced by the addition of only minute amounts of the functionalized CNCs to the LC host, Felix-2900-03, in the nematic phase. Reproduced from and adapted with permission from ref. 27 – Copyright (2021), Wiley-VCH. Perspective View Article Online View Article Online Materials Advances Fig. 9 (a) Plot showing trends of the shape compatibility-corrected maximum chirality indicator \|Ga oa,maxSXN\| for the full set of nanoshapes, now including the powerful chiral inducer MAR-GNR1, in comparison to the experimentally derived values for \|bw\| and \|bmol\|. Models of the induced N-LC phase show a complete 3601 rotation of the local director leading to a tighter pitch from left to right depending on the size, shape, and AR of the suspended nanoshape (for GNP10: p - N). (b) Visualization showing the varying degree of shape compatibility among GNRs featuring diﬀerent AR values with the 2-D projection of 5CB scaled in size to match each GNR width. Reproduced from and adapted with permission from ref. 26 – Copyright (2022), AAAS. Notes and references 21 T. Mori, A. Sharma and T. Hegmann, ACS Nano, 2016, 10, 1552–1564. 1 L. D. Barron, Chirality, 2012, 24, 879–893; S. M. Morrow, A. J. Bissette and S. P. Fletcher, Nat. Nanotechnol., 2017, 12, 410–419. 1 L. D. Barron, Chirality, 2012, 24, 879–893; S. M. Morrow, A. J. Bissette and S. P. Fletcher, Nat. Nanotechnol., 2017, 12, 410–419. 22 W. Kuhn, Annu. Rev. Phys. Chem., 1958, 9, 417–438. 23 A. Guerrero, B. Auguie´, J. Alonso-Go´mez, Z. Dzˇolic´, S. Go´mez- Gran˜a, M. M. Cid and L. M. 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The work by the authors’ teams described here was financially supported by the US National Science Foundation (NSF, DMR- 1506018 and DMR-1904091; TH), the Ohio Third Frontier (OTF) program for Ohio Research Scholars: ‘‘Research Cluster on Surfaces in Advanced Materials’’ (TH), and the Italian MIUR (PRIN project 2015XJA9NT; CZ). The authors also acknowledge Hossein Mirzakhani and Diana P. N. Gonçalves for some of the graphical designs. 17 A. P. Almeida, L. Querciagrossa, P. E. S. Silva, F. Gonçalves, J. P. Canejo, P. L. Almeida, M. H. Goghinho and C. Zannoni, Soft Matter, 2019, 15, 2838–2847. 18 P. G. de Gennes and J. Prost, The Physics of Liquid Crystals, Oxford University Press, Oxford, 1993. 19 A. Sharma, T. Mori, H.-C. Lee, M. Worden, E. Bidwell and T. Hegmann, ACS Nano, 2014, 8, 11966–11976. 20 G. Solladie´ and R. Zimmermann, Angew. Chem., Int. Ed. Engl., 1984, 23, 348. Open Access Article. Published on 04 March 2022. 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This methodology provides a broadly expandable tool to a priori predict experimental chirality trans- fer eﬃcacy data. Thus, the dense network of chiral molecules on the surface of the gold nanoshapes is the key driving force for their unusually high ability to transfer chirality to a surrounding achiral N-LC medium, fundamentally aﬀected by shape con- gruency. Yet, any further enhancement of the chirality transfer eﬃcacy can likely be accomplished by introducing nanoshapes featuring both a chiral core and an IPR2D value matching the one of the N-LC host, which is what current experiments in our labs are focusing on. Such intrinsically chiral cores have been reported for a number of anisometric nanomaterials including, In the described N-LC, this opportunity is realized by measuring the induced helical pitch and calculating the helical twisting power in an induced N*-LC medium for essentially any possible nanoshape varying in size, shape, or AR; all without 3352 \| Mater. Adv., 2022, 3, 3346–3354 © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Conflicts of interest 14 M. Grzelczak, J. Pe´rez-Juste, P. Mulvaney and L. M. Liz- Marza´n, Chem. Soc. Rev., 2008, 37, 1783–1791. Access Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 10 Y. Kim, B. Yeom, O. Arteaga, S. J. Yoo, S.-G. Lee, J.-G. Kim and N. A. Kotov, Nat. Mater., 2016, 15, 461–468. Open Access Article. Published on 04 March 2022. Downloaded on 10/24/2024 5:40:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. and N. A. Kotov, Nat. Mater., 2016, 15, 461–468 11 H.-E. Lee, H.-Y. Ahn, J. Mun, Y. Y. Lee, M. Kim, N. H. Cho, K. Chang, W. S. Kim, J. Rho and K. T. Nam, Nature, 2018, 556, 360–365. Perspective for example, in a recent report by Ben-Moshe and colleagues for bipyramidal tellurium28 or Markovich et al. for rod-like Eu3+-doped terbium phosphate nanocrystals.29 Given the recently increasing number of articles citing shape compatibility arguments,30 recognized for simple molecular LC systems by Feringa and co-workers years ago,31 we foresee that the utility of the mathematical and computational concepts described here may soon be significantly advanced by machine learning and artificial intelligence (AI) strategies, and as such support familiar ‘you cannot put a square peg in a round hole’ metaphors for molecular and nanoscale chirality transfer systems. Ultimately, a further expansion of these concepts to lyotropic LC systems32,33 that are biologically significantly more relevant as well as to applications seems all but inevitable.34,35 7 T. P. Yoon and E. N. Jacobsen, Science, 2003, 299, 1691–1693; A. Pfaltz and W. J. Drury III, Proc. Natl. Acad. Sci. U. S. 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Bergquist and T. Hegmann, ChemNanoMat, 2017, 3, 863–868. 34 K. Perera, A. Nemati, E. Mann, T. Hegmann and A. Ja´kli, ACS Appl. Mater. Interfaces, 2021, 13, 4574–4582. 35 D. P. N. Gonçalves, M. E. Pre´voˆt, S. U¨stu¨nel, T. Ogolla, A. Nemati, S. Shadpour and T. Hegmann, Liq. Cryst. Rev., 2021, 9, 1–34. 3354 \| Mater. Adv., 2022, 3, 3346–3354 © 2022 The Author(s). Published by the Royal Society of Chemistry Materials Advances Ed., 2020, 59, 17600–17606; A. B. George and K. S. Korelev, PLoS Comput. Biol., 2018, 14, e1006645. 33 L. Bergquist and T. Hegmann, ChemNanoMat, 2017, 3, 863–868. 31 N. Katsonis, A. Minoia, T. Kudernac, T. Mutai, H. Xu, H. Uji-i, R. Lazzaroni, S. De Feyter and B. Feringa, J. Am. Chem. Soc., 2008, 130, 386–387. 34 K. Perera, A. Nemati, E. Mann, T. Hegmann and A. Jakli, ACS Appl. Mater. Interfaces, 2021, 13, 4574–4582. 35 D. P. N. Gonçalves, M. E. Pre´voˆt, S. U¨stu¨nel, T. Ogolla, A. Nemati, S. Shadpour and T. Hegmann, Liq. Cryst. Rev., 2021, 9, 1–34. 32 S. 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https://openalex.org/W3117602214	https://bmjopengastro.bmj.com/content/bmjgast/7/1/e000497.full.pdf	English	null	Sirolimus for the treatment of polyposis of the rectal remnant and ileal pouch in four patients with familial adenomatous polyposis: a pilot study	BMJ open gastroenterology	2,020	cc-by	4,588	Design Patients with FAP with InSiGHT Polyposis Staging System 3 of the retained rectum or pouch received sirolimus for 6 months, dosed at plasma concentration levels of 5–8 µg/L. Primary outcomes were safety and change in marked polyp size. Secondary outcomes were change in number of polyps and effect on proliferation and apoptosis assessed by immunohistochemistry. VHR and BJM contributed equally. Results Each of the included four patients reported 4 to 18 adverse events (toxicity grades 1–3). One patient prematurely terminated the study because of adverse events. Marked polyp size decreased in 16 (80%)/20 and remained the same in 4 (20%)/20 patients. The number of polyps decreased in all patients (MD −25.75, p=0.13). Three out of four patients showed substantial induction of apoptosis or inhibition of proliferation. Received 24 July 2020 Revised 19 October 2020 Accepted 3 November 2020 Received 24 July 2020 Revised 19 October 2020 Accepted 3 November 2020 Conclusion Six months of sirolimus treatment in four patients with FAP showed promising effects especially on the number of polyps in the rectal remnant and ileal pouch, although at the cost of numerous adverse events. Trial registration number ClinicalTrials.gov ID NCT03095703. n October 23, 2024 by guest. Protected Roos VH, et al. BMJ Open Gastro For numbered affiliations see end of article. Correspondence to Dr Evelien Dekker; e.dekker@amsterdamumc.nl © Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ. Sirolimus has been widely investigated in human studies as an immunosuppressive agent in patients with renal transplantations. In patients with FAP, only low-dose sirolimus (0.05–0.1 mg/kg) treatment has been studied in two children precolectomy and showed to reduce both the size of duodenal and colonic adenomas as well as the severity in dysplasia.9 The aim of our pilot study was to investigate the safety of sirolimus and the effect on the ABSTRACT To cite: Roos VH, Meijer BJ, Kallenberg FGJ, et al. Sirolimus for the treatment of polyposis of the rectal remnant and ileal pouch in four patients with familial adenomatous polyposis: a pilot study. BMJ Open Gastro 2020;7:e000497. doi:10.1136/ bmjgast-2020-000497 development of colorectal cancer, inter- national guidelines recommend a prophy- lactic colectomy.2 3 However, after removal of the colon, adenomas and carcinomas still continue to develop in the remaining large and small intestine. Therefore, lifelong endo- scopic surveillance is recommended. Objective After prophylactic colectomy, adenomas continue to develop in the remaining intestine of patients with familial adenomatous polyposis (FAP). There is a lack of standard clinical recommendation for chemoprevention in patients with FAP. Because of promising in vivo studies, the aim of this pilot study was to investigate the safety of sirolimus and its effect on progression of intestinal adenomas. In recent years several drug therapies aiming to decrease polyp burden and poten- tially delay surgery in patients with FAP have been investigated.4 The APC mutations that initiate adenoma development enhance epithelial proliferation by activating Wnt- signalling and lead to clonal expansion of the mutated epithelial cell. This epithelial growth process is dependent on increased protein translation driven by activity of mammalian target of rapamycin (mTOR) as part of the mTOR complex 1.5 In vivo studies in Apc mutant mice showed that inhibiting mTORC1 signalling with the mTORC1 inhib- itor sirolimus strongly decreased epithelial proliferation and tumour growth in Apc mutant adenomas, while not affecting the proliferation of normal intestinal epithelial cells.5 Treatment with sirolimus not only decreased intestinal adenoma formation but even led to polyp regression.6 7 More- over, sirolimus increased survival and time to progression to dysplasia.6 8 ► ►Additional material is published online only. To view, please visit the journal online (http://dx.doi.org/10.1136/ bmjgast-2020-000497). Sirolimus for the treatment of polyposis of the rectal remnant and ileal pouch in four patients with familial adenomatous polyposis: a pilot study by copyright. on October 23, 2024 by guest. Protected http://bmjopengastro.bmj.com/ ished as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from Victorine H Roos,1 Bartolomeus J Meijer,1 Frank G J Kallenberg,1 Barbara A J Bastiaansen,1 Lianne Koens,2 Frederike J Bemelman,3 Patrick M M Bossuyt,4 Jarom Heijmans,1,5 Gijs van den Brink,1,6 Evelien Dekker ‍ ‍ 1 To cite: Roos VH, Meijer BJ, Kallenberg FGJ, et al. Sirolimus for the treatment of polyposis of the rectal remnant and ileal pouch in four patients with familial adenomatous polyposis: a pilot study. BMJ Open Gastro 2020;7:e000497. doi:10.1136/ bmjgast-2020-000497 Roos VH, et al. BMJ Open Gastro 2020;7:e000497. doi:10.1136/bmjgast-2020-000497 by copyright. on October 23, 2024 by gu http://bmjopengastro.bmj.com/ BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from by copyright. on October 23, 2024 by gu http://bmjopengastro.bmj.com/ BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from by copyright. http://bmjopengastro.bmj.com/ BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from Sirolimus for the treatment of polyposis of the rectal remnant and ileal pouch in four patients with familial adenomatous polyposis: a pilot study Sirolimus for the treatment of polyposis of the rectal remnant and ileal pouch in four patients with familial adenomatous polyposis: a pilot study SU S Demographic characteristics of patients The primary objective was to assess the safety outcomes by analysis of adverse events (by monthly telephone calls), laboratory abnormalities and regular physical examination (during hospital visits at 3 and 6 months). The second primary objective was to evaluate the effect of sirolimus on the size of five marked polyps with an adeno- matous appearance. During baseline LGI endoscopy five polyps between 3 and 10 mm were measured (using an open biopsy forceps) and photo documented. Hereafter these polyps were marked using tattoo dye (SPOT) and photographed with ink marking. Six months after the sirolimus treatment, the polyp size was estimated by the endoscopist using an open biopsy forceps. All endosco- pies were performed by two experienced endoscopists on dedicated FAP programmes. From October 2017 until December 2018, 11 patients were approached for study participation, six patients declined to participate (online supplemental figure 1). Five patients underwent a baseline LGI endoscopy. One of these patients could, despite an IPSS 3, not be included in the study because polyps were too small for marking. Of the four remaining patients, one patient withdrew study participation after 3 months because of adverse events and underwent a LGI endoscopy 2 days after withdrawal. All four patients were Caucasian, two were male and median age was 50 (range 42–60) years. Patients 1, 2 and 3 had undergone a total proctocolectomy with ileal pouch-anal anastomosis, while patient 4 had undergone a subtotal colectomy with ileorectal anastomosis. All patients had an APC mutation in the premutation cluster region (5′ to 1250). Patients 3 and 4 had a previous history of smoking and none of them had a history of desmoids. October 23, 2024 by guest. Protected Secondary objectives were to evaluate the effect of the sirolimus treatment on the number of polyps (only counting these with an adenomatous aspect on optical diagnosis) and global polyp burden at baseline and 6 months. The global polyp burden was assessed by the endoscopist and valued −2 (much better), −1 (better), 0 (same), 1 (worse) or 2 (much worse) relative to the baseline LGI endoscopy. Furthermore, quality of life Statistical analysis Descriptive statistics were used for the demographic char- acteristics, adverse events, change in marked polyp size, number of polyps, global polyp burden and HRQOL questionnaire results. SPSS for Windows software (V.21.0, Chicago, Illinois, USA) was used for the analysis. Study design and patient population l d l Inclusion and exclusion criteria of our prospective pilot study are defined in the online supplemental table 1. We included adult patients with classical FAP and a confirmed APC mutation having IPSS 3 rectal or pouch polyposis . All patients provided written informed consent, and ethical approval was obtained from the Academical Medical Center institutional review board. Participants who withdrew from the study were not replaced. When these participants had been using sirolimus for at least 1 month, a lower gastrointestinal (LGI) endoscopy was arranged within 6 weeks after withdrawal. by copyright. on October 23, 2024 by guest. Protected http://bmjopengastro.bmj.com/ lished as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from Finally, five biopsies of normal appearing mucosa and five biopsies of adenomatous tissue at baseline and after 6 months of sirolimus treatment were taken. Immuno- histochemistry was performed in paraffin-embedded slides according to routine methodology. Per biopsy one image at 10× magnification was processed using Olympus BX51 microscope and quantified with ImageJ (V.1.52a, National Institutes of Health). In a blinded manner, per microscopic field, positive-stained epithelial cells were counted and divided by the amount of crypts present at the microscopic field for pS6 (CST #9205) and cleaved caspase 3 (CST #9661). For Ki67 (DAKO, Glostrup) posi- tive staining was quantified as area in ImageJ and divided by haematoxylin positive staining within the same epithe- lial selected area . INTRODUCTION © Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ. Familial adenomatous polyposis (FAP) is caused by a mutation in the adenomatous polyposis coli (APC) gene and characterised by the development of hundreds to thou- sands of colorectal adenomas. When left untreated, the risk of developing colorectal cancer is nearly 100% with a mean age at diagnosis of 45 years.1 To prevent the y y p The aim of our pilot study was to investigate the safety of sirolimus and the effect on the 1 by copyright. on October 23, 2024 http://bmjopengastro.bmj.com/ BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from Study intervention Eligible patients received sirolimus treatment (2 mg one time per day) for a duration of 6 months. Sirolimus was provided free of charge by Pfizer BV. After 7 to 10 days sirolimus blood levels were measured. If not within the target range of 5–8 µg/L (based on experiences in the field on renal transplantation in the Academic Medical Center), dosing adjustments were made. In case of dosing adjustments, sirolimus level testing was performed 7 to 10 days hereafter until the target range was achieved. If the sirolimus levels were within the target range, level meas- urements were performed at 3 months and 6 months. Drug compliance was assessed by pill count review at 3 and 6 months. Open access was evaluated at baseline, 3 and 6 months using health related quality of life (HRQOL) questionnaires (EORTC QLQ-C30, EORTC QLQ-CR29, SF-36 and EQ-5D-5L). progression of intestinal adenomas in adult patients with FAP with InSiGHT Polyposis Staging System (IPSS) 3 of the retained rectum or pouch, using both clinical as well as molecular outcomes. by copyright. on October 23, 2024 by guest. Protected http://bmjopengastro.bmj.com/ astroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from The size of the marked polyps and total number of polyps were assessed by two independent reviewers (VHR and BJM) scoring the matched still images and videos per patient before and after treatment both blinded for the order. Since the independent reviewers had the same level of experience, the mean size per polyp and mean number of polyps were calculated and presented adjoining the endoscopists review. Clinical outcomes A more detailed overview of the global polyp burden assessed by the endoscopist is shown in the online supplemental table 3. Interestingly, the aspect of several polyps showed a very distinct morphological change after sirolimus treatment demonstrating a more flattened appearance with a central dimple. The morphological change appeared to suggest polyp regression (figure 2A). observations (median difference −12.50, p=0.13, online supplemental figure 3B). The global polyp burden had improved in patients 1, 2 and 3 and had not changed in patient 4. A more detailed overview of the global polyp burden assessed by the endoscopist is shown in the online supplemental table 3. Interestingly, the aspect of several polyps showed a very distinct morphological change after sirolimus treatment demonstrating a more flattened appearance with a central dimple. The morphological change appeared to suggest polyp regression (figure 2A). Each patient reported 4 to 18 adverse events (toxicity grades 1–3). One patient prematurely terminated the study because of adverse events. Size marked polyps decreased in 16 (80%)/20 and remained the same in 4 (20%)/20. The total number of polyps decreased in all patients. Three out of four patients showed substantial induction of apoptosis or inhibition of proliferation. Each patient reported 4 to 18 adverse events (toxicity grades 1–3). One patient prematurely terminated the study because of adverse events. Size marked polyps decreased in 16 (80%)/20 and remained the same in 4 (20%)/20. The total number of polyps decreased in all patients. Three out of four patients showed substantial induction of apoptosis or inhibition of proliferation. The HRQOL questionnaires reported increase in urinary frequency, blood and mucus in the stool, dysgeusia, flatulence, faecal incontinence, stool frequency, decrease in sexual function, increase in fatigue, dyspnoea, insomnia and diarrhoea (online supplemental figure 4). Furthermore, the global health status had decreased in patients 2, 3 and 4. During the study period sirolimus dosing varied from 1.5 mg to 4 mg per day and sirolimus levels varied from 2.87 µg/L to 11.0 µg/L (online supplemental figure 2). A median of six (range 3–8) blood tests for sirolimus level testing were needed per patient. Clinical outcomes Four to 16 adverse events per included patient were reported during 6 months of sirolimus treatment ranging 2 Roos VH, et al. BMJ Open Gastro 2020;7:e000497. doi:10.1136/bmjgast-2020-000497 by copyright. on October 23, 2024 http://bmjopengastro.bmj.com/ BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from by copyright. http://bmjopengastro.bm BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from Figure 1 (A) Marked polyp size before and after sirolimus treatment, shown per patient, according to the matched still images assessed by two independent reviewers. (B) Total number of polyps before and after sirolimus treatment, shown per patient, according to the video observations assessed by two independent reviewers blinded for the order. by copyright. on October 23, 2024 by guest. Protected http://bmjopengastro.bmj.com/ shed as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from Figure 1 (A) Marked polyp size before and after sirolimus treatment, shown per patient, according to the matched still images assessed by two independent reviewers. (B) Total number of polyps before and after sirolimus treatment, shown per patient, according to the video observations assessed by two independent reviewers blinded for the order. from toxicity grade 1 to 2 (online supplemental table 2). Frequencies of adverse events seemed dose-dependent until 3 mg and concentration dependent until 6 µg/L. The events occurred after a median of 59 days (range 2–154). Patient 3 used 2 mg of sirolimus and experienced 18 adverse events during the first 3 months, with toxicity grades varying from 1 to 3, and prematurely termi- nated participation. The adverse events described were predominantly gastrointestinal disorders (29%), related to study investigations (12%) and skin disorders (12%). One serious adverse event was observed in patient 2: the discovery of a desmoid tumour in the abdominal wall (2.7 × 1.7 × 8.0 cm) located near a scar. One year prior to the start of the study no desmoid tumour was observed on a CT scan, performed 2 months after a redo of the pouch because of a dysfunctional pouch. The family history of the patient was positive for desmoid disease. This patient wished to finish the study and subsequently started sulindac treatment (150 mg two times per day), after which the desmoid tumour showed regression. observations (median difference −12.50, p=0.13, online supplemental figure 3B). The global polyp burden had improved in patients 1, 2 and 3 and had not changed in patient 4. Roos VH, et al. BMJ Open Gastro 2020;7:e000497. doi:10.1136/bmjgast-2020-000497 Immunohistochemistry outcomes According to the video observations, all five marked polyps decreased in size in patients 1 and 2, to a lesser extent in patient 3 (4(80%)/5) and patient 4 showed a decrease in only two polyps (figure 1A). These were also shown to a lesser extent in the procedural observations (9 (45%)/20 decrease, 11 (55%)/20 remained the same, online supplemental figure 3A). ctober 23, 2024 by guest. Protected In all patients, adenoma biopsies were evaluated by an experienced pathologist (LK) before and after treatment and demonstrated low-grade dysplasia. As expected, phosphorylation of mTOR downstream signalling target ribosomal protein S6 was decreased in adenomatous tissue compared with adjacent healthy biopsies at base- line as detected with a phosphorylation-specific S6 anti- body. After treatment with sirolimus, phosphorylation of the ribosomal S6 protein decreased in the adenomatous All patients showed a decrease in the total number of polyps both in the video observations (median differ- ence −25.75, p=0.13, figure 1B) as in the procedural 3 Roos VH, et al. BMJ Open Gastro 2020;7:e000497. doi:10.1136/bmjgast-2020-000497 by copyright. on October 23, 2024 http://bmjopengastro.bmj.com/ BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from by copyright. http://b BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from p Figure 2 (A) Aspect of adenoma before and after sirolimus treatment, showing a central depression in the adenoma in patient 1 and demonstrating a more flattening aspect of an adenoma after treatment in patient 3. (B–D) Representative image of phosphorylated S6, Ki67 and cleaved caspase 3 throughout adenomatous tissue, shown for patient 2. (E) Left panel: quantification of pS6 positive cells per 10× microscopic field. Middle panel: quantification of Ki67 relative to haematoxylin staining for selected epithelium within a 10× microscopic field. Right panel: quantification of cleaved caspase 3 positive cells per 10× microscopic field. by copyright. J Open Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded f by copyright. on October 23, 2024 by guest. Protected http://bmjopengastro.bmj.com/ Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from Figure 2 (A) Aspect of adenoma before and after sirolimus treatment, showing a central depression in the adenoma in patient 1 and demonstrating a more flattening aspect of an adenoma after treatment in patient 3. (B–D) Representative image of phosphorylated S6, Ki67 and cleaved caspase 3 throughout adenomatous tissue, shown for patient 2. Immunohistochemistry outcomes (E) Left panel: quantification of pS6 positive cells per 10× microscopic field. Middle panel: quantification of Ki67 relative to haematoxylin staining for selected epithelium within a 10× microscopic field. Right panel: quantification of cleaved caspase 3 positive cells per 10× microscopic field. reduced the number of polyps, could improve the global polyp burden and decreased the marked polyp size in some patients, in contrast to expected stable or progres- sive disease.9 Although sirolimus dosing was in compa- rable therapeutic range, patients in our study suffered from considerably more adverse events. According to the literature in renal transplant patients, adverse events are influenced by sirolimus levels of >15 µg/L.10 In this study, measured concentrations were lower than 11.0 µg/L and only showed dose and concentration dependency up to a certain level. Furthermore, the majority of the adverse events were transient in nature while adminis- tration of sirolimus was continued, suggesting no direct dose or concentration relationship using the target range of 5–8 µg/L. It is unknown if the desmoid tumour that occurred was already present at the start of the study. However, the recent operation and positive family history suggest this to be a result of the nature of the underlying disease. tissue in all four patients, suggesting that the sirolimus dose was adequate for mTORC1 target engagement and inhibition (figure 2B,E). The effect of mTORC1 inhibi- tion on proliferation was assessed by immunostaining of Ki67 in the intestinal epithelium. Changes in prolifera- tion were heterogeneous throughout the biopsies of all patients. Nevertheless, the largest and most consistent reduction of proliferation was found in patients 1 and 2 corresponding to the decrease in adenoma numbers (figure 2C,E). Furthermore, apoptosis was addressed by cleaved caspase 3 staining and showed most apoptosis in patients 2 and 3 (figure 2D,E). on October 23, 2024 by guest. Protected Roos VH, et al. BMJ Open Gastro 2020;7:e000497. doi:10.1136/bmjgast-2020-000497 ORCID iD ORCID iD Evelien Dekker http://orcid.org/0000-0002-4363-0745 ORCID iD Evelien Dekker http://orcid.org/0000-0002-4363-0745 REFERENCES 1 Bussey HJR. Familial polyposis coli. family studies, histopathology, differential diagnosis, and results of treatment, 1975. 1 Bussey HJR. Familial polyposis coli. family studies, histopathology, differential diagnosis, and results of treatment, 1975. Author affiliations 1 2 Balmaña J, Castells A, Cervantes A, et al. Familial colorectal cancer risk: ESMO clinical practice guidelines. Ann Oncol 2010;21 Suppl 5:v78–81. i 1Department of Gastroenterology and Hepatology, Amsterdam UMC - Location AMC, University of Amsterdam, Cancer Center Amsterdam, Amsterdam, North Holland, The Netherlands 1Department of Gastroenterology and Hepatology, Amsterdam UMC - Location AMC, University of Amsterdam, Cancer Center Amsterdam, Amsterdam, North Holland, The Netherlands 3 Syngal S, Brand RE, Church JM, et al. Acg clinical guideline: genetic testing and management of hereditary gastrointestinal cancer syndromes. Am J Gastroenterol 2015;110:223–62. 2Department of Pathology, Amsterdam UMC - Location AMC, University of Amsterdam, Amsterdam, North Holland, The Netherlands y 4 Kemp Bohan PM, Mankaney G, Vreeland TJ, et al. Chemoprevention in familial adenomatous polyposis: past, present and future. Fam Cancer 2020:1–11. 3Department of Internal Medicine, Division of Nephrology, Amsterdam UMC - Location AMC, University of Amsterdam, Amsterdam, North Holland, The Netherlands 5 Faller WJ, Jackson TJ, Knight JR, et al. Mtorc1-Mediated translational elongation limits intestinal tumour initiation and growth. Nature 2015;517:497–500. 4Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Amsterdam UMC - Location AMC, University of Amsterdam, Amsterdam, North Holland, The Netherlands 6 Hardiman KM, Liu J, Feng Y, et al. Rapamycin inhibition of polyposis and progression to dysplasia in a mouse model. PLoS One 2014;9:e96023. 6 Hardiman KM, Liu J, Feng Y, et al. Rapamycin inhibition of polyposis and progression to dysplasia in a mouse model. PLoS One 2014;9:e96023. 5Department of Internal Medicine and Hametology, Amsterdam UMC - Location AMC, University of Amsterdam, Amsterdam, North Holland, The Netherlands 6Roche Innovation Center Basel, Roche, Basel, Basel-Stadt, Switzerland 5Department of Internal Medicine and Hametology, Amsterdam UMC - Location AMC, University of Amsterdam, Amsterdam, North Holland, The Netherlands 6Roche Innovation Center Basel, Roche, Basel, Basel-Stadt, Switzerland 7 Miller SJ, Heist KA, Feng Y, et al. Multimodal imaging of growth and rapamycin-induced regression of colonic adenomas in APC mutation-dependent mouse. Transl Oncol 2012;5:313–20. 7 Miller SJ, Heist KA, Feng Y, et al. Multimodal imaging of growth and rapamycin-induced regression of colonic adenomas in APC mutation-dependent mouse. Transl Oncol 2012;5:313–20. 8 Koehl GE, Spitzner M, Ousingsawat J, et al. Rapamycin inhibits oncogenic intestinal ion channels and neoplasia in APC(Min/+) mice. Oncogene 2010;29:1553–60. DISCUSSION Data availability statement Data are available upon reasonable request. Data availability statement Data are available upon reasonable request. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. In conclusion, maintenance therapy with sirolimus at a target plasma concentration of 5–8 µg/L is sufficient to inhibit mTORC1 in intestinal adenomas, reduces the number of polyps and leads to a reduction in prolifera- tion. This recapitulates the effect of sirolimus treatment observed in Apc mutant mice. To overcome the relatively poor tolerability of sirolimus using this therapeutic target range in these patients, strategies to reduce side effects, such as lowering the sirolimus target range in a phase IIb study, use of an mTOR inhibitor with better tolerability or ideally the development of a gut-targeted inhibitor of mTOR could be considered. Nevertheless, our prom- ising results suggest that these efforts could lead to a new chemopreventive approach for patients with FAP. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. DISCUSSION The current study is the first to evaluate the effect of sirolimus treatment in adults with FAP having IPSS 3 staged rectal remnant or pouch polyposis using both clinical as well as molecular outcomes. Being a pilot study, power was limited and the study had no comparator group. Nevertheless, outcomes were well defined and prespeci- fied and assessment of outcomes was blinded. Inline with the case report of Yuksekkaya et al we found that sirolimus Immunohistochemistry showed an decreased expres- sion of S6 phosphorylation in adenomatous tissue in all four patients, comparable to the studies in an Apc Roos VH, et al. BMJ Open Gastro 2020;7:e000497. doi:10.1136/bmjgast-2020-000497 4 BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 2 by copyright. on October 23, 2024 by g http://bmjopengastro.bmj.com/ BMJ Open Gastroenterol: first published as 10.1136/bmjgast-2020-000497 on 29 December 2020. Downloaded from Open access intellectual content. Final approval of the article was given by VHR, BJM, FGJK, BAJB, FJB, PMMB, JH, GRdB and ED. intellectual content. Final approval of the article was given by VHR, BJM, FGJK, BAJB, FJB, PMMB, JH, GRdB and ED. mutant mouse model of FAP.2 The reduction of phos- phorylated S6 during sirolimus treatment suggests that the target plasma concentration resulted in adequate target engagement and mTORC1 inhibition in the intes- tinal adenomas. Effects on proliferation however were heterogeneous. Patients 1 and 2 showed more promising results on adenoma numbers and proliferation rate than patients 3 and 4. Patient 3 had stopped sirolimus treat- ment for several days before colonoscopy, potentially explaining the lack of impaired proliferation. Patient 4 did not seem to benefit from sirolimus treatment. Although we observed increased apoptotic cell death in adenomas, this varied per patient and may have been influenced by sample variance. Funding Sirolimus was kindly provided free of charge by Pfizer BV. Funding Sirolimus was kindly provided free of charge by Pfizer BV. Competing interests ED: Endoscopic equipment on loan of FujiFilm, a research grant from FujiFilm. Honorarium for consultancy from FujiFilm, Olympus, Tillots, GI Supply and CPP-FAP and speakers' fee from Olympus, Roche and GI Supply. Competing interests ED: Endoscopic equipment on loan of FujiFilm, a research grant from FujiFilm. Honorarium for consultancy from FujiFilm, Olympus, Tillots, GI Supply and CPP-FAP and speakers' fee from Olympus, Roche and GI Supply. Patient consent for publication Not required. Provenance and peer review Not commissioned; externally peer reviewed. Roos VH, et al. BMJ Open Gastro 2020;7:e000497. doi:10.1136/bmjgast-2020-000497 Acknowledgements We would like to thank Christine Cohen, research coordinator, for her contributions. Acknowledgements We would like to thank Christine Cohen, research coordinator, for her contributions. Acknowledgements We would like to thank Christine Cohen, research coordinator, for her contributions. 9 Yuksekkaya H, Yucel A, Gumus M, et al. Succesful use of sirolimus. Am J Gastroenterol 2016;111:1040–1. 9 Yuksekkaya H, Yucel A, Gumus M, et al. Succesful use of sirolimus. Am J Gastroenterol 2016;111:1040–1. Contributors VHR, BJM, FGJK, BAJB, FJB, GvdB and ED helped in conception and design. Analysis and interpretation of the data were done by VHR, BJM, PMMB, JH, GRdB and ED. Drafting of the article was done by VHR and BJM. FGJK, BAJB, FJB, PMMB, JH, GRdB and ED performed critical revision of the article for important on October 23, 2024 by guest. Protected com/ 10 Stenton SB, Partovi N, Ensom MHH. Sirolimus: the evidence for clinical pharmacokinetic monitoring. Clin Pharmacokinet 2005;44:769–86. ber 23, 2024 by guest. Protected 5 Roos VH, et al. BMJ Open Gastro 2020;7:e000497. doi:10.1136/bmjgast-2020-000497
https://openalex.org/W3195681072	https://europepmc.org/articles/pmc8431985?pdf=render	English	null	Absent Internal Carotid Artery With Intact Circle of Willis	Curēus	2,021	cc-by	3,087	Review began 07/15/2021 Review ended 08/02/2021 Published 08/11/2021 © Copyright 2021 Sonne et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 4.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Open Access Case Report Open Access Case Report DOI: 10.7759/cureus.17090 Absent Internal Carotid Artery With Intact Circle of Willis James W. Sonne , Helen Kaiser , Shanna Williams 1 1 1 1. Biomedical Sciences, University of South Carolina School of Medicine Greenville, Greenville, USA 1. Biomedical Sciences, University of South Carolina School of Medicine Greenville, Greenville, USA Corresponding author: James W. Sonne, jamessonne@gmail.com Introduction The internal carotid arteries bifurcate from the common carotid arteries to traverse the carotid canals and eventually provide blood supply, along with the vertebral arteries, to the brain by way of the Circle of Willis [1]. Branches directly from the internal carotid arteries supply a number of critical structures including the pituitary gland from the superior hypophyseal artery, and the orbit and eye from the ophthalmic artery. Collateral circulation from the bilateral internal carotid arteries and vertebral arteries provides redundant irrigation to the brain in cases of stenosis or temporary, acute occlusion during regular head movements. Developmentally, the internal carotid arteries arise from the third aortic arch within the third pharyngeal arch. This third pharyngeal arch is also responsible for the development of a portion of the hyoid bone, the stylopharyngeus muscle, and the glossopharyngeal nerve (CN IX) responsible for motor and sensory innervation of portions of the visceral neck. The third aortic arch combines with the dorsal aorta of the aortic sac of the developing heart to eventually form the ascending, distal portion of the internal carotid artery [2]. As with any developmental event, functional or morphological errors can result. In the case of the internal carotid artery, hypoplasia or even complete agenesis has been known to occur [3]. Because of the numerous and sometimes tortuous branches of the cervical neck and internal carotid artery, the absence of the internal carotid artery can lead to compensation through hyperplasias, rare anastomoses, and fistulas [4]; while also being correlated with issues of cerebral circulation including aneurysm or thromboembolism [5]. In this article, we describe a case of left-sided agenesis of the internal carotid artery verified by absent ipsilateral carotid canal identified during routine cadaveric dissection for medical education. Hyperplasia of the vertebral, basilar, and contralateral internal carotid arteries was present; but no other morphological variations or remnant arteries were identified, and a complete Circle of Willis was present. While previous cases have been reported in the scientific literature, these features represent a unique presentation that does not appear to be previously described [4, 6-7]. Furthermore, we discuss potential anastomotic pathways to the orbit, eye, and hypophysis, along with the clinical relevance of these circulatory patterns. Abstract The internal carotid arteries are one of the primary suppliers of the Circle of Willis and cerebral blood flow, but the rare case of agenesis of the internal carotid artery can impair the functional redundancies of cerebral blood supply. In this study, routine, medical education-focused cadaveric dissection of an 80-year-old male cadaver (cause of death was ventricular tachycardia) was performed. A case of agenesis of the left internal carotid artery and the carotid canal was identified. Upon investigation, we found that the compensatory pattern of irrigation in the Circle of Willis did not conform to previously described cases in the scientific literature. Further literature review suggested that such agenesis can be associated with a wide range of conditions from stroke, migraine, tinnitus, and Horner’s syndrome. Due to the altered blood flow pattern, we caution the reading physician regarding the potential for ischemia and iatrogenic damage, particularly of the pituitary gland and eye. We suggest the use of neuroangiographic imaging in cases of agenesis of an internal carotid artery. Categories: Medical Education, Neurosurgery, Anatomy Keywords: agenesis, internal carotid artery, circle of willis, cerebral blood flow, hypophysis How to cite this article Sonne J W, Kaiser H, Williams S (August 11, 2021) Absent Internal Carotid Artery With Intact Circle of Willis. Cureus 13(8): e17090. DOI 10.7759/cureus.17090 © Copyright 2021 Sonne et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 4.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Case Presentation Case Presentation Routine dissection of 10 human cadavers was performed in the educational gross anatomy laboratory of the authors’ institution between August 2020 and January 2021. Rotations of M1 medical students, assisted by M4s and the anatomy module faculty performed the dissections guided by provided laboratory images. During the final unit of the module, head and neck dissection was performed following thoracic dissection How to cite this article Sonne J W, Kaiser H, Williams S (August 11, 2021) Absent Internal Carotid Artery With Intact Circle of Willis. Cureus 13(8): e17090. DOI 10.7759/cureus.17090 How to cite this article Sonne J W, Kaiser H, Williams S (August 11, 2021) Absent Internal Carotid Artery With Intact Circle of Willis. Cureus 13(8): e17090. DOI 10 7759/cureus 17090 and prior to craniotomy and brain removal. During routine dissection, a notable absence of the left internal carotid artery was identified (Figure 1) in an 80-year-old, male cadaver with ventricular tachycardia listed as the cause of death. The left common carotid artery branched from the aortic arch to ascend within the carotid sheath, eventually giving off all notable branches of the external carotid artery without exception. However, no bifurcation to form the left internal carotid artery was present. The gross dilation typically attributed to the carotid sinus was present at the level of the superior thyroid artery. Components of the third pharyngeal arch, including the stylopharyngeus and glossopharyngeal nerves, were identified bilaterally from a retropharyngeal view. The hyoid bone was present without notable variation. of the superior thyroid artery. Components of the third pharyngeal arch, including the stylopharyngeus and glossopharyngeal nerves, were identified bilaterally from a retropharyngeal view. The hyoid bone was present without notable variation. FIGURE 1: Dissection of the left neck. Dissection of the left neck revealing the complete agenesis of the left internal carotid artery. (Line art courtesy of Sara DeHaan.) Upon craniotomy and removal of the brain, the vertebral and basilar arteries were notably enlarged, as was the contralateral (right) internal carotid artery. The left carotid canal was absent in the left middle cranial fossa, while the sphenoid foramen was easily identified. Aside from marked hyperplasia of the vertebral, basilar, and right internal carotid artery, the Circle of Willis and related branches were present without notable variation (Figure 2). No additional anastomoses or persistent fetal intracranial arteries were identifiable. FIGURE 2: Ventral view of the brain with Circle of Willis. View of the intact Circle of Willis with absent left internal carotid artery. (Line art courtesy of Sara DeHaan.) 759/cureus.17090 FIGURE 1: Dissection of the left neck. Dissection of the left neck revealing the complete agenesis of the left internal carotid artery. (Line art courtesy of Sara DeHaan.) FIGURE 1: Dissection of the left neck. Dissection of the left neck revealing the complete agenesis of the left internal carotid artery. (Line art courtesy of Sara DeHaan.) Upon craniotomy and removal of the brain, the vertebral and basilar arteries were notably enlarged, as was the contralateral (right) internal carotid artery. The left carotid canal was absent in the left middle cranial fossa, while the sphenoid foramen was easily identified. 2021 Sonne et al. Cureus 13(8): e17090. DOI 10.7759/cureus.17090 Discussion In the case of bilateral agenesis of the internal carotid artery, the blood supply from dural branches of the middle meningeal artery is feasible, as anastomoses at this point have been known to occur [6, 8]. Clinically, the absence of the internal carotid artery has been correlated with a number of conditions, likely contingent upon the precise nature of the absence and effectiveness of any collateral circulation developed. Pulsatile tinnitus, ischemic stroke, migraine, Horner’s syndrome, and subarachnoid hemorrhage have all been reported in the scientific literature [9]. Reported single cases associated with other congenital conditions have included Goldenhar syndrome [4] and Klippel-Trenaunay syndrome [10], although the statistical correlation with such syndromes is unknown. Depending upon the exact nature of the blood supply of the hypophysis, the agenesis of the complete internal carotid artery may cause the pituitary gland to become increasingly susceptible to ischemic damage. Also, it seems likely that developmental disorders of the third pharyngeal arch would likely be associated with agenesis of the internal carotid artery and structures of similar embryologic origin, although that was not the case described herein. In addition, the variation of the internal carotid artery can impact blood flow during manual therapy intervention in physical rehabilitation [11]. The absence of the internal carotid artery is of critical importance for surgical considerations. While some patients with this condition may present asymptomatically, the sufficiency of cerebral blood flow may become dependent upon the vertebral artery or the contralateral, intact internal carotid artery. During procedures such as endarterectomy for the removal of atherosclerotic plaque, cerebral blood flow may become severely impacted having drastic consequences. Understanding blood supply to the pituitary gland is also critical due to the relatively common occurrence of hypophyseal tumors [9] and the limited redundancy in irrigation. As the blood supply to the orbit may be complicated and potentially traverse unusual locations such as the cavernous sinus and the dura mater, or form anastomoses with the middle meningeal artery, surgeries or thrombic events may inadvertently impact such a patient’s vision or pituitary function. As a single case, this report has clear limitations, including its lack of ability to determine prevalence. The routine nature of the dissection for medical education prohibited prolonged and detailed investigation required to identify collateral blood supply noted here in the Discussion. Discussion In this report, we have described a case of a complete aplasia of the left internal carotid artery with an intact Circle of Willis. A non-systematic search of relevant literature was performed using keywords including “internal carotid artery”, “aplasia”, and/or “agenesis” on the NIH NLM NCBI “PubMed” database and Google Scholar, with relevant articles identified by title and abstract. Furthermore, “Bergman’s Comprehensive Encyclopedia of Human Anatomic Variation” [6] was consulted. The case we present here differs from identified variants previously described in the scientific literature [4, 6-7]. The case presented includes a complete Circle of Willis, no persistent developmental branches or unusual intracranial anastomoses, or does it include an intracranial portion of the absent left internal carotid artery. This excluded association with the reported Types A, B, C, and E. Instead, the left portion of the Circle of Willis continues from the middle cerebral artery to merge directly with the posterior communicating artery posteriorly and the anterior cerebral artery anteriorly. In this case, there were no cavernous or intraosseous/petrous portions of the left internal carotid artery. This precluded the possibility of Type D and Type F variants. Without any portion of the internal carotid artery, including the cervical, petrous, or cavernous portions, the question regarding blood supply of the orbit, eye, and hypophysis arise. Typically, the hypophysis is supplied from the superior and inferior hypophyseal arteries which branch from the cerebral and cavernous portions of the internal carotid artery. The orbit and eye are supplied through the ophthalmic artery, branching from the cerebral portion. Of note, no ophthalmic artery was observed branching from the Circle of Willis or any cerebral artery, suggesting the morphologically normal left orbit and eye were supplied from another source. We presume this source in this case to be a branch of the maxillary artery ascending through the inferior orbital fissure; however, it is reportedly more common to find a middle meningeal artery origin of the ophthalmic artery [2]. Another possibility involves a persistent trigeminal artery from development, which was not present in this case. We presume the source of blood supply to the pituitary was likely from branches of the contralateral internal carotid artery within the cavernous sinus, as anastomoses with the contralateral pituitary blood supply are common [6]. Aside from marked hyperplasia of the vertebral, basilar, and right internal carotid artery, the Circle of Willis and related branches were present without notable variation (Figure 2). No additional anastomoses or persistent fetal intracranial arteries were identifiable. FIGURE 2: Ventral view of the brain with Circle of Willis. View of the intact Circle of Willis with absent left internal carotid artery. (Line art courtesy of Sara DeHaan.) FIGURE 2: Ventral view of the brain with Circle of Willis. View of the intact Circle of Willis with absent left internal carotid artery. (Line art courtesy of Sara DeHaan.) 2 of 4 2021 Sonne et al. Cureus 13(8): e17090. DOI 10.7759/cureus.17090 A literature review identified reports of agenesis of the internal carotid arteries, noting various types “A- F” [4, 7]. Types A and B describe a broken or incomplete Circle of Willis. Type C represents bilateral underdevelopment of the internal carotid arteries, while type E is bilateral hypoplasia of the internal carotid arteries. Type D includes an anastomosis of the cavernous portion of the internal carotid arteries. Type F includes a rete mirabile network of vasculature connecting the external carotid artery with a short internal carotid artery within the cranium. Additional types reported include persistent segmental or developmental arteries that often branch from the basilar artery to form an internal carotid artery within the cranium. 2021 Sonne et al. Cureus 13(8): e17090. DOI 10.7759/cureus.17090 Discussion Comprehensive, multi-center retrospective studies of neuroangiographic images may provide detailed information regarding the prevalence of agenesis of one or both internal carotid arteries and allow for statistical association with clinical presentations and medical conditions. Collectively, the three professional anatomists involved in this case have dissected an estimated total of 500 unique cadavers, and this is the first instance in which an internal carotid artery and carotid canal were not developed. 3 of 4 2021 Sonne et al. Cureus 13(8): e17090. DOI 10.7759/cureus.17090 References 1. Sethi D, Gofur EM, Munakomi S: Anatomy, Head and Neck, Carotid Arteries. StatPearls Publishing, Treasure Island, FL; 2021. 1. Sethi D, Gofur EM, Munakomi S: Anatomy, Head and Neck, Carotid Arteries. StatPearls Publishing, Treasure Island, FL; 2021. 1. Sethi D, Gofur EM, Munakomi S: Anatomy, Head and Neck, Carotid Arteries. StatPearls Publishing, Treasure Island, FL; 2021. 2. Rosen RD, Bordoni B: Embryology, Aortic Arch. StatPearls Publishing, Treasure Island, FL; 2021. 3. Rosen IW, Mills DF, Nadel HI, Kaiserman DD: Angiographic demonstration of congenital absence of both internal carotid arteries. Case report. J Neurosurg. 1975, 42:478-482. 10.3171/jns.1975.42.4.0478 4. Ottaviano G, Calzolari F, Martini A: Goldenhar syndrome in association with agenesia of the internal caroti artery. Int J Pediatr Otorhinolaryngol. 2007, 71:509-512. 10.1016/j.ijporl.2006.11.003 y y g j jp 5. Given CA 2nd, Huang-Hellinger F, Baker MD, Chepuri NB, Morris PP: Congenital absence of the internal carotid artery: case reports and review of the collateral circulation. Am J Neuroradiol. 2001, 22:1953-1959. 6. Zink WE, Komotar RJ, Meyers PM: Internal carotid aplasia/hypoplasia and intracranial saccular aneurysms: series of three new cases and systematic review of the literature J Neuroimaging 2007 17:141-147 5. Given CA 2nd, Huang-Hellinger F, Baker MD, Chepuri NB, Morris PP: Congenital absence of the internal carotid artery: case reports and review of the collateral circulation. Am J Neuroradiol. 2001, 22:1953-1959. , g g , , p , g carotid artery: case reports and review of the collateral circulation. Am J Neuroradiol. 2001, 22:1953-1959. 6. Zink WE, Komotar RJ, Meyers PM: Internal carotid aplasia/hypoplasia and intracranial saccular aneurysms: series of three new cases and systematic review of the literature. J Neuroimaging. 2007, 17:141-147. 10.1111/j.1552-6569.2007.00092.x 6. Zink WE, Komotar RJ, Meyers PM: Internal carotid aplasia/hypoplasia and intracranial saccular aneurysms: series of three new cases and systematic review of the literature. J Neuroimaging. 2007, 17:141-147. 10.1111/j.1552-6569.2007.00092.x 7. Bergman RA, Tubbs RS, Shoja MM, Loukas M: Bergman's comprehensive encyclopedia of human anatomic variation.. John Wiley & Sons, Inc., Hoboken, NJ; 2016. 8. Li S, Hooda K, Gupta N, Kumar Y: Internal carotid artery agenesi Neuroradiol J. 2017, 30:186-191. 10.1177/1971400917692162 8. Li S, Hooda K, Gupta N, Kumar Y: Internal carotid artery agenesis: a case report and review of literature . Neuroradiol J. 2017, 30:186-191. 10.1177/1971400917692162 9. Cohen JE, Gomori JM, Leker RR: Internal carotid artery agenesis: diagnosis, clinical spectrum, associated conditions and its importance in the era of stroke interventions. Neurol Res. 2010, 32:1027-1032. Acknowledgements We would like to recognize the contribution of the body donor and family of the decedent for their contribution to science and medical education. In addition, we would like to acknowledge the following students in alphabetical order who assisted in cadaveric dissection of this body: Adriana Rush, Ana Balcazar, Anna Hayden, Ben Dalkin, Hunter Owen, Jack Mobley, J.J. Jenkins, Madeline Holt, Maggie Sulzbach, Margaret Adams, Michael Colpoys, Sarah Baum, Shannon Smith, Tommy Tedeschi, Wade Miller, and Yomi Sanni. Finally, the authors would like to thank Sara DeHaan (@artbysdehaannn) for medical illustrations. Disclosures Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. Conclusions This cadaveric case report describes a seemingly unique variant of the cerebral blood flow in association with agenesis of one of the internal carotid arteries and carotid canal. We performed a scholarly literature review, and suggest that such a condition can have drastic implications for cranial surgery and ischemic events, including the pituitary gland and eye. Multi-center studies of angiographic imaging are recommended to determine prevalence and associated clinical presentation. 2021 Sonne et al. Cureus 13(8): e17090. DOI 10.7759/cureus.17090 References 10.1179/016164110X12767786356273 10. Goldstein SJ, Lee C, Young AB, Guidry GJ: Aplasia of the cervical internal carotid artery and malformation of the circle of Willis associated with Klippel-Trenaunay syndrome. Case report. J Neurosurg. 1984, 61:786- 789. 10.3171/jns.1984.61.4.0786 11. Thomas LC, Rivett DA, Bateman G, Stanwell P, Levi CR: Effect of selected manual therapy interventions for mechanical neck pain on vertebral and internal carotid arterial blood flow and cerebral inflow. Phys Ther. 2013, 93:1563-1574. 10.2522/ptj.20120477 4 of 4
https://openalex.org/W2546135632	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0164867&type=printable	English	null	Improved Auditory Nerve Survival with Nanoengineered Supraparticles for Neurotrophin Delivery into the Deafened Cochlea	PloS one	2,016	cc-by	9,832	RESEARCH ARTICLE OPEN ACCESS Citation: Wise AK, Tan J, Wang Y, Caruso F, Shepherd RK (2016) Improved Auditory Nerve Survival with Nanoengineered Supraparticles for Neurotrophin Delivery into the Deafened Cochlea. PLoS ONE 11(10): e0164867. doi:10.1371/journal. pone.0164867 Cochlear implants electrically stimulate spiral ganglion neurons (SGNs) in order to provide speech cues to severe-profoundly deaf patients. In normal hearing cochleae the SGNs depend on endogenous neurotrophins secreted by sensory cells in the organ of Corti for survival. SGNs gradually degenerate following deafness and consequently there is consid- erable interest in developing clinically relevant strategies to provide exogenous neurotro- phins to preserve SGN survival. The present study investigated the safety and efficacy of a drug delivery system for the cochlea using nanoengineered silica supraparticles. In the present study we delivered Brain-derived neurotrophic factor (BDNF) over a period of four weeks and evaluated SGN survival as a measure of efficacy. Supraparticles were bilater- ally implanted into the basal turn of cochleae in profoundly deafened guinea pigs. One ear received BDNF-loaded supraparticles and the other ear control (unloaded) supraparticles. After one month of treatment the cochleae were examined histologically. There was signifi- cantly greater survival of SGNs in cochleae that received BDNF supraparticles compared to the contralateral control cochleae (repeated measures ANOVA, p = 0.009). SGN survival was observed over a wide extent of the cochlea. The supraparticles were well tolerated within the cochlea with a tissue response that was localised to the site of implantation in the cochlear base. Although mild, the tissue response was significantly greater in cochleae treated with BDNF supraparticles compared to the controls (repeated measures ANOVA, p = 0.003). These data support the clinical potential of this technology particularly as the supraparticles can be loaded with a variety of therapeutic drugs. of Washington, UNITED STATES Received: January 24, 2016 Accepted: October 3, 2016 Published: October 27, 2016 Copyright: © 2016 Wise 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. Andrew K. Wise1,2,3, Justin Tan3, Yajun Wang4, Frank Caruso4, Robert K. Shepherd1,2,3 Andrew K. Wise1,2,3, Justin Tan3, Yajun Wang4, Frank Caruso4, Robert K. Shepherd1,2,3 1 The Bionics Institute, 384–388 Albert Street, East Melbourne, Melbourne, Australia, 2 The Department of Medical Bionics, University of Melbourne, Melbourne, Australia, 3 Department of Otolaryngology, University of Melbourne, Melbourne, Australia, 4 ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, and the Department of Chemical and Biomolecular Engineering, the University of Melbourne, Melbourne, Australia a1111 * awise@bionicsinstitute.org * awise@bionicsinstitute.org Introduction Hearing loss is one of the most common sensory deficits affecting over 5.3% of people world- wide (approximately 360 million people: World Health Organization, 2012). Deafness can have a significant impact on communication in a hearing world and affect the development of language in children [1], with social, vocational and mental health implications throughout life [2]. The number of people impacted by deafness is expected to rise as the population ages. Sensorineuralhearing loss (SNHL) is the most common form of deafness and typically results from damage to the delicate sensory hair cells within the cochlea, or loss of their synap- tic connections with the spiral ganglion neurons (SGNs). The only therapeutic option for peo- ple with profound to severe SNHL is a cochlear implant; a bionic device that restores hearing function via electrical stimulation of the remaining SGNs to effectively bypass the lost sensory modality. However, one major consequence of SNHL is the progressive degeneration of the SGNs that occurs in both humans [3, 4] and animal deafness models [5–7]. A primary cause of SGN degeneration is the loss of the endogenous supply of neurotrophins, in particular Brain- Derived Neurotrophic Factor (BDNF) and Neurotrophin 3 (NT-3), which are normally pro- duced by hair cells and supporting cells in the organ of Corti [8–13]. Both BDNF and NT-3 are required for normal neural development and innervation of cochlear hair cells with NT-3 par- ticularly important for synaptogenesis [14, 15]. As the SGNs are the target neurons for the cochlear implant a functional population of SGNs is essential for the implant to perform effec- tively. For example, a recent study has shown a strong correlation between word recognition scores and SGN counts indicating that a greater population of SGNs results in better cochlear implant performance [16]. As such, clinically relevant strategies that can prevent the progres- sive degeneration of SGN with cochlear implantation have received substantial interest. Exogenous administration of neurotrophins using implantable mini-pumps has been shown to improve the survival of SGNs in animal deafness models [17–24] with the SGN peripheral fibres becoming larger and more numerous compared to untreated cochleae [20, 25]. However, the supply of neurotrophins from pump-based devices is finite, necessitating the need for pumps to be refilled or replaced and thus leading to concerns over the long-term safety of these devices as a result of infection [26]. Data Availability Statement: All relevant data are within the paper. Funding: This work was funded by National Health and Medical Research Council Project Grant APP1005071 and APP1064375, Australian Research Council under the Australian Laureate Fellowship scheme (120100030), and National Institutes of Health (USA) HHS-N-263-2007- 00053-C. Competing Interests: The authors have declared that no competing interests exist. 1 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Nanoengineered Drug Delivery for Improved Auditory Nerve Survival Auditory brainstem responses Only animals with otoscopically normal external ears were used in this study. Prior to any sur- gical procedure the hearing status was assessed under anesthesia (ketamine, 60mg/kg (Parnell Australia) and xylazine, 4mg/kg (Ilium, Australia); intramuscular injection). Click-evoked auditory brainstem responses (ABRs) were measured to assess the hearing status of the guinea pigs before and after deafening using procedures describedpreviously [20, 35]. The anaesthe- tized guinea pigs were placed on a heat pad with the temperature maintained at 37°C in a sound attenuated room and needle electrodes were placed at the skull vertex, at the nape of the neck and on the abdomen. ABRs were measured to acoustic clicks delivered by a calibrated speaker at intensities up to 100 dB peak equivalent (p.e.) sound pressure level (SPL). The ABR was amplified, recorded to computer and averaged over 200 trials that were presented at stimu- lus intensities ranging from 0 dB to 100 dB p.e. SPL. Hearing threshold was determined and only guinea pigs with normal hearing thresholds in both ears (ABR threshold <50 dB p.e. SPL) were used in the study. Experimental Animals Six young adult pigmented guinea pigs of either sex (mean 550 g) were used in this study. Both ears were used resulting in a total of 12 cochleae. All procedures were approved by the St. Vincent’s Hospital Animal Research & Ethics Committee (ref# 014/12-r1) in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals, and conformed to the Code of Practice of the National Health and Medical Research Council of Australia. Nanoengineered Drug Delivery for Improved Auditory Nerve Survival approach has advantages and limitations, the use of nanoengineeredcarriers offers promise of delivering therapeutic levels of neurotrophins in a safe manner that is effective over the long term. Recently we have developed a new drug carrier system that uses mesoporous silica nanopar- ticles as building blocks to form larger (~ 500 μm) supraparticles (SPs) [50]. In vitro experi- ments showed that the SPs could be loaded with high payloads of BDNF and a pilot in vivo experiment showed that BDNF loaded SPs could be surgically implanted into the cochlea of the guinea pig. In the present study we have evaluated the efficacyof BDNF-loaded SPs to protect SGNs from deafness-induceddegeneration and assessed the biocompatibility of the SP carrier system by quantifying the tissue response following chronic implantation. The results showed that SPs were biocompatible and able to deliver a therapeutic level of BDNF that resulted in significant SGN survival that was observedthroughout a wide extent of the cochlea compared to deafened control cochleae. This finding highlights the clinical suitability of the supraparticle system that can overcome some of the significant safety concerns of pump-based drug delivery devices yet yield similar therapeutic effects in terms of the level and extent of SGN survival throughout the cochlea, an outcome that has proven difficultto achieve in other drug delivery strategies. Introduction Although the long-term survival-promoting effects following the cessation of exogenous neurotrophin delivery are yet to be established there is some evidence that improved SGN survival may persist for at least a short period of time (weeks) following the removal of the exogenous neurotrophin supply [27, 28]. However, this is not a universal finding with one previous study showed no sustained survival effect fol- lowing cessation of neurotrophin delivery [18]. It is therefore likely that long-term neurotro- phin delivery strategies will be required for the survival effects to be maintained over time. Encouragingly, SGN survival can be enhanced when exogenous neurotrophins are applied in concert with electrical stimulation from a cochlear implant (with SGNs also exhibiting lower electricalthresholds) [29–32] meaning that even at low levels, neurotrophin delivery might remain therapeutically beneficialwhen combined with cochlear implant use. Although there are potential benefits of neurotrophin therapy for clinical application a sig- nificant factor limiting translation is the need for a drug delivery mechanism that provides a long-term supply of the neurotrophins in a safe and effective manner. Given the potential infection risks of implantable pump devices [26] and the limitations of systemic delivery in crossing the blood-labyrinth barrier, research has focused on localised neurotrophins delivery strategies that are clinically translatable and that can be combined with a cochlear implant. These include cell-based therapies [32–34], gene therapies [35–41], electrode coating materials [42–45] and biocompatible carrier systems including nanoparticles [46–49]. Although each 2 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Supraparticle sterilization, NT loading and NT release properties Supraparticles were sterilized by soaking them in 100 μL of ethanol (80 vol/vol%) for 4 hours prior to rinsing with 100 μl of Milli-Q water six times. SPs were then washed once in 0.1 M phosphate buffered saline (PBS). Eight SPs were loaded for each experimental animal by plac- ing them in an Eppendorf tube containing 15 μl of BDNF (Geneway, BDNF Human Protein, Cat. # 10-663-45078) solution (1 mg/ml of BDNF) and incubating them at ambient room tem- perature for three days with occasional shaking. Immediately prior to implantation the BDNF- loaded SPs (BDNF-SPs) were rinsed once in 20 μl of Milli-Q water and placed in sterile saline prior to implantation. The SPs possess large pore volumes and high surface areas, and therefore allow large amounts of BDNF to be loaded. In previous in vitro elution studies we have reported that each SP could absorb approximately 1.3 μg of BDNF during the loading proce- dure and approximately 400 ng of BDNF could be released via a linear release profile over the first month [50]. The porous characteristics of the supraparticles are thought to improve the bioeffectivenessof the BDNF by immobilizing the protein and limiting its breakdown in the in vivo environment [53, 54]. Deafening Procedure Animals were deafened ototoxically using a procedure which has been previously shown to produce a severe bilateral SNHL [30, 35, 51]. Under gaseous anesthesia (1–2% isoflurane in O2, 1L/min) the right jugular vein was exposed and cannulated. Frusemide (130mg/kg; Ilium, Aus- tralia) diluted in warm Hartmann’s solution was slowly injected. The vein was tied off and the 3 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Nanoengineered Drug Delivery for Improved Auditory Nerve Survival incision sealed with cyanoacrylate.The ototoxic aminoglycoside kanamycin sulfate (420mg/kg; Sigma-Aldrich, USA) dissolved in 3ml Hartmann’s solution was then injected subcutaneously (s.c.). Approximately four days after the deafening procedure ABRs were re-measured as describedabove, to confirm deafness. An increase in hearing thresholds of >50dB indicated severe to profound SNHL. All thresholds recorded were >100dB p.e. SPL, which was the maxi- mum intensity presented. Supraparticle Preparation The SPs were synthesized as describedpreviously [50]. Briefly, mesoporous silica nanoparticles (diameter, ca. 400 nm) [52] were dispersed in water at a concentration of 5 wt% and formed a stable colloidal suspension. Droplets (0.5 μl) of the suspension solution were applied to a hydrophilic surface (paraffin film) before being dried with air flow. Under capillary force, the mesoporous silica colloids self-assemble into a compact structure to form SPs. The SPs were then annealed at 923°K to enhance mechanical stability of the SPs. The SPs had a bimodal pore structure (~2–3 nm and 15–30 nm) and the space within the SP, between the densely packed nanoparticles, was ~100–200 nm, resulting in a porous structure with a high surface area. The SPs used in this study had a diameter of ca. 500 μm. PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Bilateral implant surgery Schematic illustration of the top view looking down on the cochlea depicting the approximate location where the SPs were implanted (grey circles). The cochlear regions that were analysed for SGN density (region 1 to region 8; R1-R8) and tissue response to implantation (grey region on dotted lines–Ai-iii and Bi-iii) are shown. Tissue response data was averaged across region A and B. (B) A mid modiolar micrograph of an implanted cochlea showing the cochlear regions in which SGN density within Rosenthal’s canal was analysed (R1-R8). doi:10 1371/journal pone 0164867 g001 doi:10.1371/journal.pone.0164867.g001 and the analgesic Temgesic (Reckitt-Benckiser, UK) (50 mg/kg; s.c.) were given after surgery and on the next day to aid recovery. Bilateral implant surgery One week following deafening the animal underwent bilateral cochlear implant surgery using aseptic surgical techniques. Guinea pigs were anaesthetized with an intramuscular injection of ketamine (60 mg per kg body weight) and xylazine (4 mg per kg body weight). Using a post- auricular approach, the bulla was exposed and a small hole was drilled to expose the basal turn of the cochlea. A cochleostomy (approximately 800 μm) was made using a 0.8 mm diamond drill and gentle suction was applied to remove bone debris from the cochlea (Fig 1). A total of 8 SPs were placed into the basal turn of the cochlea using a 21 gauge polyurethane catheter (Optiva, Medex Medical, UK). One cochlea was implanted with BDNF-SPs and the other cochlea was implanted with unloaded SPs (control-SPs) using identical surgical techniques. The side implanted with the BDNF-SPs was randomized. Following implantation, a small piece of muscle was placed over the cochleostomy in order to seal the cochlea. The wound was closed by suturing surrounding muscles in two layers and closing the skin incision with staples. Hartmann’s solution (10ml/kg; s.c.), the antibiotic Baytril (Bayer, Germany) (0.10mg/kg; s.c.), PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 4 / 17 Nanoengineered Drug Delivery for Improved Auditory Nerve Survival Fig 1. Schematic illustration of the top view looking down on the cochlea depicting the approximate location where the SPs were implanted (grey circles). The cochlear regions that were analysed for SGN density (region 1 to region 8; R1-R8) and tissue response to implantation (grey region on dotted lines–Ai-iii and Bi-iii) are shown. Tissue response data was averaged across region A and B. (B) A mid modiolar micrograph of an implanted cochlea showing the cochlear regions in which SGN density within Rosenthal’s canal was analysed (R1-R8). doi:10.1371/journal.pone.0164867.g001 Fig 1. Schematic illustration of the top view looking down on the cochlea depicting the approximate location where the SPs were implanted (grey circles). The cochlear regions that were analysed for SGN density (region 1 to region 8; R1-R8) and tissue response to implantation (grey region on dotted lines–Ai-iii and Bi-iii) are shown. Tissue response data was averaged across region A and B. (B) A mid modiolar micrograph of an implanted cochlea showing the cochlear regions in which SGN density within Rosenthal’s canal was analysed (R1-R8). Fig 1. PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Nanoengineered Drug Delivery for Improved Auditory Nerve Survival thresholded pixels located within the osseous spiral lamina to obtain a percentage value. Data was averaged across the four sections. Spiral ganglion neuron density measurement All quantification was carried out by a single observer blinded to the experimental groups. Sec- tions were viewed and imaged using an Axio Lab microscope and software (Zeiss, Germany). In the hematoxylin and eosin-stained sections the area of Rosenthal’s canal was measured for each cochlear region (Region 1 to 8; see Fig 1) using ImageJ V1.46. SGNs were identified within Rosenthal’s canal and counted. Only SGNs exhibiting a clear nucleus were counted using previ- ously published techniques [32, 33, 51, 55, 56]. Data was collected from five non-continuous sections with a separation of 72 μm ensuring that no cell was counted more than once. The density of the SGNs was determined for each cochlear region as an averaged from five sections. In the apical cochlear regions (regions 6–8) Rosenthal’s canal typically narrows and therefore SGN density data for regions 6–8 were combined. Tissue response measurements In order to quantify the immune response to the implanted SPs, the extent of fibrosis and new bone growth in the cochlea was measured in hematoxylin and eosin-stained sections at three different locations separated by a distance of at least 400 μm. The tissue response was quanti- fied as the percentage of the area of the scala tympani occupied by any inflammatory cells or fibrous tissue and new bone using techniques previously describedby [32] and similar to those implemented by [57, 58]. Briefly, an image of the scala tympani was captured and the area mea- sured. The ‘Moments’ algorithm in Image J was used to threshold the image to identify the tis- sue response. The area of scala tympani was measured and the proportion of the scala tympani occupied by the tissue response was calculated. Data was measured from a total of six positions within the cochlea and with three data points per cochleae averaged for the lower basal region (Ai-Aiii) and three data points averaged for the upper basal region (Bi-Biii) (see Fig 1A). Statistics Statistical comparisons of SGN density and tissue response were made by comparing data from the cochleae implanted with BDNF-SPs to the data in the contralateral control cochlea that were implanted with control-SPs. A repeated measures (RM) analysis of variance (ANOVA) using p<0.05 level of significance was used to determine statistical significance and post hoc comparisons were made using the Holm Sidak method. PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Tissue collection and preparation Following the 28 day treatment period, the animals were given an overdose of pentobarbital (150mg/kg; intraperitoneal) and transcardially perfusedwith 0.9% NaCl (37°C) followed by 10% Neutral Buffered Formalin (10% NBF; 4°C). The cochleae were dissected and a small hole was made in the apex. The round and oval windows were incised with a 25 gauge needle and the cochleae post-fixed in 10% NBF for one hour on a shaker at room temperature. The cochleae were then placed in 10% ethylenediamine tetraacetic acid (EDTA) in PBS at room temperature for decalcification.After decalcification,cochleae were cryo-protected in 15% and then 30% sucrose solution before they were embedded in Tissue-Tek O.C.T. cryosectioning compound (Sakura, Japan) as describedpreviously [51] and stored at -80°C. Cochleae were sectioned at 12μm using a CM 1900 UV cryostat (Leica, Germany) at -22°C in the modiolar plane and mounted onto Superfrost-Plus slides (Menzel-Gläser, Braunschweig, Germany). A representative series of cochlear sections were stained with Mayer’s haemotoxylin and Putt’s eosin (H&E) for general qualitative examination, SGN density measurements within Rosenthal’s canal and quantification of the tissue response. A representative series of mid modiolar sections were also processed for immunohis- tochemistry. Standard immunofluorescent protocols were followed using antibodies against heavy chain neurofilament (NF-H; Merck Millipore, Australia) to stain the SGNs and periph- eral fibres using AlexaFluor secondary antibodies for visualisation (Molecular Probes, USA). Slides were mounted in media containing DAPI and imaged on a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Germany). Peripheral nerve fibres located in the osseous spiral lamina in the upper basal turn (R2) were imaged from four non-consecutive mid modiolar sections (72 μm separation). Images were analysed in ImageJ V1.46 (NIH, USA, http://rsb.info.nih.gov/ ij/index.html) by thresholding neurofilament labelled pixels located using the inbuilt “moment” threshold filter [32]. Nerve fibres were quantified by determining the area of 5 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Spiral ganglion neuron survival A representative series of cochlear sections were stained with hematoxylin and eosin to quantify the effect of BDNF-SP treatment on SGN survival. Fig 3 shows representative exam- ples of cochlear regions 1–5 (R1-5) taken from a cochlea implanted with BDNF-SPs (left panel) and from the contralateral control cochlea implanted with Control-SPs (right panel). Aminoglycoside deafening resulted in bilateral threshold shift of >50dB to acoustic clicks (data not shown) and complete loss of the organ of Corti throughout the cochlea (Fig 3). In order to quantify SGN survival the density of SGNs was calculated by measuring the area of Rosenthal’s canal (dotted lines in Fig 3) and counting the number of SGNs. Fig 3A and 3B shows a section taken from the lower basal turn (region 1) for a BDNF-SP treated (left) and Control-SP treated (right) cochlea at low (A) and higher magnification (B) of the SGNs. There was a significantly greater density of SGNs in cochleae that were treated with BDNF-SPs com- pared to the Control-SP cochleae. Importantly, the greater SGN survival was evident through- out the cochlea as can be seen in Fig 3C to 3F showing representative data from cochlear regions 2–5. A representative series of cochlear sections were stained with hematoxylin and eosin to quantify the effect of BDNF-SP treatment on SGN survival. Fig 3 shows representative exam- ples of cochlear regions 1–5 (R1-5) taken from a cochlea implanted with BDNF-SPs (left panel) and from the contralateral control cochlea implanted with Control-SPs (right panel). Aminoglycoside deafening resulted in bilateral threshold shift of >50dB to acoustic clicks (data not shown) and complete loss of the organ of Corti throughout the cochlea (Fig 3). In order to quantify SGN survival the density of SGNs was calculated by measuring the area of Rosenthal’s canal (dotted lines in Fig 3) and counting the number of SGNs. Fig 3A and 3B shows a section taken from the lower basal turn (region 1) for a BDNF-SP treated (left) and Control-SP treated (right) cochlea at low (A) and higher magnification (B) of the SGNs. There was a significantly greater density of SGNs in cochleae that were treated with BDNF-SPs com- pared to the Control-SP cochleae. Importantly, the greater SGN survival was evident through- out the cochlea as can be seen in Fig 3C to 3F showing representative data from cochlear regions 2–5. Spiral ganglion neuron survival Mid modiolar cochlear sections were stained with a neuronal antibody (neurofilament) that specifically labels SGNs and their nerve fibres. Example data from all of the cochleae used in this study is shown in Fig 2. Images show the SGNs within Rosenthal’s canal (dotted lines) in the upper basal turn (region 2) for each guinea pig (GP_01 to 06 labeled within each image). There is a clear difference in SGN survival with the cochleae that received BDNF-SP exhibit- ing more SGNs compared to the contralateral cochleae that received Control-SP. To ascer- tain the effects of BDNF-SP treatment on peripheral fibre survival analysis of the peripheral nerve fibres in the upper basal region (region 2) was carried out by determining the propor- tion of the osseous spiral lamina occupied by the nerve fibres. The nerve fibres occupied a greater proportion of the osseous spiral lamina in cochleae implanted with BDNF-SPs PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 6 / 17 Nanoengineered Drug Delivery for Improved Auditory Nerve Survival Fig 2. Example cochlear sections of the BDNF-SP and Control-SP treated cochleae for all six animals used in this study (GP_01 to 06). Images show the upper basal turn (region 2) and sections are stained with a neuronal marker (neurofilament: green) and a nucleus marker (DAPI: blue). Rosenthal’s canal containing the SGN is depicted by the white dotted line in each image. It is clearly evident that there is greater SGN survival in the BDNF-SP treated cochleae compared to the contralateral cochleae that received Control-SP treatment. This is consistent across the entire cohort of animals used in the study. Fig 2. Example cochlear sections of the BDNF-SP and Control-SP treated cochleae for all six animals used in this study (GP_01 to 06). Images show the upper basal turn (region 2) and sections are stained with a neuronal marker (neurofilament: green) and a nucleus marker (DAPI: blue). Rosenthal’s canal containing the SGN is depicted by the white dotted line in each image. It is clearly evident that there is greater SGN survival in the BDNF-SP treated cochleae compared to the contralateral cochleae that received Control-SP treatment. This is consistent across the entire cohort of animals used in the study. doi:10.1371/journal.pone.0164867.g002 (19.1% ± 1.3 SEM) compared to control-SPs (13.6% ± 1.3 SEM) (two tailed paired t-test, p = 0.002). p = 0.002). PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Spiral ganglion neuron survival Analysis of the SGN density data showed a statistically greater density of SGNs in the cochleae treated with BDNF- SPs (two way repeated measures ANOVA, p = 0.009) using ‘cochlear region’ and ‘neurotrophin treatment’ as factors. Post hoc analysis indicated that a sig- nificant difference in SGN density was observedin all cochlear regions except the most apical regions (region 6–8) (Holm-Sidak, p<0.005, p<0.05; Fig 4). 7 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Nanoengineered Drug Delivery for Improved Auditory Nerve Survival Nanoengineered Drug Delivery for Improved Auditory N ne.0164867 October 27, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 8 / 17 0164867 October 27 2016 PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 8 / 17 Nanoengineered Drug Delivery for Improved Auditory Nerve Survival Fig 3. Representative examples of cochlear sections obtained from a cochlea treated with BDNF-SPs (left) and Control-SPs (right) for cochlear Regions 1–5 (A-F respectively). (A) shows the lower basal region (Region 1) with Rosenthal’s canal outlined (dotted outline). There was a flattening of the sensory epithelium and complete loss of the organ of Corti (arrow) that was symmetrical between ears. There was a tissue response (; Region Ai) to the presence of the SPs that was observed in the scala tympani with the osseous spiral lamina depicted with a dotted line. (B) Higher magnification image of SGNs (arrows) in Rosenthal’s canal in Region 1 from BDNF-SP treated cochlea (left) and from a Control-SP treated cochlea (right). There was a greater density of SGNs in the BDNF-SP treated cochlea compared to the control cochlea. (C) Cochlear section taken at Region 2 shows the Rosenthal’s canal (dotted line) and the fibrotic tissue response (; Region Bi) for the BDNF-SP (left) and Control-SP (right) cochleae. (D–F) Cochlear section taken at Region 3–5 shows the Rosenthal’s canal (dotted line) for the BDNF-SP (left) and Control-SP (right) cochleae. There was no tissue response in these cochlear regions. Scale bar (A and C-F = 100 μm) and (B = 50 μm). doi:10.1371/journal.pone.0164867.g003 doi:10.1371/journal.pone.0164867.g003 Tissue Response A prerequisite for the clinical viability of the SP drug delivery system is that it is safe for implantation in humans. In order to determine SP safety the foreign body tissue response was measured within the cochlea (see Fig 1). Fig 3 shows examples of the tissue response through- out the cochlea to the implantation of the SPs. The tissue response was restricted to the scala tympani near the site of implantation in the basal cochlear region (region A and B; see Fig 1). The tissue response was made up of loose fibrotic tissue, small areas of osteoid or bone, with the occasional presence of active inflammatory reaction as indicated by the presence of lym- phocytes neutrophils and multinucleated giant cells. The tissue response was quantified by measuring the proportion of the scala tympani occu- pied by the new tissue and the data is presented in Fig 4B. The tissue response was most promi- nent in the scala tympani Region A, in close proximity to the cochleostomy and the location of the SPs within the cochlea. There was a significant interaction between cochlear Region and SP Treatment (two way repeated measures ANOVA, p = 0.029). Post hoc analysis indicated that the tissue response was significantly greater in Region A compared to Region B (Holm-Sidak, Fig 4. (A) Average of the SGN density measured in each cochlear region after one month of treatment with BDNF-SP (black) and the Control-SPs (Grey). There was a significantly greater density of SGNs in cochleae treated with BDNF-SPs compared to the Control-SPs (two way ANOVA, p = 0.009) with post hoc analysis indicated (Holm-Sidak, *p<0.005, p<0.05). Error bars ± 1 SEM. (B) Analysis of the cochlear tissue response measured in the scala tympani (ST) in cochlear Regions A and B showed a tissue response for Region A (near the site of the cochleostomy) that was significantly larger than the tissue response in Region B (Post Hoc Holm-Sidak; *p<0.001). The tissue response measured in Region A in the BDNF-SP treated cochleae was greater than that in the Control-SP treated cochlea (Post Hoc Holm-Sidak; p = 0.003). doi:10.1371/journal.pone.0164867.g004 Fig 4. (A) Average of the SGN density measured in each cochlear region after one month of treatment with BDNF-SP (black) and the Control-SPs (Grey). Effects of BDNF-SPs SGN survivalwas observedthroughout a wide spatial extent of the cochlea in all but the most api- cal cochlear regions with SGN densities that were consistent with neural densities in normal hearing guinea pigs from previous studies [20, 30]. Many of the other clinically translatable approaches to deliver neurotrophins to the cochlea describedabove (e.g. gene therapy, cell-based therapy, electrode coatings and hydrogels placed on the round window) have observedSGN sur- vival effects typically restricted to the basal cochlear regions, in close proximity to the drug deliv- ery device. The results of the current study showed that there was no significant difference in SGN survivalbetween BDNF-SPs and Control-SPs in the apical cochlear region (region 6–8). It is known that SGNs in the apical cochlear regions are less susceptible to degeneration following aminoglycoside exposure, even when there is complete destructionof the organ of Corti, indicat- ing that apical SGNs are less susceptible to deafness-induceddegeneration [61]. Therefore, a lack of a statistical difference in SGN survivalin apical regions does not necessarily indicate that a therapeutic level of BDNF was not present. Nevertheless, the results from this study provide direct evidence that a therapeutic level of BDNF was present in the basal to upper middle cochlear regions as demonstrated here leading to an improved SGN survival in these regions. Discussion This study examined the safety and efficacyof intracochlear implantation of mesoporous silica SPs that were designed as a carrier for BDNF delivery in the inner ear. We implanted BDNF-SP or Control-SPs into the basal turn of deafened guinea pigs and, following a four week treatment period, examined the cochleae histologically to measure SGNs survival and SP biocompatibil- ity. The results showed significant SGN survival along a wide extent of the cochleae that were treated with the BDNF-SPs compared to the contralateral cochleae treated with Control-SPs. There was a significantly greater density of peripheral fibres as indicated by a greater propor- tion of nerve fibres within the osseous spiral lamina in the BDNF-SP treated cochleae. The SPs were implanted via a cochleostomy and this procedure resulted in a tissue response that was largely localised to the basal turn, close to the site of the cochleostomy. However, there was a small but significant increase in the tissue response in cochleae that received BDNF-SP com- pared to Control-SPs. Nanoengineered Drug Delivery for Improved Auditory Nerve Survival p<0.001) and was significantly greater in the cochleae implanted with the BDNF-SPs com- pared to the cochleae implanted with the Control-SPs (Holm-Sidak, p = 0.003). Effects of Deafness Animals were deafened with the aminoglycoside kanamycin and the loop diuretic frusemide. All animals were profoundly deaf and exhibited ABR thresholds over 100 dB SPL p.e. (data not shown). Histological analysis confirmed that the organ of Corti had completely collapsed, indi- cating that, similar to previous studies, the deafening procedure resulted in complete loss of hair cells and degeneration of the supporting cells, such that there was typically a completely flattened epithelium (Fig 3) [18, 20, 30, 35, 37, 59]. The effects of this deafening technique are symmetrical with SGN loss similar between ears [60] indicating that the contralateral cochlea serves as a reliable control for experimental techniques designed to improve SGN survival. Tissue Response There was a significantly greater density of SGNs in cochleae treated with BDNF-SPs compared to the Control-SPs (two way ANOVA, p = 0.009) with post hoc analysis indicated (Holm-Sidak, *p<0.005, p<0.05). Error bars ± 1 SEM. (B) Analysis of the cochlear tissue response measured in the scala tympani (ST) in cochlear Regions A and B showed a tissue response for Region A (near the site of the cochleostomy) that was significantly larger than the tissue response in Region B (Post Hoc Holm-Sidak; *p<0.001). The tissue response measured in Region A in the BDNF-SP treated cochleae was greater than that in the Control-SP treated cochlea (Post Hoc Holm-Sidak; p = 0.003). doi:10.1371/journal.pone.0164867.g004 9 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Biocompatibility of Supraparticles In order to determine the safety of the SP for drug delivery the tissue response to implantation of BDNF-SPs and Control-SPs was quantified. The tissue response was primarily located in the 10 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Nanoengineered Drug Delivery for Improved Auditory Nerve Survival lower basal turn (Region A), principally around the site of the cochleostomy and was restricted to a small proportion of the scala tympani. Further along the cochlea, in the middle turns (Region B) the tissue response, if present, occupied an even smaller proportion of the scala tympani. The tissue response was only observedin the scala tympani, with the other cochlear compartments (the scala vestibuli and scala media) devoid of any tissue response. These find- ing provide evidence that the SPs were well tolerated in vivo. There was a significantly greater tissue response in Region A of the BDNF-SP treated cochleae compared to the Control-SP treated cochleae. An enhanced tissue response following intracochlear BDNF delivery has been reported before [30, 62] and may reflect the use of human BDNF protein delivered to the guinea pig cochlea in this and previous studies. The extent of tissue response measured in the Control-SP cohort was consistent with that observedin previous studies that have performed a cochleostomy in the basal turn [38, 57] where the surgical exposure and bone dust generated created a local tissue response consisting of new bone growth and loose fibrotic tissue. We were unable to obtain histological evidence of the presence of the mesoporous silica SPs within the cochlea. It is likely that the SPs remained present at the time of tissue harvesting but could not be detected in our sections due to the histological processing of the tissue (decalcifi- cation, cochlear infiltration, freezing, sectioning and washing). Our in vitro studies confirm that the SPs remain viable for over 70 days in saline [50]. The longer-term fate of the SPs in vivo, when implanted into the cochlea, is still to be deter- mined. The SPs will eventually breakdown and be cleared by the body. The SPs are comprised of colloidal silica, which is water soluble, easily absorbed and rapidly excreted. Colloidal silica is used clinically as a dietary supplement. In vitro studies have shown that silica particles have no or very low cytotoxic effects in various cell lines [63]. Biocompatibility of Supraparticles In vivo experiments have shown that silica nanoparticles are biocompatible, biodegradable, and bioexcretable [64, 65] and do not affect cochlear function even when used as an intracellular carrier for hair cells and SGNs [66]. It is likely that the dissolved silica will be cleared via the cochlear vasculature. The FDA has recently approved the human trial of silica nanoparticles that are used for imaging lymph nodes in cancer patients (NCT02106598; ClinicalTrials.gov). PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Advantages of Supraparticle Carriers Previous research has shown that nanoengineeredparticles (~2 μm in size) could load and deliver BDNF over a sustained duration [46]. The current study has used a SP carrier system with characteristics that make it uniquely suited for drug delivery to the cochlea. Firstly, the rel- atively large size of the SPs (~500 μm) meant that they were easily handled during surgical implantation into the cochlea and would ensure that they could not disperse far from the implantation site. Dispersal of smaller particles or cells from the cochlea to the central nervous system and/or the vestibular end organs is problematic because it reduces the therapeutic load to the target SGNs within the cochlea. A number of researchers have used hydrogels or foams to minimise the dispersal of particles or cells from the cochlea [46, 67]. Secondly, the SPs pro- vide sustained released of BDNF [50] meaning that longer durations of drug delivery are possi- ble. Thirdly, the SPs can be loaded with high payloads of therapeutics and deliver therapeutic levels of these drugs. Fourthly, the immobilization of the BDNF in the pores of the SPs serves to protect the protein from denaturing in vivo [53], and thus maintains its bioactivity [54] whereas protein instability over time is a problem when stored in mini-pumps [68]. This is important as neurotrophins have a relatively rapid half-life in vivo [68–70]. The results show- ing significant SGN survival support the conclusion that the released BDNF was biologically effective. Fifthly, the SP technology enables the use of multiple drugs, each loaded in separate SPs that can potentially have release characteristics tailored for best effect for each drug type. PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 11 / 17 Nanoengineered Drug Delivery for Improved Auditory Nerve Survival Finally, the SP system offers the clinician the option of implanting the SPs into the cochlea, for instance along with a cochlear implant, or implanting them onto to round window membrane (external to the cochlea). The round window membrane is permeable to small molecules and drugs [71, 72]. A current clinical approach to deliver therapeutic compounds to the cochlea to treat hearing loss is to inject drugs into the middle ear cavity and rely on passive diffusion of the drug across the round window membrane. However, with this technique much of the drug is quickly lost, thus limiting the efficacyof the technique for delivering drugs to the cochlea. Advantages of Supraparticle Carriers Implantation of drug-loadedSPs onto the round window membrane would be expected to improve drug entry into the cochlea [49]. Conclusion This study has shown that SPs can provide a safe and effective strategy to delivery therapeutics to the inner ear for SGN survival, with survival effects observedover a wide extent of the cochlea. The SPs were well tolerated by the cochlea providing evidence that the technique is clinically relevant. Future studies to evaluate the supraparticle distribution and the pharmaco- kinetics of the released therapeutic would pave the way for clinical translation of the drug deliv- ery strategy. The technology could potentially be combined with a cochlear implant to improve implant performance. Clinical Relevance for Cochlear Implants Due to the progressive nature of many types of SNHL it is highly likely that delivery techniques providing localised drug delivery over extended durations will be most successful. Because the cochlea becomes accessible during implantation surgery, it provides the opportunity of implanting a therapeutic delivery device inside the cochlea, along with the electrode array. The features of the SP delivery system (as discussed above) make it a suitable drug delivery strategy for combination with cochlear implantation in order to improve nerve survival and/or to pro- tect residual hearing. There is a strong correlation in SGN survival and cochlear implant performance with greater numbers of surviving SGNs resulting in improved cochlear implant performance [16]. The development of clinically translatable neurotrophin delivery strategies that increase SGN survival and the survival of their peripheral fibres, and reduce the electricalthresholds [30, 41, 73] would therefore be likely lead to improvements in cochlear implant function in contempo- rary devices. Furthermore, ongoing SGN degeneration and cell loss is a significant impediment to the clinical implementation of current focussing strategies that aim to provide more precise neural activation within the cochlea [56, 74, 75]. Technology that prevents SGN and nerve fibre loss, and promotes SGN fibre regrowth, may improve the clinical outcomes of future stimulation strategies designed to improve the precision of neural activation within the cochlea. Acknowledgments Thanks to Associate Professor James Fallon, Ms. Nicole Critch, Ms. Prudence Nielsen, Mr. Damian Robb and Dr Sue Peirce for their contributions to this research. References 1. Geers A, Moog J, Schick B. Acquisition of spoken and signed English by profoundly deaf children. J Speech Hear Disord. 1984; 49(4):378–88. Epub 1984/11/01. PMID: 6503244. 2. Lin FR, Metter EJ, O’Brien RJ, Resnick SM, Zonderman AB, Ferrucci L. Hearing loss and incident dementia. Arch Neurol. 2011; 68(2):214–20. 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Aminoglycoside-induced degeneration of adult spiral ganglion neurons involves differential modulation of tyrosine kinase B and p75 neurotrophin receptor signaling. Am J Pathol. 2006; 169(2):528–43. PMID: 16877354. doi: 10.2353/ajpath.2006.060122 13. Zilberstein Y, Liberman MC, Corfas G. Inner hair cells are not required for survival of spiral ganglion neurons in the adult cochlea. J Neurosci. 2012; 32(2):405–10. Epub 2012/01/13. 32/2/405 [pii] doi: 10. 1523/JNEUROSCI.4678-11.2012 PMID: 22238076. 14. Wang Q, Green SH. Author Contributions Conceptualization: AKW JT YW FC RS. Data curation: AKW. 12 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0164867 October 27, 2016 Nanoengineered Drug Delivery for Improved Auditory Nerve Survival Formal analysis: AKW. Funding acquisition: AKW RS. Investigation: AKW. Methodology:AKW JT YW FC RS. Project administration: AKW RS FC. Resources: AKW RS FC. Supervision:AKW RS FC. Visualization: AKW JT YW FC RS. Writing – original draft: AKW RS. Writing – review& editing: AKW. Formal analysis: AKW. Funding acquisition: AKW RS. Investigation: AKW. Methodology:AKW JT YW FC RS. Project administration: AKW RS FC. Resources: AKW RS FC. Supervision:AKW RS FC. Visualization: AKW JT YW FC RS. Writing – original draft: AKW RS. Writing – review& editing: AKW. Formal analysis: AKW. Funding acquisition: AKW RS. Investigation: AKW. Methodology:AKW JT YW FC RS. Project administration: AKW RS FC. Resources: AKW RS FC. Supervision:AKW RS FC. Visualization: AKW JT YW FC RS. Writing – original draft: AKW RS. Writing – review& editing: AKW. Formal analysis: AKW. Funding acquisition: AKW RS. Funding acquisition: AKW RS. Investigation: AKW. Resources: AKW RS FC. Supervision:AKW RS FC. Visualization: AKW JT YW FC RS. Writing – original draft: AKW RS. Writing – review& editing: AKW. 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https://openalex.org/W2919851414	https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-019-5465-z	English	null	In silico characterization of multiple genes encoding the GP63 virulence protein from Leishmania braziliensis: identification of sources of variation and putative roles in immune evasion	BMC genomics	2,019	cc-by	13,939	Castro Neto et al. BMC Genomics (2019) 20:118 https://doi.org/10.1186/s12864-019-5465-z Castro Neto et al. BMC Genomics (2019) 20:118 https://doi.org/10.1186/s12864-019-5465-z Open Access In silico characterization of multiple genes encoding the GP63 virulence protein from Leishmania braziliensis: identification of sources of variation and putative roles in immune evasion Artur L. Castro Neto1,2, Adriana N. A. L. M. Brito2, Antonio M. Rezende2, Franklin B. Magalhães3 and Osvaldo P. de Melo Neto2* Artur L. Castro Neto1,2, Adriana N. A. L. M. Brito2, Antonio M. Rezende2, Franklin B. Magalhães3 and Osvaldo P. de Melo Neto2* Artur L. Castro Neto1,2, Adriana N. A. L. M. Brito2, Antonio M. Rezende2, Franklin B. Magalhães3 and Osvaldo P. de Melo Neto2* * Correspondence: opmn@cpqam.fiocruz.br * Correspondence: opmn@cpqam.fiocruz.br Abstract The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: opmn@cpqam.fiocruz.br 2Instituto Aggeu Magalhães, Fundação Oswaldo Cruz (Fiocruz-Pernambuco) Recife, Pernambuco, Brazil Full list of author information is available at the end of the article * Correspondence: opmn@cpqam.fiocruz.br 2Instituto Aggeu Magalhães, Fundação Oswaldo Cruz (Fiocruz-Pernambuco), Recife, Pernambuco, Brazil Full list of author information is available at the end of the article © 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. Abstract Background: The leishmaniasis are parasitic diseases caused by protozoans of the genus Leishmania, highly divergent eukaryotes, characterized by unique biological features. To survive in both the mammalian hosts and insect vectors, these pathogens make use of a number of mechanisms, many of which are associated with parasite specific proteases. The metalloprotease GP63, the major Leishmania surface antigen, has been found to have multiple functions required for the parasite’s survival. GP63 is encoded by multiple genes and their copy numbers vary considerably between different species and are increased in those from the subgenus Viannia, including L. braziliensis. Results: By comparing multiple sequences from Leishmania and related organisms this study sought to characterize paralogs in silico, evaluating their differences and similarities and the implications for the GP63 function. The Leishmania GP63 genes are encoded on chromosomes 10, 28 and 31, with the genes from the latter two chromosomes more related to genes found in insect or plant parasites. Those from chromosome 10 have experienced independent expansions in numbers in Leishmania, especially in L. braziliensis. These could be clustered in three groups associated with different mRNA 3′ untranslated regions as well as distinct C-terminal ends for the encoded proteins, with presumably distinct expression patterns and subcellular localizations. Sequence variations between the chromosome 10 genes were linked to intragenic recombination events, mapped to the external surface of the proteins and predicted to be immunogenic, implying a role against the host immune response. Conclusions: Our results suggest a greater role for the sequence variation found among the chromosome 10 GP63 genes, possibly related to the pathogenesis of L. braziliensis and closely related species within the mammalian host. They also indicate different functions associated to genes mapped to different chromosomes. For the chromosome 10 genes, variable subcellular localizations were found to be most likely associated with multiple functions and target substrates for this versatile protease. Keywords: Leishmania braziliensis, GP63, Virulence proteins * Correspondence: opmn@cpqam.fiocruz.br 2Instituto Aggeu Magalhães, Fundação Oswaldo Cruz (Fiocruz-Pernambuco), Recife, Pernambuco, Brazil Full list of author information is available at the end of the article © 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. Background of the parasite, and provides the Leishmania with a faster entry into the macrophage, through the activation of a host tyrosine phosphatase [7, 9, 10]. GP63 has also been shown to be released through exosomes into the extracellular medium and this may facilitate its uptake by the macro- phage even before the internalization of the Leishmania parasite [11]. Lack of GP63 drastically reduces the Leishmania’s ability to establish and maintain an infection, since the hosts are more likely to induce an innate immunity inflammatory response [12]. Within the host cell cytoplasm, GP63 has been shown to cleave the transcriptional factor AP-1, which regulates the production of pro-inflammatory cytokines and nitric oxide by the macrophage [11, 13]. GP63 was also shown to be associated with the inactivation of the mTOR kinase, leading to the inhibition of protein synthesis in the macrophage and pro- viding an ideal environment for the proliferation of the pathogen [14]. g The leishmaniasis are parasitic infectious diseases caused by flagellated protozoa belonging to the genus Leishmania, family Trypanosomatidae, and which are transmitted by sandflies of the genera Phlebotomus or Lutzomyia. These diseases are found as two major clinical forms, named as cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL), with a global incidence for each in the range of hundreds of thousands of cases per year [1]. Multiple Leishmania species are associated with the leishman- iasis and distinct species, closely related or not, are responsible for the disease in different parts of the world. Those belonging to the subgenus Viannia are restricted to the New World (including L. braziliensis and L. guyanensis), have evolved separately from better known species belonging to the subgenus Leishmania (L. major, L. infantum, L. mexicana and others) and are associated with the mucocutaneous leishmaniasis (MCL), a more aggressive variation of CL [2]. Early studies have shown that GP63 is more abundantly expressed in the promastigote stage of the Leishmania life cycle, the proliferative stage within the insect vector. This expression may peak during metacyclogenesis, when the parasite prepares to infect the mammalian hosts, and is subsequently reduced again upon differentiation into amas- tigotes, the intracellular stage that multiplies within the mammalian macrophages [7, 15, 16]. The abundant GP63 expression in promastigotes indicates relevant functions also in the insect vector, presumably needed for survival and proliferation. Background Indeed, a potential involvement in the degradation of protein components that would lead to the adhesion of the parasite in the insect gut epithelium has been shown [17, 18]. Due to its wide substrate specificity, GP63 may also perform a nutritional role for the parasite, acting as an endopeptidase [19, 20], or even protect the Leishmania against the insect defences [19]. gg [ ] As successful pathogens, the various Leishmania species have developed effective mechanisms to escape the mam- malian host immune response and proliferate [3, 4]. Some of these evasion mechanisms are dependent on proteases, which help ensure that the parasites can invade the mammalian tissue, survive, differentiate and multiply [5]. The GP63 protease, also known as leishmanolysin or major surface protease (MSP), was first discovered in 1980 as the major surface antigen of the promastigote form of many species of Leishmania [6]. It was later found to be bound to the cell membrane through a GlycosylPhospha- tidylInositol (GPI) anchor and was also identified as an important virulence factor. This is a zinc-dependent me- talloproteinase, which belongs to the peptidase family M8 and the metzincin class and includes conserved features such as the motif HEXXHXXGXXH and a pro-peptide lo- cated in the protein’s N-terminal region that renders the proenzyme inactive during translation and is removed during its maturation and activation. The GP63 proteins also include an N-terminal signal sequence which directs them to the endoplasmic reticulum and to the Leishmania secretory pathway [7, 8]. Concerning the GP63 gene organization, there is a no- ticeable variation in the number of gene copies encoding these proteins among different Leishmania species. In L, major these genes are present in more than one chromo- some and multiple copies have been detected arranged in tandem [21], with the same multi-copy arrangement also found in L. infantum and L. braziliensis [22]. Noteworthy, however, is the substantial increase in the number of gp63 genes reported for L. braziliensis and other species belonging to the subgenus Viannia, when compared with the subgenus Leishmania. This was reported in early stud- ies [23–25] and has been confirmed more recently by results derived from a screening for cosmids harboring multiple GP63 genes from L. braziliensis [26], as well as by genome sequencing data for different Leishmania spe- cies [27–29]. No clear biological reasons are known, how- ever, to explain this expansion in the GP63 gene copy number. Castro Neto et al. BMC Genomics (2019) 20:118 Page 2 of 17 Castro Neto et al. BMC Genomics (2019) 20:118 Page 2 of 17 Background The results of the search for each HMM are summarized in Additional file 2: Table S2 and confirm the gene count number for GP63 genes derived from the L. braziliensis genome sequencing, 38 genes, lower than earlier estimates based on the hybridization studies [23]. y Next, we considered that the shot gun nature of the se- quencing strategy used for the assembly of the best genome available from a Viannia species, from the L. braziliensis 2904, might have led to the grouping of similar GP63 genes together, causing in turn a reduction in the number of genes found. In order to obtain as many natural GP63 sequences as possible, and therefore have a clearer idea of the true number of genes present in the L. braziliensis gen- ome, we opted to amplify these genes using primers directed to conserved regions of representative genes iden- tified in the genome analysis. The PCR strategy used to amplify the GP63 genes can be seen in Fig. 1, superimposed on a schematic representation of a typical GP63. The scheme highlights conserved elements found on all GP63 sequences, such as the zinc-binding motif, multiple cysteine residues, the GPI anchor site and a nearly universally con- served motif of seven consecutive amino acids we named KDELMAP. Six oligonucleotides annealing to sequences encoding the N-terminal ends of the GP63 sequences were used as 5′ primers, considering the variation previously observed within the N-terminus of the various GP63 genes and in order to maximize the number of genes amplified. As 3′ primers, two sets of two oligonucleotides annealing to the more conserved KDELMAP or the GPI anchor site motifs were alternatively used. Individual PCR reactions were set up with different pairs of oligonucleotides, always with a single 5′ and a single 3′ primer. After amplification, cloning and sequencing a total of 40 different GP63 gene fragments were obtained, with thirty-four of those having sequences different from the ones described in the data- bases. The new GP63 DNA fragments obtained by PCR are listed in Additional file 3: Table S3, which also includes the set of oligonucleotide primers used to amplify each se- quence. In all 31 new gene fragments were found, since some were duplicates (also indicated in the table). The new sequences have all been submitted to GenBank and were compared with the already known GP63 genes from L. Background Here, aiming to contribute further to the under- standing of the role of GP63 in Leishmania pathogenesis GP63 has been found to play multiple roles during Leishmania infection in mammals, starting in the extra- cellular environment where it acts inactivating the complement cascade, by cleaving C3b into iC3b. This inactivation prevents the formation of the membrane attack complex (MAC), despite allowing the opsonisa- tion of the Leishmania, mediated by iC3b, and facilitat- ing its phagocytosis. GP63 can also facilitate the binding of the parasite to the macrophage through fi- bronectin receptors, cleaving proteins from the host’s extracellular matrix. Within the macrophages, it also acts to reduce the production of TNF, IL-12 and nitric oxide, which contributes to the protection and survival Page 3 of 17 Castro Neto et al. BMC Genomics (2019) 20:118 Page 3 of 17 Castro Neto et al. BMC Genomics (2019) 20:118 in general but with a focus on Viannia species, we sought to investigate the GP63 gene expansion further, using a range of in silico tools. We started by better defining the extent of GP63 gene diversity in L. braziliensis, followed by an in-depth analysis of the similarities and differences between different genes from this and related Leishmania species. The GP63 genes were first grouped according to their chromosomal localization followed by phylogenetic comparisons between different trypanosomatid species. Further grouping according to sequence similarities or dif- ferences within non-coding and coding elements was also carried out, in order to define putative functional distinc- tions. Possible mechanisms associated with the gene expan- sion due to DNA recombination were then investigated and variations in sequence mapped on the GP63 structure and linked with predictions of immunogenic potential. Our results are consistent with a selective expansion of a subset of GP63 genes in L. braziliensis that might be linked to mammalian pathogenesis and might be required for a bet- ter protection against the host immune system. the search. The genome of L. braziliensis strain 2904 depos- ited on TriTrypDB lists 39 GP63 genes and, in total, the HMMs identified the presence of 38 related sequences. A single gene (LbrM.10.1720) was not recovered using these models and indeed its coding sequence did not provide an alignment with a score high enough to be considered as a GP63. Background braziliensis and these analyses will be discussed further below. They are consistent with a higher copy number for the L. braziliensis GP63 genes than pre- dicted based on the genome sequencing alone and more in agreement with the original estimates based on Southern-blots, although no precise quantification Genomic analysis of known Leishmania GP63 genes Genomic analysis of known Leishmania GP63 genes To clarify the relationship between the multiple GP63 genes in Leishmania, we opted to review their chromo- somal organization within the major lineages of pathogenic Leishmania. For L. major, the best studied of the available Leishmania genome sequences, three sets of GP63 genes were found distributed in chromosomes 28 (one gene), 31 (one gene) and 10 (four genes) [21]. Based on the sequences available at the Tritryp database, a similar organization is also observed for L. infantum (summarized in Fig. 2) and L. mexicana (not shown), represented by two and one genes for chromosome 28 in L. infantum and L. mexicana, respectively, and one gene for chromosomes 31 in both species, considering that the L. mexicana chromo- some 30 is equivalent to the L. major chromosome 31. Five GP63 genes are also found in chromosome 10 for both species and in agreement with previously reported data for L. infantum [22]. p p y p q The significantly greater number of L. braziliensis GP63 genes from chromosome 10 is supported by our PCR data where primers sets directed to the chromosome 10 genes were able to amplify more genes than the ones originally used for their synthesis. For example, a primer pair designed to amplify the gene LbrM.10.0470 allowed the amplification of eight different gene fragments (G0510B2; G0560B1; G0560B2; G1610B3; G1610B4; G1610B5; G1610B6; G1620B1) and similarly, the primer pair directed to gene LbrM.10.0540 amplified fragments from six differ- ent genes (G0510C1; G0510C2; G0540C1; G0560C4; G1640C1; G1640C2). In contrast, two sets of PCR reac- tions directed to a single GP63 gene from chromosome 31, using the same 5′ primer and two distinct 3′ primers, only led to the amplification of the same gene, LbrM.31.2260 (Additional file 3: Table S3). Indeed, we believe that most of the six GP63 genes annotated from the L. braziliensis chromosome 31 might not exist and in fact are either pseudogenes or derived from genome assembly errors. Only one of those genes (LbrM.31.2260) has GP63-related protein features, such as the propeptide domain (HEXXH), In L. braziliensis, based on the available genomic data for the 2904 strain, major differences in the organization of the GP63 genes are observed when they are compared with those found in species from the Leishmania subgenus. Results Search for new L. braziliensis GP63 paralogs The early studies based on hybridization assays [23, 24] had suggested that the total number of GP63 genes found within Leishmania species belonging to the Viannia sub- genus is greater than the number of genes available at the TriTrypDB database and identified after the L. braziliensis genome sequencing and annotation. Recent data based on next generation sequencing have also suggested major vari- ations in copy number of GP63 genes between species within the same subgenus, Leishmania or Viannia, that have not yet been included on the annotated genomes [28–30]. Here, to begin to understand the true diversity of the L. braziliensis GP63 genes, we first sought to reevaluate the available L. braziliensis GP63 gene sequences consider- ing that the automatic annotation methods might have missed further genes. We therefore performed a reanalysis of the L. braziliensis genome sequences and searched for possible new GP63 paralogs that might not have been annotated. To do this we performed a search in the L. braziliensis genome using the Hidden Markov Models (HMMs) methodology [31], carried out after a grouping of the entire proteome set from different Leishmania species (described in methods). Nine subsets of GP63 sequences were created using the OrthoMCL tool in order to group these sequences and allow the search to be performed, as shown in Additional file 1: Table S1, with the number of genes in each subset varying in size from 56 to only two. All nine subsets were used to build HMMs and these were then applied for the search of new paralogs in the pre- dicted proteome from L. braziliensis 2904. In general, all HMMs were able to find the GP63 sequences assigned to each subset, however no new paralogs were found during Castro Neto et al. BMC Genomics (2019) 20:118 Page 4 of 17 Fig. 1 GP63 general gene features. The scheme highlights conserved elements found on most GP63 sequences, such as the signal peptide, propeptide, zinc-binding motif [8], multiple cysteine residues, the GPI anchor site and a nearly universally conserved motif of seven consecutive amino acids we named KDELMAP. The PCR strategy used to amplify the GP63 genes is also shown with products varying from 1 Kb (up to the KDELMAP region) and 1,6 Kb (finishing on the GPI anchor site) Fig. 1 GP63 general gene features. Results The scheme highlights conserved elements found on most GP63 sequences, such as the signal peptide, propeptide, zinc-binding motif [8], multiple cysteine residues, the GPI anchor site and a nearly universally conserved motif of seven consecutive amino acids we named KDELMAP. The PCR strategy used to amplify the GP63 genes is also shown with products varying from 1 Kb (up to the KDELMAP region) and 1,6 Kb (finishing on the GPI anchor site) contrast, six GP63 genes or gene fragments are found on chromosome 31, again generally flanked by orthologues to the same genes found flanking the GP63 gene found in the L. major and L. infantum chromosome 31. Even more noteworthy, however, are the 33 GP63 genes found clus- tered on chromosome 10. Again, these are localized to the same region seen harboring the other Leishmania chromo- some 10 genes, as confirmed by the presence of neighbor- ing sequences encoding orthologues to those found flanking the L. major and L. infantum GP63 genes from chromosome 10. However, the precise gene organization cannot be properly defined and many of the genes sequenced are assembled in relatively short contigs, as indi- cated in the scheme from Fig. 2c. Again, this might be due to the high similarity between the gene sequences and the nature of the sequencing strategy which might have pre- vented a proper assembly of repeated sequences. is possible either way. Despite the fact that both the gen- omic and PCR data used for our analyses are derived from the same L. braziliensis 2904 strain, the question of cultur- ing in different laboratories being responsible for the differences observed regarding gene copy number and identification between the two sets of results can be raised. Nevertheless, considering the limited time frame for the culturing procedures, these could only have any impact on gene or chromosomal duplication events and would not lead to the different gene sequences that were found through PCR and/or DNA sequencing. Genomic analysis of known Leishmania GP63 genes First, no GP63 gene is found on chromosome 28, as highlighted before for other Viannia species [32], despite the presence of orthologues to the same genes flanking the single GP63 sequence from L. major and L. infantum. In Castro Neto et al. BMC Genomics (2019) 20:118 Page 5 of 17 A B C Fig. 2 (See legend on next page.) A B A B C Fig. 2 (See legend on next page.) C Castro Neto et al. BMC Genomics (2019) 20:118 Page 6 of 17 (See figure on previous page.) Fig. 2 Genomic organization of Leishmania GP63 genes. Genomic organization of GP63 genes from Leishmanis sp., showing the general distribution, location and synteny of these genes along chromosomes 10, 28 and 31. a Synteny analysis on chromosome 28 showing the localization of the GP63 gene in L. major and L. infantum and its absence in the equivalent position from the L. braziliensis chromosome 28. b Distribution of GP63 genes in Leishmania sp., chromosome 31. Pseudogenes from the L. braziliensis genome are highlighted in gray. c Expansion of GP63 genes in the L. braziliensis chromosome 10, when compared to L. major and L. infantum, within the same chromosome position, as indicated by the flanking genes on the left (some unrelated genes are also included). The horizontal arrows indicate the transcription sense of the genes and the yellow, black and red colors define the 3’ UTR groups identified for the L. major and L. infantum genes. The diagram for L. infantum does not include the preliminary annotation derived from the recent resequencing of its genome [30]. For the L. braziliensis genes, identical colors group those with similar 3’UTRs, as classified in Table 1 and shares a high similarity (85%) with the L. major and L. infantum chromosome 31 genes. The LbrM.31.2200, LbrM.31.2220, LbrM.31.2230, LbrM.31.2240 and LbrM.31. 2250 genes have stop codons in the middle of their se- quences and/or in alignments showed identical N-terminal or C-terminal regions to LbrM.31.2260 (data not shown). It is possible that these genes may represent parts of LbrM.31.2260 not properly assembled and this in agree- ment with our PCR data finding only LbrM.31.2260. Over- all these results are consistent with the expansion in the number of GP63 genes in L. braziliensis, and other species belonging to the Viannia subgenus, being mainly directed to the chromosome 10 genes. the genes located on chromosome 10. Noteworthy are the T. GP63 evolutionary analyses Sequences encoding GP63 related genes are also found in other trypanosomatids and more distantly related kineto- plastids and these include multiple genes from T. brucei, T. cruzi, and others. The number of genes in these para- sites is quite variable. T. cruzi has over 150 GP63 genes annotated in the TriTrypDB database, but with many pseudogenes among them. T. brucei and C. fasciculata have a smaller amount with 10 and 18 genes respectively. This multiplicity of GP63 genes along the various kineto- plastids lineages reinforce the multiple roles this protein has, independent of the life cycle of the organism involved. Here, we next sought to assess how the Leishmania GP63 genes are related to those found in more distantly related kinetoplastids and whether some function can be inferred based on which genes are found in each organism. To do this we built a phylogenetic tree comparing the most divergent and representative sequences from the three major sets of Leishmania GP63 genes (from chromosomes 10, 28 and 31) with genes from different Trypanosoma species (T. brucei, T. cruzi and T. theileri) and more distantly related organisms. These included species that parasitize reptiles (L. tarentolae) and plants (Phytomonas sp.), have monoxenous life-cycles in insects (Crithidia fasciculata and Leptomonas pyrrhocoris) and are free living (Bodo saltans). As shown in Fig. 3, the phylogenetic analysis could separate the GP63 genes mapped to the Leishmania chromosome 10 from those genes mapped to chromosomes 28 and 31. We also could observe a clear separation between the Leishmania subgenus based on Genomic analysis of known Leishmania GP63 genes cruzi and T. brucei GP63 sequences more closely asso- ciated with the GP63 genes from B. saltans and T. theileri. Also, when we observe the clustering of the genes present in chromosomes 28 and 31 from Leishmania, they gener- ally show more proximity to the genes from L. pyrrhocoris, C. fasciculata and Phytomonas sp. Nevertheless, one L. pyrrhocoris and two C. fasciculata genes are more closely related to those from the L. braziliensis chromosome 10. Overall, the gene clusters shown in the tree highlight the higher similarity between the Leishmania sp. genes from chromosomes 28 and 31 with the GP63 genes found in organisms that live in insects only or parasitize plants. For instance, the 38 annotated GP63 genes from Phytomonas are more closely related to the Leishmania chromosome 28 GP63. It is then possible to hypothesize that these genes might me more involved in the insect stage of the parasite life cycle. Genes more closely related to the chromosome 10 GP63 genes can be found in the insect parasites L. pyrrhocoris and C. fasciculate, but in general these genes seem to have suffered a sub- stantial expansion within Leishmania species. Evaluation of the sequence diversity of the Leishmania GP63 genes from chromosome 10 Bs: Bodo saltans, Tt: Trypanosoma theileri, Tb: Trypanosoma brucei, Tc: Trypanosoma cruzi, Lp: Leptomonas pyrrhocoris, Cf: Crithidia fasciculata, Lb: Leishmania braziliensis M2904, Lmj: Leishmania major, Lmx: Leishmania mexicana, Li: Leishmania infantum, Lt: Leishmania tarentolae Fig. 3 Phylogenetic tree comparing selected Leishmania GP63 paralogs with sequences from more distantly related organisms. The tree demonstrates the proximity between the GP63 genes from the Leishmania chromosomes 28 and 31, with GP63 genes from monoxenes trypanosomatids genes, such as Leptomonas pyrrhocoris and Crithidia fasciculata, as well as, genes from a plant tripanosomatid, like Phytomonas sp. Values for highly supported nodes have been replaced by black and white squares, which represents the Bayesian posterior probabilities and bootstrap support for PhyML, respectively. Bs: Bodo saltans, Tt: Trypanosoma theileri, Tb: Trypanosoma brucei, Tc: Trypanosoma cruzi, Lp: Leptomonas pyrrhocoris, Cf: Crithidia fasciculata, Lb: Leishmania braziliensis M2904, Lmj: Leishmania major, Lmx: Leishmania mexicana, Li: Leishmania infantum, Lt: Leishmania tarentolae chromosome 10 GP63 genes is not dependent on their mammalian hosts. We also included in this analysis the new L. braziliensis sequences generated by us through the PCR approach. The phylogenetic tree shown in Fig. 4 sum- marizes the results from these analyses based on align- ments using the full-length sequences for all proteins (or the full-length PCR fragments). For clarity, only the most divergent representative sequences were used to build this tree, with those very similar or nearly identical to the ones shown purportedly removed from the final figure. In the original analysis all chromosome 10 sequences from the selected species were used but with similar results (not shown). Within each of the three Leishmania subgenera analysed, all GP63 sequences from chromosome 10 are more closely related to sequences from the same or related species than to sequences found in species belonging to the other subgenera. Even within the Leishmania clades, the L. infantum genes (in red) seemed to be more closely related to each other than to their L. major counterparts, although for the two Viannia species (L. braziliensis and L. guyanensis) analysed genes (in green) more closely related between the two species were found. These results are in agreement with independent expansions on the number of the chromosome 10 GP63 sequences in each clade, with major expansions occurring for both Sauroleishmania (in blue) and Viannia species. For the latter species, at least, the start of this expansion may have preceded the split between L. braziliensis and L. Evaluation of the sequence diversity of the Leishmania GP63 genes from chromosome 10 Considering the expansion of the chromosome 10 GP63 genes in the Leishmania lineage in general, and even more so in L. braziliensis and other Viannia species, we then opted to investigate the origins of their diversity further. To do this we compared the full extent of the chromosome 10 GP63 sequences from relevant Leishmania species using as outgroups selected genes from chromosomes 28 and 31. For this analysis, we also included sequences from L. tarentolae (based on the published genome sequence [33]), where a similar expansion in the chromosome 10 GP63 genes was noticed, with 49 genes found in this chromosome while only one gene was found in chromo- some 31 and another in chromosome 33. L. tarentolae is currently classified within the Sauroleishmania subgenus, but it is likely to be more closely related to the Leishmania subgenus than to Viannia [2, 34]. The relevance in includ- ing the L. tarentolae sequences is due to the fact that it does not parasitize mammals, only lizards, meaning that any potential role in pathogenesis associated with the Castro Neto et al. BMC Genomics (2019) 20:118 Page 7 of 17 Fig. 3 Phylogenetic tree comparing selected Leishmania GP63 paralogs with sequences from more distantly related organisms. The tree demonstrates the proximity between the GP63 genes from the Leishmania chromosomes 28 and 31, with GP63 genes from monoxenes trypanosomatids genes, such as Leptomonas pyrrhocoris and Crithidia fasciculata, as well as, genes from a plant tripanosomatid, like Phytomonas sp. Values for highly supported nodes have been replaced by black and white squares, which represents the Bayesian posterior probabilities and bootstrap support for PhyML, respectively. Bs: Bodo saltans, Tt: Trypanosoma theileri, Tb: Trypanosoma brucei, Tc: Trypanosoma cruzi, Lp: Leptomonas pyrrhocoris, Cf: Crithidia fasciculata, Lb: Leishmania braziliensis M2904, Lmj: Leishmania major, Lmx: Leishmania mexicana, Li: Leishmania infantum, Lt: Leishmania tarentolae Fig. 3 Phylogenetic tree comparing selected Leishmania GP63 paralogs with sequences from more distantly related organisms. The tree demonstrates the proximity between the GP63 genes from the Leishmania chromosomes 28 and 31, with GP63 genes from monoxenes trypanosomatids genes, such as Leptomonas pyrrhocoris and Crithidia fasciculata, as well as, genes from a plant tripanosomatid, like Phytomonas sp. Values for highly supported nodes have been replaced by black and white squares, which represents the Bayesian posterior probabilities and bootstrap support for PhyML, respectively. Evaluation of the sequence diversity of the Leishmania GP63 genes from chromosome 10 guyanensis but has subsequently continued and may be an on- going process. Castro Neto et al. BMC Genomics (2019) 20:118 Page 8 of 17 Fig. 4 Phylogeny of Leishmania GP63 paralogs from chromosome 10. Phylogenetic tree comparing multiple chromosome 10 GP63 genes from selected Leishmania species. The tree highlights the separation of the GP63 genes according to the three main Leishmania subgenera (Sauroleishmania, Leishmania and Viannia). Values for highly supported nodes have been replaced by black and white squares, which represents the Bayesian posterior probabilities and bootstrap support for PhyML, respectively. Lb: Leishmania braziliensis M2904, Lmj: Leishmania major, Lmx: Leishmania mexicana, Li: Leishmania infantum, Lt: Leishmania tarentolae, Lg: Leishmania guyanensis Fig. 4 Phylogeny of Leishmania GP63 paralogs from chromosome 10. Phylogenetic tree comparing multiple chromosome 10 GP63 genes from selected Leishmania species. The tree highlights the separation of the GP63 genes according to the three main Leishmania subgenera (Sauroleishmania, Leishmania and Viannia). Values for highly supported nodes have been replaced by black and white squares, which represents the Bayesian posterior probabilities and bootstrap support for PhyML, respectively. Lb: Leishmania braziliensis M2904, Lmj: Leishmania major, Lmx: Leishmania mexicana, Li: Leishmania infantum, Lt: Leishmania tarentolae, Lg: Leishmania guyanensis Fig. 4 Phylogeny of Leishmania GP63 paralogs from chromosome 10. Phylogenetic tree comparing multiple chromosome 10 GP63 genes from selected Leishmania species. The tree highlights the separation of the GP63 genes according to the three main Leishmania subgenera (Sauroleishmania, Leishmania and Viannia). Values for highly supported nodes have been replaced by black and white squares, which represents the Bayesian posterior probabilities and bootstrap support for PhyML, respectively. Lb: Leishmania braziliensis M2904, Lmj: Leishmania major, Lmx: Leishmania mexicana, Li: Leishmania infantum, Lt: Leishmania tarentolae, Lg: Leishmania guyanensis Gene recombination in GP63 sequences from chromosome 10 Through analyzes of the alignments generated in this study, we identified that specific regions of certain GP63 gene se- quences were very similar to equivalent regions from other GP63 genes which otherwise were more divergent. For ex- ample, certain small motifs generally seen only on the L. braziliensis Group 1 genes were also found in one or more of the group 2 genes and vice-versa, an indication of gene recombination. Indeed, the locus for these genes is reported as having high plasticity [23, 37] and the data from the literature shows that this gene family can be influenced by mosaic or fragmental gene conversion [26, 38]. Here, in order to understand why the expansion of the GP63 genes occurs mainly on chromosome 10, we performed an in-silico search for recombination events targeting these genes so as to better evaluate whether their variability was related to intragenic recombination. The software chosen to find the recombination events (RDP4) uses several tools to determine events such as the likely position of recombination breakpoints and the identity of sequences most closely related to the gene being evaluated. In this study we only considered recombination events that were detected by at least two of the tools tested. Therefore, we decided to perform a gene recombination analysis with all the GP63 genes of L. braziliensis present in databases and the ones generated by us through PCR. We first targeted the chromosome 31 GP63 genes, but no recombination events were detected by the software. In contrast, when the 38 PCR sequences from chromosome 10 were analyzed 30 (or 79%) were reported as recombinant genes. Regarding the database genes, 32 recombination events were found, Next, we attempted to group the L. braziliensis chromo- some 10 genes based first on similarities and differences within the putative 3’UTRs. Thirty genes were analyzed based on the sequences available from the reference gen- ome sequence and these were classified into six groups according to their 3’UTR, with the first two groups repre- sented by 8 and 15 genes, respectively, while the remaining groups included only one or two genes (Table 1 – also col- ored differently in Fig. 2). When these 3’UTRs were com- pared with the three L. infantum and L. major groups no clear similarities were found with any of the L. braziliensis groups, likely due to the large sequence variation observed between species from the two distinct subgenera. Identification of functional differences between the various Leishmania GP63 genes from chromosome 10 infantum Group 1 proteins are character- ized by a longer C-terminus enriched in hydrophobic and positively charged residues and lack the typical asparagine required for the GPI anchor. In contrast, the shorter C-terminus from the Group 2 and 3 proteins include the GPI anchor site and end in a stretch of mostly hydropho- bic amino acids (Fig. 5). and 3 can be seen, since they share a nearly identical C-terminus that includes the GPI anchor attachment motif (DGGN [36]). The Group 1 genes from L. braziliensis also share conserved elements with the L. major/L. infantum Group 1, such as the lack of a typical GPI anchor site and the presence of a hydrophilic set of amino acids followed by a hydrophobic region resembling a transmembrane segment. The motif “MRQWRERMTALATVT” found in the L. braziliensis sequences is also very similar to the “MQRWNDRMAGLATAA” motif found in L. infantum LinJ.10.0510 gene. Only the L. braziliensis Group 2 genes then, characterized by a shorter C-terminus missing entirely the GPI anchor site or related hydrophobic sequences, do not seem to have counterparts in L. infantum nor in L. major. Neverthe- less, it seems likely that, as observed in L. infantum and L. major, the different groups of L. braziliensis chromo- some 10 GP63 genes are also differentially regulated during the parasite growth in culture and this is in agreement with the different 3’UTRs seen associated with each group. Identification of functional differences between the various Leishmania GP63 genes from chromosome 10 Identification of functional differences between the various Leishmania GP63 genes from chromosome 10 infantum and L. major, three growth stage-specific pat- terns of expression were observed for the then known GP63 genes, with one gene constitutively expressed, a sec- ond gene (or genes) expressed during the log phase of promastigote growth and the third gene expressed only in stationary phase cells and/or amastigotes [15, 25]. Here, by comparing their coding sequences and 3’ UTRs with those from the available genomes, we were able to map those genes within the annotated genome sequences (indi- cated in Fig. 2): LinJ.10.0510 and LmjF.10.0470 are the So far not much is known regarding possible functional differences between the various GP63 genes found within any specific trypanosomatid. In Leishmania, with the goal of defining functional differences between multiple GP63, and even prior to the completion of the first Leishmania genomes, early studies investigated the expression pattern of selected genes attempting to identify differences in ex- pression during the parasite life cell cycle [8, 15, 35]. In L. Castro Neto et al. BMC Genomics (2019) 20:118 Castro Neto et al. BMC Genomics (2019) 20:118 Page 9 of 17 Page 9 of 17 constitutively expressed genes (here named Group 1 – colored in black in the figure); LinJ. 10.0490/LinJ.10.0500 and LmjF. 10.0460/LmjF.10.0465 are equivalent to the previously described log phase promastigote genes (Group 2 – highlighted in yellow); LinJ. 10.0520/LinJ.10.0530 from L. infantum and the L. major LmjF.10.0480 gene corres- pond to the stationary phase/amastigote specific GP63 (Group 3 – colored in red). The Group 1 genes are char- acterized by unique 3’ UTRs and 3′ intergenic regions ab- sent from the remaining chromosome 10 genes, while the Group 2 and 3 genes share very similar sequences within the first ~ 400 nucleotides of their 3’UTRs, although these subsequently diverge into two distinct patterns that correl- ate with the two groups (these genes and their groups are highlighted by different colors in the scheme from Fig. 2). We also looked at differences within the coding sequences that could be typical of GP63 genes belonging to any par- ticular group. As previously reported for L. major [25], a clear distinction is observed between the C-terminus of the Group 1 proteins and those from Group 2 and 3. Both L. major and L. Gene recombination in GP63 sequences from chromosome 10 We also looked at the amino acid sequences, looking for features in common for L. braziliensis genes sharing similar UTRs. No association with features such as signal peptide, transmem- brane domains and isoelectric points was found, however distinct C-terminus were observed which also separated the L. braziliensis into six groups, with a clear association observed between each 3’UTR group and nearly all of the proteins’ C-terminal ends (Table 1). Since Groups 4, 5 and 6 consists of truncated proteins they will not be considered further here, but for the remaining three groups their C-terminal ends were also compared with those seen to be associated with the L. infantum and L. major groups. A clear association between the two proteins from the L. bra- ziliensis Group 3 and the L. major/L. infantum Groups 2 Castro Neto et al. BMC Genomics (2019) 20:118 Page 10 of 17 730 740 750 760 770 780 790 800 ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| LinJ.10.0500 556 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0490 556 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0520 720 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0530 555 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0510 557 PPYVEVCQAN VKGAKDFAGD SDSSSSAGDA ADRAAMQRWN DRMAGLATAA MVLLGMVLSL MALVVVWLLL LTCPWWCCKF 570 580 590 600 610 620 630 640 ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| LmjF.10.0460 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLAVAL--- ---------- ---------- LmjF.10.0465 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLTVAL--- ---------- ---------- LmjF.10.0480 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLAVAL--- ---------- ---------- LmjF.10.0470 561 PYVEVCQGNV QAAKDFDGDS DSSSSSSDAA DKAAIERWNE RMAGLATAAT VLLGVVLSLM ALVVVWLLLV SCPRWCCKVG 710 720 730 740 750 ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| .... Gene recombination in GP63 sequences from chromosome 10 LbrM.10.0510 569 KGLIDFERDA ADTAAMRQWR ERMTALATVT AALLGIVLAA MAGLVVGLLV ISLS LbrM.10.0530 448 KGVIDFEGDA ADTAAMRRWR ERMTALATVT AALLGIVLAA MAGLAVWLLL ISLP LbrM.10.0550 569 KGVIDFERDA ADTAAMRQWS ERMYVLATVT AVLLGIVLAA MAGLVSV--- ---- LbrM.10.0560 570 KGVIDFERDA ADTAAMRQWS ERMTALATVT AVLLGIVLAA MAGLVVGLLV ISLS LbrM.10.0580 569 KGLIDFERDA ADTAAMRQWS ERMYVLATVT AVLLGIVLAA MAGLVVGLLV ISLP LbrM.10.0600 558 KGVIDFEGDA ADTAAMRRWR ERMTALATVT AALLGIVLAA MAGLAVWLLL ITIP LbrM.10.0610 568 KGLIDFERDA ADTAAMRQWS ERMYVLATVT AVLLGIVLAA MAGLVVGLLV ISLS LbrM.10.1580 514 KGLIDFEGDA ADTAAMRQWS ERMYVLATVT AVLLGIVLAA MAGLVVGLLV ISLS LbrM.10.0470 562 KGLIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0480 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0500 370 KGVIDFERDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0520 560 KGVIDFERDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0540 542 KGVIDFERDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0570 435 KGVIDFEGDA ADTAAA---- ---------- ---------- ---------- ---- LbrM.10.1570 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1590 692 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1610 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1620 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1630 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1640 696 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1650 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1660 562 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1680 386 KGLIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0590 556 QGAT-----S SGNAAAGRRG PRAAATALLV AALLAIACA- ---------- ---- LbrM.10.1690 478 QGAT-----S SGNAAAGRRG PRAAATALLV AALLAIACA- ---------- ---- Group 1 Group 2 Group 3 Leishmania braziliensis C C Leishmania braziliensis Constitutive C-terminal motif Predicted GPI anchor site Stage specific C-terminal motif Fig. 5 Leishmania major, L. infantum and L. braziliensis C-terminal alignments and groups. Alignment showing the difference in the C-terminuses of the chromosome 10 GP63 sequences having divergent 3’ UTRs. a and b shows the L. major and L. infantum C-terminus sequences, respectively, their groups based on their expression profiles and the predicted GPI anchor site (boxed in green). The motif identified in the constitutively expressed L. infantum group 1 sequence also found in the L. braziliensis group 1 C-terminus is boxed in red, while the motif found in the L. infantum and L. major stage specific genes from Groups 2 and 3 nearly identical to a similar motif from the L. braziliensis group 3 genes is boxed in orange. c Alignment of the C-terminal ends of the different L. braziliensis chromosome 10, with the grouping described in the text and the conserved elements also seen in L. major and L. infantum indicated as for A and B sometimes with more than one event for the same gene (Fig. 6 and Additional file 4: Table S4). Gene recombination in GP63 sequences from chromosome 10 Gene duplication and recombination events then are possibly the major source of the novel GP63 sequences seen in the chromo- some 10 from L. braziliensis and closely related sequences. domains (N-terminus, Central domain and C-terminus) and with features typical of the catalytic modules of zinc proteases [39]. After observing the recombination events and sequence variations between the multitudes of L. brazi- liensis GP63 genes, we decided to investigate where these variations are found along the 3D structure of the protein. Through three-dimensional protein structure predictions, we were able to model the structure of eight divergent GP63 sequences with high modeling scores, as can be seen in Fig. 7. We then mapped on the models the most variable motifs identified by the previous multiple alignments (highlighted in blue in the structures shown in the figure). Gene recombination in GP63 sequences from chromosome 10 LbrM.10.0510 569 KGLIDFERDA ADTAAMRQWR ERMTALATVT AALLGIVLAA MAGLVVGLLV ISLS LbrM.10.0530 448 KGVIDFEGDA ADTAAMRRWR ERMTALATVT AALLGIVLAA MAGLAVWLLL ISLP LbrM.10.0550 569 KGVIDFERDA ADTAAMRQWS ERMYVLATVT AVLLGIVLAA MAGLVSV--- ---- LbrM.10.0560 570 KGVIDFERDA ADTAAMRQWS ERMTALATVT AVLLGIVLAA MAGLVVGLLV ISLS LbrM.10.0580 569 KGLIDFERDA ADTAAMRQWS ERMYVLATVT AVLLGIVLAA MAGLVVGLLV ISLP LbrM.10.0600 558 KGVIDFEGDA ADTAAMRRWR ERMTALATVT AALLGIVLAA MAGLAVWLLL ITIP LbrM.10.0610 568 KGLIDFERDA ADTAAMRQWS ERMYVLATVT AVLLGIVLAA MAGLVVGLLV ISLS LbrM.10.1580 514 KGLIDFEGDA ADTAAMRQWS ERMYVLATVT AVLLGIVLAA MAGLVVGLLV ISLS LbrM.10.0470 562 KGLIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0480 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0500 370 KGVIDFERDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0520 560 KGVIDFERDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0540 542 KGVIDFERDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0570 435 KGVIDFEGDA ADTAAA---- ---------- ---------- ---------- ---- LbrM.10.1570 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1590 692 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1610 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1620 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1630 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1640 696 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1650 569 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1660 562 KGVIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.1680 386 KGLIDFEGDA ADTAAV---- ---------- ---------- ---------- ---- LbrM.10.0590 556 QGAT-----S SGNAAAGRRG PRAAATALLV AALLAIACA- ---------- ---- LbrM.10.1690 478 QGAT-----S SGNAAAGRRG PRAAATALLV AALLAIACA- ---------- ---- Group 1 Group 2 Group 3 Leishmania major Leishmania infantum Leishmania braziliensis Group 1 Group 2 Group 3 Group 1 Group 2 Group 3 Constitutive C-terminal motif Predicted GPI anchor site Stage specific C-terminal motif A B C Fig. 5 Leishmania major, L. infantum and L. braziliensis C-terminal alignments and groups. Alignment showing the difference in the C-terminuses of the chromosome 10 GP63 sequences having divergent 3’ UTRs. a and b shows the L. major and L. infantum C-terminus sequences, respectively, their groups based on their expression profiles and the predicted GPI anchor site (boxed in green). The motif identified in the constitutively expressed L. infantum group 1 sequence also found in the L. braziliensis group 1 C-terminus is boxed in red, while the motif found in the L. infantum and L. major stage specific genes from Groups 2 and 3 nearly identical to a similar motif from the L. braziliensis group 3 genes is boxed in orange. c Alignment of the C-terminal ends of the different L. braziliensis chromosome 10, with the grouping described in the text and the conserved elements also seen in L. major and L. Gene recombination in GP63 sequences from chromosome 10 infantum indicated as for A and B 570 580 590 600 610 620 630 640 ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| LmjF.10.0460 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLAVAL--- ---------- ---------- LmjF.10.0465 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLTVAL--- ---------- ---------- LmjF.10.0480 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLAVAL--- ---------- ---------- LmjF.10.0470 561 PYVEVCQGNV QAAKDFDGDS DSSSSSSDAA DKAAIERWNE RMAGLATAAT VLLGVVLSLM ALVVVWLLLV SCPRWCCKVG Leishmania major Group 1 Group 2 Group 3 A 730 740 750 760 770 780 790 800 ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| LinJ.10.0500 556 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0490 556 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0520 720 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0530 555 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0510 557 PPYVEVCQAN VKGAKDFAGD SDSSSSAGDA ADRAAMQRWN DRMAGLATAA MVLLGMVLSL MALVVVWLLL LTCPWWCCKF 570 580 590 600 610 620 630 640 ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| LmjF.10.0460 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLAVAL--- ---------- ---------- LmjF.10.0465 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLTVAL--- ---------- ---------- LmjF.10.0480 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLAVAL--- ---------- ---------- LmjF.10.0470 561 PYVEVCQGNV QAAKDFDGDS DSSSSSSDAA DKAAIERWNE RMAGLATAAT VLLGVVLSLM ALVVVWLLLV SCPRWCCKVG Leishmania major Leishmania infantum Group 1 Group 2 Group 3 Group 1 Group 2 Group 3 A B 730 740 750 760 770 780 790 800 ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| LinJ.10.0500 556 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0490 556 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0520 720 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0530 555 PPYVEVCQGN VQAAKD---- -GGNAAAGRR GPRAA----- ------ATAL LVAALLAVAL ---------- ---------- LinJ.10.0510 557 PPYVEVCQAN VKGAKDFAGD SDSSSSAGDA ADRAAMQRWN DRMAGLATAA MVLLGMVLSL MALVVVWLLL LTCPWWCCKF LmjF.10.0460 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLAVAL--- ---------- ---------- LmjF.10.0465 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLTVAL--- ---------- ---------- LmjF.10.0480 560 PYVEVCQGNV QAAKD----- ---------G GNTAAGRRGP RAAATALLVA ALLAVAL--- ---------- ---------- LmjF.10.0470 561 PYVEVCQGNV QAAKDFDGDS DSSSSSSDAA DKAAIERWNE RMAGLATAAT VLLGVVLSLM ALVVVWLLLV SCPRWCCKVG Leishmania infantum Group 1 Group 2 Group 3 Group 1 Group 2 Group 3 B B Leishmania infantum 710 720 730 740 750 ....\|....\| ....\|....\| ....\|....\| ....\|....\| ....\|....\| .... Protein structural modeling, mapping of variable regions and B-cell epitope prediction Based on the crystallized structure of a membrane GP63 from L. major promastigotes, GP63 was identified as a compact protein consisting predominantly of β sheet secondary structure elements divided into three distinct Page 11 of 17 Castro Neto et al. BMC Genomics (2019) 20:118 Table 1 Leishmania braziliensis GP63 3’ UTR and C-terminal end gene groups. Table showing the Gp63 gene groups from chromosome 10 defined according to the 3’ UTR and C-terminuses sequence similarity Group L. braziliensis 3’UTRs L. braziliensis chromosome 10 GP63 paralogs 1 LbrM.10.0510, LbrM.10.0530, LbrM.10.0560, LbrM.10.0580, LbrM.10.0600, LbrM.10.0610, LbrM.10.1540, LbrM.10.1580 LbrM.10.0510, LbrM.10.0530, LbrM.10.0550, LbrM.10.0560, LbrM.10.0600, LbrM.10.0610, LbrM.10.1540, LbrM.10.1580 2 LbrM.10.0470, LbrM.10.0480, LbrM.10.0500, LbrM.10.0520, LbrM.10.0540, LbrM.10.0570, LbrM.10.1570, LbrM.10.1590, LbrM.10.1610, LbrM.10.1620, LbrM.10.1630, LbrM.10.1640, LbrM.10.1650, LbrM.10.0600, LbrM.10.1680 LbrM.10.0470, LbrM.10.0500, LbrM.10.0520, LbrM.10.0540, LbrM.10.0570, LbrM.10.1570, LbrM.10.1590, LbrM.10.1610, LbrM.10.1620, LbrM.10.1630, LbrM.10.1640, LbrM.10.1650, LbrM.10.1660, LbrM.10.1680 3 LbrM.10.0590, LbrM.10.1690 LbrM.10.0590, LbrM.10.1690 4 LbrM.10.0550, LbrM.10.1720 LbrM.10.1720 5 LbrM.10.0490, LbrM.10.1700 LbrM.10.0490, LbrM.10.1700 6 LbrM.10.1550 LbrM.10.1550 As can be observed, most of the variable regions were posi- tioned externally on the structures. within the Lbr10.0590 polypeptide (not shown). Over- all, these results are consistent with variable regions localizing externally and being more capable of indu- cing an immune response. y We next sought to evaluate how the various GP63 sequence would be recognized by the B cells from the mammalian immune system and also to predict their ability to induce the production of specific antibodies. Linear and conformational B-cell epitope predictions were carried out using the various chromosome 10 GP63 sequences from L. braziliensis. The linear epitope predictions returned 56 epi- topes from the sequences of the modeled proteins. Regard- ing their localization within the various structures, from the total of 56 epitopes, 41 were mapped to the proteins’ exter- nal regions, whereas six were localized internally and nine could not be evaluated due to the comparative nature of the tridimensional structure modeling (Table 2). The mod- eling is based on a mature GP63 crystallized structure, which lost part of its N-terminal region, during the protein posttranslational modification, which prevented the assess- ment of epitopes localized to this region. Out of the 41 lin- ear epitopes localized externally, 33 coincide with motifs that display sequence variation, while 8 are found in regions conserved between the different GP63 sequences. Considering only the epitopes that were predicted to localize internally, four coincide with variable sequences while two were associated with conserved regions. Protein structural modeling, mapping of variable regions and B-cell epitope prediction As for the prediction of conformational B-cell epitopes, the ana- lysis returned 40 epitopes, with 32 mapped to the proteins’ external region. Twenty-three of those were in motifs with sequence variation, while nine were in conserved regions. Regarding the eight epitopes localized internally, five coin- cided with variable sequence motifs and three were in con- served motifs. We also identified 14 motifs that were present in both linear and conformational epitope predic- tions (not shown). As above, most of the epitopes coin- cided with variable motifs localized externally, as shown in Table 2. Noteworthy also is the fact that a peptide from L. infantum GP63 that has been previously shown to react strongly with sera from dogs infected with visceral leishmaniasis [40] is nearly identical to one of the L. braziliensis B-cell epitopes predicted by our analysis Discussion The in-silico analysis carried out here highlights the strong selective pressure for the expansion in copy number of the chromosome 10 GP63 genes within Leishmania species, and in particular in the Viannia and Sauroleishmania sub- genera. The increased number of the chromosome 10 GP63 genes in different Leishmania species evolved inde- pendently generating a wide range of paralogs, which display sequence variations and may be generated by recombination. This expansion seems to be an ongoing process that might be related to pathogenesis or defense mechanisms directed to the vertebrate host and the parallel expansion in both Viannia and Sauroleishmania species is something that must be taken into account. Such expansion of multiple genes arranged in tandem, originating from duplication and recombination events, demonstrates the adaptability of Leishmania species to the environment, associated with the evolutionary pressure suffered by the GP63 genes [28]. As a result, the presence of these multi-copy arrays may lead to speciation [41] or indicate the possible need for stage-specific genes [28]. As previ- ously highlighted [42], an expansion in GP63 sequences also occurred independently in other trypanosomatids, such as T. cruzi and T. brucei, and this led to novel GP63 domains which might be associated with species-specific or group-specific functions. It is likely then that this expan- sion in Leishmania GP63 genes might be related to novel aspects of the pathogenesis of these parasites to the verte- brate hosts, but this still needs to be better defined. When multiple strains from a single species is considered the overall GP63 diversity might be even greater, as recently evaluated [43], and this might be associated with possibly different virulence phenotypes and clinical outcomes for the disease. The recent release of a new L. infantum gen- ome based on data using two distinct methodologies of Page 12 of 17 Castro Neto et al. BMC Genomics (2019) 20:118 next generation sequencing, and showing a higher copy if we extrapolate the expression for Group 1 based on the A B Fig. 6 Leishmania braziliensis GP63 recombination events. Representative figure indicating recombination events identified using the RDP4 software. The p-values calculated for the recombination events are indicated as well as the parental and recombinant regions of the chosen gene. Discussion The graph shows the gene that underwent a recombination event (in blue) and other two genes that most likely were involved in this recombination, named major parent (in yellow) and minor parent (in purple), identified by pairwise alignment. Despite their definition as major or minor parent, any one of these genes may be involved in the recombination. a Predicted recombination event between two PCR genes, G1620B1 and G0510G4, with the recombinant gene (G0540C1) showing a 100% identity in most of the N-terminus region with G0510G4. b Predicted recombination event between two annotated genes, LbrM10.1630 and LbrM10.0600, showing the putative recombinant gene (LbrM10.0590) with an almost 100% pairwise identity in most of its N-terminal and central regions with the gene LbrM10.0600. A representative GP63 gene structure is shown in the bottom of the figure, discriminating N-terminal, Central and C-terminal regions A A B B Fig. 6 Leishmania braziliensis GP63 recombination events. Representative figure indicating recombination events identified using the RDP4 software. The p-values calculated for the recombination events are indicated as well as the parental and recombinant regions of the chosen gene. The graph shows the gene that underwent a recombination event (in blue) and other two genes that most likely were involved in this recombination, named major parent (in yellow) and minor parent (in purple), identified by pairwise alignment. Despite their definition as major or minor parent, any one of these genes may be involved in the recombination. a Predicted recombination event between two PCR genes, G1620B1 and G0510G4, with the recombinant gene (G0540C1) showing a 100% identity in most of the N-terminus region with G0510G4. b Predicted recombination event between two annotated genes, LbrM10.1630 and LbrM10.0600, showing the putative recombinant gene (LbrM10.0590) with an almost 100% pairwise identity in most of its N-terminal and central regions with the gene LbrM10.0600. A representative GP63 gene structure is shown in the bottom of the figure, discriminating N-terminal, Central and C-terminal regions Fig. 6 Leishmania braziliensis GP63 recombination events. Representative figure indicating recombination events identified using the RDP4 software. The p-values calculated for the recombination events are indicated as well as the parental and recombinant regions of the chosen gene. The graph shows the gene that underwent a recombination event (in blue) and other two genes that most likely were involved in this recombination, named major parent (in yellow) and minor parent (in purple), identified by pairwise alignment. Discussion Despite their definition as major or minor parent, any one of these genes may be involved in the recombination. a Predicted recombination event between two PCR genes, G1620B1 and G0510G4, with the recombinant gene (G0540C1) showing a 100% identity in most of the N-terminus region with G0510G4. b Predicted recombination event between two annotated genes, LbrM10.1630 and LbrM10.0600, showing the putative recombinant gene (LbrM10.0590) with an almost 100% pairwise identity in most of its N-terminal and central regions with the gene LbrM10.0600. A representative GP63 gene structure is shown in the bottom of the figure, discriminating N-terminal, Central and C-terminal regions if we extrapolate the expression for Group 1 based on the data with the L. major and L. infantum genes [15, 25], they are likely to be expressed constitutively with likely func- tions during the mammalian infection. In contrast, for the Group 3 genes, with only two paralogs, their expression might be restricted to the promastigote stage of the para- site life cycle, therefore with minor or no relevant function next generation sequencing, and showing a higher copy number of GP63 genes for this species [30], also high- lights the need for better quality genomes in order to properly define the true diversity of these genes for multiple Leishmania and trypanosomatid species. The expansion observed in the chromosome 10 genes are concentrated in the Group 1 and Group 2 genes and, Castro Neto et al. BMC Genomics (2019) 20:118 Page 13 of 17 Fig. 7 Comparative protein modelling of selected Leishmania braziliensis GP63 paralogs from chromosome 10. Modelled structures of eight representative GP63 paralogs from the L. braziliensis chromosome 10. The segments labelled in blue indicate the variable motifs in each protein, while the segments yellow represent the predicted linear B-cell epitopes and the green markings show variable motifs coinciding with the predicted epitopes 3 paralogs from chromosome 10. Modelled structures of eight representative in blue indicate the variable motifs in each protein, while the segments show variable motifs coinciding with the predicted epitopes Fig. 7 Comparative protein modelling of selected Leishmania braziliensis GP63 paralogs from chromosome 10. Modelled structu GP63 paralogs from the L. braziliensis chromosome 10. Discussion The segments labelled in blue indicate the variable motifs in each protein yellow represent the predicted linear B-cell epitopes and the green markings show variable motifs coinciding with the predicted linked to the mRNA 3’UTRs and sorting out the molecular mechanisms associated will be a major endeavor. An im- portant question that emerges regarding the expression of the genes from Groups 1 and 2 is related to the multiple paralogs. Are multiple genes belonging to the same group expressed simultaneously or some are expressed more effi- ciently than others or alternatively? This will also need to be investigated further. in the mammalian host. For all three groups their expres- sion will have to be confirmed but the large sequence variations observed for both Groups 1 and 2 genes, con- centrated on potentially immunogenic regions localized on the surface of the GP63 molecules, also imply related expression patterns during the mammalian stage of the Leishmania life cycle. As previously shown in L. major and L. mexicana [44], these expression patterns should be Castro Neto et al. BMC Genomics (2019) 20:118 Page 14 of 17 Page 14 of 17 Table 2 Leishmania braziliensis GP63 B-cell epitope prediction. Table showing the distribution of the predicted epitopes along the studied GP63 paralogs Total Number of Epitopes Conserved Region Sequence Variation Region Type of Epitopes Protein Region 41 8 33 Linear External 6 2 4 Linear Internal 32 9 23 Conformational External 8 3 5 Conformational Internal Table 2 Leishmania braziliensis GP63 B-cell epitope prediction. Table showing the distribution of the predicted epitopes along the studied GP63 paralogs activity [42]. In a recent study targeting L. braziliensis GP63 sequences, structural differences have also been found to be associated with sequence variability, implying functional differences, such as during substrate binding, which may affect the interaction with the host [47]. Alter- natively, the variability in protein structure could mainly affect recognition by the host immune system and pro- mote infection mainly because the host would need to produce different antibodies to neutralize a single group of proteins. Our results, showing variability concentrated on antigenic regions on the protein’s surface, is in agree- ment with previously reported data based on L. major and L. infantum sequence analysis where regions of GP63 sequence variation were mapped to the surface of the pro- tein and were associated with immunodominant epitopes [37]. Conclusions Our results suggest a greater role for the sequence variation found among the chromosome 10 GP63 genes for the pathogenesis of L. braziliensis and closely related species within the mammalian host. The variation in sequence and the expansion in number of these GP63 genes have occurred independently in different Leishmania lineages, is associated with intragenic recombination events and has a likely role against the host immune response. They also indicate different functions associated to genes mapped to different chromosomes and, for the chromosome 10 genes at least, variable subcellular localizations likely associated with multiple functions and target substrates for this versatile protease. In early studies performed with L. guyanensis, it was suggested that new GP63 genes may be generated by events of mosaicism through recombination between 5′ and 3’ UTRs and protein coding regions [24], and mosai- cism in GP63 sequences was subsequently also found in L. braziliensis genes [26]. The data obtained by us corrobor- ate with other studies investigating GP63 recombination that found it to target mainly the N-terminal and C-ter- minal regions of the gene [37, 38]. The impact of the GP63 sequence variability in its structure has been in- vestigated in a wider scale, comparing Trypanosoma and Leishmania sequences, and found to be associated with variability in its zinc binding site and presumably Discussion However, more studies are needed to better under- stand the recognition of different GP63 paralogs by the host immune system. Another relevant question remaining deals with the functional roles for the distinct groups and how these might be associated with sequence differences between the paralogs. A possible link with the proteins’ subcellular localization is presumed based on the differences in the C-terminus of the subsets identified and the presence or absence of a typical GPI anchor. These differences regard- ing the presence or absence of GPI anchor sites have been suggested before based on comparisons between the L. major and L. infantum GP63 sequences [8]. Here, the C-terminal Group 2 of L. braziliensis GP63 sequences lacks the GPI anchor signal entirely, which is consistent with proteins that are directly secreted into the extracellu- lar medium, as previously reported for L. mexicana GP63 [45]. This release into the extracellular environment might contribute at the early stages of infection, due to the abil- ity of GP63 to digest the extracellular matrix proteins, fa- cilitating parasite mobility and invasion [46]. Alternatively, these proteins might be selectively transferred to exosomes and later to the macrophages in order to influence its me- tabolism and promote Leishmania growth [11]. For the L. braziliensis Group 1 proteins, they all share a C-terminus having a likely transmembrane domain with no clear GPI anchor site. Lack of a typical GPI anchor site however, with a more likely transmembrane domain identified, was also seen in the L. major Group 1 gene, which was nevertheless seen to have a GPI anchor [25]. The distinct C-terminal ends nevertheless clearly suggest critical differences in sub- cellular localization for the distinct GP63 groups, but these need further experimental confirmation. Overall the data presented here highlights novel and relevant aspects related to the expansion of GP63 genes in L. braziliensis and related Viannia species and raises spe- cific issues regarding the role of GP63 in the parasite pathogenesis during the infection in mammals. It is pos- sible that species belonging to the subgenus Viannia may have added a new level of complexity to GP63 function and this may somehow be related to the capacity of some species to cause the more aggressive mucocutaneous form of the disease. The new questions raised here then, when solved, shall provide novel and relevant knowledge regard- ing the very unique mechanisms of pathogenesis associ- ated with these parasites. B-cell epitope prediction Linear B-cell epitope predictions were performed for the protein sequences used in the 3D modeling step. The pre- dictions were carried out using the following programs: AAP12 [60], BCPred12 [61] and BepiPred [62]. Only epi- topes predicted by at least two programs, with lengths equal to or greater than 10 amino acids and with scores greater than 0.8 were considered as positive predictions on AAP12 and BCpred12. Epitopes with scores over 0.5 obtained by BepiPred were also included in the analysis. In addition to the linear prediction, a conformational prediction of epitopes was also performed to evaluate if the protein structures were also able to generate inter- action with the immune system. The conformational epi- topes were predicted by the CBTOPE web server [63], where only epitopes with more than 10 amino acids and a score above 4 were considered for this study. After the prediction, an assessment was performed to map the localization of all the epitopes on the modeled proteins. Parasites and culture conditions In this study, we used Leishmania (Viannia) braziliensis (MHOM/BR/75/M2904) in its promastigote form. This is a reference strain from the Evandro Chagas Institute, Belém, Brazil. The cells were cultured at 26 °C in Schneider (Sigma) pH 7.2 supplemented with 20% fetal Page 15 of 17 Page 15 of 17 Castro Neto et al. BMC Genomics (2019) 20:118 cruzi [taxid: 353153], Trypanossoma brucei [taxid: 185431], Trypanossoma theileri [taxid: 67003] and Bodo saltans [taxid: 75058]. Another tree was made with selected GP63 sequences used in the previous analyses plus the ones obtained by PCR from L. braziliensis as well as GP63 sequences from Leishmania guyanensis [taxid: 5670]. For all trees, the selected sequences were aligned by MAFFT (default settings) and the alignments automatically edited by Trimal [52] to keep just phylogenetically informative sites. ProtTest [53] was then used to predict the best evolu- tionary model which was subsequently used as a setting to build phylogenetic trees with PhyML, applying the Max- imum Likelihood (ML) method [54], and MrBayes, apply- ing the Bayesian method [55, 56]. The branch support for the ML tree was given by non-parametric bootstrap ana- lysis using 1000 replicates. The Bayesian inferred trees were determined by 5,000,000 chains to check for convergence and a 100% burn-in was discarded. The aligned nucleotide sequences from L. braziliensis, obtained from the Tri- TrypDB database and through PCR, were analyzed for recombination using the RDP4 program [57]. bovine serum (FBS), antibiotics (Streptomycin / Penicillin 0.1%) and 0.1% Hemin. bovine serum (FBS), antibiotics (Streptomycin / Penicillin 0.1%) and 0.1% Hemin. PCR, cloning and sequencing Approximately, 108 L. braziliensis promastigotes were used for total genomic DNA extraction using DNAzol (Invitrogen) and standard procedures. PCR reactions for the amplification of the GP63 sequences were performed using Phusion® High-Fidelity DNA Polymerase (New Eng- land Biolabs), following the manufacturer’s protocol and with the oligonucleotides used as primers listed in the Additional file 5: Table S5. After amplification, cloning and sequencing of the PCR products, a nomenclature was cre- ated for the newly generated sequences in order to identify from which set of primers they were derived, whether from those encoding the KDELMAP or GPI regions, and defin- ing which annotated GP63 gene it most closely resembles. The newly generated sequences derived from the PCR am- plifications were deposited on the GenBank and all acces- sion numbers are listed in Additional file 3: Table S3. Modelling of GP63 homologs and searches for non- conserved regions First, the predicted proteomes of the following organisms were downloaded from TritrypDB in August 25, 2014: L. braziliensis strain 2903 [taxid: 1295825], L. braziliensis strain 2904 [taxid: 420245], L. infantum [taxid: 435258], L. major [taxid: 347515], L. donovani [taxid: 981087], L. mexicana [taxid: 929439] and L. tarentolae [taxid: 5689]. GP63 genes were then identified within the downloaded proteomes, considering only genes annotated as GP63, encoding proteins longer than 30 amino acids and with no more than one stop codon per sequence. All of the protein sequences derived from genes that met these inclusion cri- teria were submitted to the analysis of the OrthoMCL program [48], and grouped according to homology using the Markov Cluster algorithm [49]. Protein sequences from each group were aligned using the MAFFT software (default settings) [50] and the multiple alignments used as input for the hmmbuild, version 3.0, a tool from the HMMER package [51] to build Hidden Markov Models (HMMs). The models were then used with the hmmsearch tool to search for new paralogs within the L. braziliensis strain 2904 proteome. A cutoff of 0.001 for hit significance (e-value < = 0.001) was applied. Eight of the most variable paralogs from different L. braziliensis C-terminal groups were chosen for the three-dimensional modelling. The modelling was per- formed for the amino acid sequences previously obtained from TriTrypDB and applying the SWISS MODEL plat- form [58]. When the models were completed, their qual- ities were assessed through Procheck [59]. Specific regions of the protein models were then evaluated using the initial alignment information, highlighting the non-conserved re- gions which were characterized by amino acid exchanges. GP63 phylogenetic analysis and detection of recombination events A phylogenetic tree was built with GP63 protein sequences from genes encoded within chromosomes 10, 28 and 31 from diverse Leishmania species and more distantly related organisms. These include the Phytomonas sp. isolate Hart11 [taxid: 134014], Crithidia fasciculata [taxid: 5656], Leptomonas pyrrhocoris [taxid: 157538], Trypanossoma Page 16 of 17 Page 16 of 17 Castro Neto et al. BMC Genomics (2019) 20:118 Castro Neto et al. BMC Genomics Competing interests Competing interests The authors declare that they have no competing interests. 20. Santos ALS, Branquinha MH, D’Avila-Levy CM. The ubiquitous gp63-like metalloprotease from lower trypanosomatids: in the search for a function. An Acad Bras Cienc. 2006;78:687–714. Funding The Leishmania work in Dr. de Melo Neto’s lab was more recently funded with grants provided by the Brazilian funding agencies FACEPE (APQ-0239-2.02/12 and APQ-1662-2.02/15), CNPq (480899/2013–4, 313934/2013–4 and 401282/ 2014–7) and CAPES (23038.007656/2011–92). Studentships for the graduate students (ALCN, and ANALMB) were provided by CAPES and FACEPE. 13. Contreras I, Gómez MA, Nguyen O, Shio MT, McMaster RW, Olivier M. Leishmania-induced inactivation of the macrophage transcription factor AP- 1 is mediated by the parasite metalloprotease GP63. PLoS Pathog. 2010;6: e1001148. 14. Jaramillo M, Gomez MA, Larsson O, Shio MT, Topisirovic I, Contreras I, et al. Leishmania repression of host translation through mTOR cleavage is required for parasite survival and infection. Cell Host Microbe. 2011;9: 331–41. Received: 6 June 2018 Accepted: 21 January 2019 Received: 6 June 2018 Accepted: 21 January 2019 Additional file 2: Table S2. GP63 genes identified by HMM from the L. braziliensis M2904 proteome. Table showing the number of GP63 genes identified by each HMM after the search for new paralogs within the L. braziliensis M2904 genome sequences. (DOCX 11 kb) References 1. Pace D. Leishmaniasis. J Inf Secur. 2014;69(Suppl 1):10–8. Additional file 3: Table S3. Set of oligonucleotides, GP63 sequences obtained by PCR and their respective GenBank accession number. (DOCX 15 kb) 2. Akhoundi M, Kuhls K, Cannet A, Votýpka J, Marty P, Delaunay P, et al. A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies. PLoS Negl Trop Dis. 2016;10:1–40. 3. Kaye P, Scott P. 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Ellis M, Sharma DK, Hilley JD, Coombs GH, Mottram JC. Processing and trafficking of Leishmania mexicana GP63. Analysis using GPI8 mutants
https://openalex.org/W1535621869	https://www.intechopen.com/citation-pdf-url/37364	English	null	Protein Phosphatases Drive Mitotic Exit	InTech eBooks	2,012	cc-by	6,831	1. Introduction Mitosis is the final stage of the cell cycle that results in the formation of two independent daughter cells with an equal and identical complement of chromosomes (Figure 1). This requires a complex series of events such as nuclear envelope breakdown, spindle formation, equal chromosome segregation, packaging of chromosomes into daughter nuclei and constriction of the plasma membrane at the cell equator, which is subsequently abscised to generate two independent daughter cells. For mitosis to be successful, these events need to occur in a strict order and be spatiotemporally controlled, which is primarily mediated by protein phosphorylation (Dephoure et al., 2008). In human cells more than one thousand proteins show increased phosphorylation during mitosis (Dephoure et al., 2008). These phosphorylation events are mediated by mitotic protein kinases such as cyclin-dependent kinases (Cdks), Auroras, Polo-like kinases (Plks), Mps1, Neks and NimA (Ma and Poon, 2011). In mammalian cells, the majority of phosphorylation events and thus mitotic progression is driven by the activity of Cdk1, which is the main subtype of Cdks (Dephoure et al., 2008). Its activity during mitosis is due to binding cyclin B1 and phosphorylation of a residue in the T-loop. Mitotic exit involves two stages: (1) membrane ingression, which begins during anaphase following chromosome segregation and involves the breakdown of mitotic structures including the mitotic spindle. It also involves the physical constriction of the cell membrane between segregating chromosomes at the cell equator to generate a thin intracellular bridge between nascent daughter cells. This is followed by (2) membrane abscission at a specific location along the intracellular bridge to generate two independent daughter cells (Figure 1). During mitotic exit, cells also decondense their chromosomes and re-assemble interphase structures such as the nuclear envelope and endoplasmic reticulum. Again, these events need to occur in a strict ordered sequence and requires the reversal of Cdk1-mediated phosphorylation events. Cdk1 is inactivated upon anaphase and is largely dependent on proteasomal-mediated degradation of cyclin B1 by the anaphase promoting complex/cyclosome (APCCdc20) (Peters, 2006). However, downregulation of Cdk is not sufficient for mitotic exit in human cells. Thus, mitotic phosphatases are also thought to contribute to both the inactivation of Cdk1 at the onset of anaphase and to the mitotic exit process in higher eukaryotes. Consistent with this idea, in the early stages of mitotic exit, Cdk1 is transiently inhibited by phosphorylation prior to the degradation of cyclin B1 (D'Angiolella et al., 2007). Protein Phosphatases Drive Mitotic Exit Megan Chircop Children’s Medical Research Institute, The University of Sydney, Australia 10 Protein Phosphatases Drive Mitotic Exit Megan Chircop Children’s Medical Research Institute, The University of Sydney, Australia 10 www.intechopen.com 1. Introduction It is possible that the transient phosphorylation of Cdk1 is also due to inhibition of the Cdc25C phosphatase by the PP2A phosphatase, which is the same www.intechopen.com 216 Protein Kinases phosphatase that keeps Cdc25C inactive during interphase (Forester et al., 2007). However, recent evidence indicates that the Cdc14B phosphatase dephosphorylates Cdc25C resulting in its inhibition and consequent phosphorylation of Cdk1 (Tumurbaatar et al., 2011). Moreover, Cdk substrates are dephosphorylated in an ordered sequence from anaphase to cytokinesis (Bouchoux and Uhlmann, 2011). Thus, mitotic exit further depends on the activation of protein phosphatase(s). Indeed, mitotic exit is blocked in cells lacking Cdk1 activity when protein phosphatase activity is suppressed (Skoufias et al., 2007). Fig. 1. Schematic illustration of the stages of mitosis. The relative abundance of phosphorylation events is shown above each mitotic stage and the major cellular events occurring at each stage are shown below. These events are known to be regulated by phosphorylation/dephosphorylation. Mitotic exit begins during anaphase and involves two sequential stages: (1) membrane ingression that generates a cleavage furrow followed by (2) membrane abscission of the intracellular bridge that connects the two nascent daughter cells. Chromosomes/nuclei shown in blue. Midbody shown in red. Fig. 1. Schematic illustration of the stages of mitosis. The relative abundance of Fig. 1. Schematic illustration of the stages of mitosis. The relative abundance of phosphorylation events is shown above each mitotic stage and the major cellular events occurring at each stage are shown below. These events are known to be regulated by phosphorylation/dephosphorylation. Mitotic exit begins during anaphase and involves two sequential stages: (1) membrane ingression that generates a cleavage furrow followed by (2) membrane abscission of the intracellular bridge that connects the two nascent daughter cells. Chromosomes/nuclei shown in blue. Midbody shown in red. Fig. 1. Schematic illustration of the stages of mitosis. The relative abundance of phosphorylation events is shown above each mitotic stage and the major cellular events occurring at each stage are shown below. These events are known to be regulated by phosphorylation/dephosphorylation. Mitotic exit begins during anaphase and involves two sequential stages: (1) membrane ingression that generates a cleavage furrow followed by (2) membrane abscission of the intracellular bridge that connects the two nascent daughter cells. Chromosomes/nuclei shown in blue. Midbody shown in red. 1. Introduction Although there is a large body of knowledge about the phosphoproteins and protein kinases involved in mitosis and how they are regulated, the specific dephosphorylation events and the involvement of specific phosphatases in mitosis has only recently become appreciated. Studies are now revealing how the timely execution of mitotic events depends on the delicate interplay between protein kinases and phosphatases. To date, most reviews have focused on the role of protein dephosphorylation at the mitotic spindle and specifically how it regulates chromosome alignment (metaphase) and segregation (anaphase) (Bollen et al., 2009, De et al., 2009). This chapter will focus on providing an updated overview of the protein dephosphorylation events that occur during the later stages of mitosis (anaphase – cytokinesis) that contribute to driving mitotic exit and the generation of two independent daughter cells. More specifically, this chapter will provide insights into the protein phosphatases responsible for these dephosphorylation events and how they are regulated in mammalian cells. 2. Mitotic phosphatases in mammalian cells In Saccharomyces cerevisiae (Shou et al., 1999, Visintin et al., 1999) and Schizosaccharomyces pombe (Cueille et al., 2001, Trautmann et al., 2001), mitotic exit and co-ordination of the final stage of mitosis, cytokinesis, are driven by the dual serine-threonine and tyrosine-protein www.intechopen.com 217 Protein Phosphatases Drive Mitotic Exit phosphatase, Cdc14. Thus the action of Cdc14 is, in part, to counteract Cdk activity by dephosphorylating Cdk substrates (Visintin et al., 1998). Cdc14 is tightly regulated both spatially and temporally (Stegmeier and Amon, 2004, Queralt and Uhlmann, 2008) as well as being a part of several feedback loops that contribute to a rapid metaphase-anaphase transition (Holt et al., 2008). We have gained a detailed molecular picture of the way that the Cdc14 phosphatase orchestrates mitotic exit in yeast (reviewed in (Stegmeier and Amon, 2004, Queralt and Uhlmann, 2008)). However, much less is known about the protein dephosphorylation events and the responsible phosphatases that reverse Cdk phosphorylation and thus drive mitotic exit in eukaryotes. Homologues of Cdc14 exist in most if not all eukaryotes, but they do not seem to have the same central function in late mitosis as in budding yeast (Trautmann and McCollum, 2002). In Caenorhabditis elegans, depletion of CeCDC-14 by RNAi causes defects in cytokinesis; however, this is most likely due to failure to form an intact central spindle (Gruneberg et al., 2002). The human genome encodes two Cdcl4 homologues, Cdc14A and Cdc14B and both can rescue Cdc14 yeast phenotypes (Queralt and Uhlmann, 2008), suggesting functional conservation. However, neither Cdc14A nor Cdc14B are required for mitotic exit in higher eukaryotes (Berdougo et al., 2008) although they do seem to be required to generally dephosphorylate Cdk targets (Mocciaro and Schiebel, 2010). This indicates that they have overlapping functions or that additional mitotic exit phosphatases are required. Instead, recent reports suggest that Cdc14s might act by reversing the activating phosphorylations on Cdc25 phosphatases, thereby indirectly contributing to the regulation of Cdk activity in human cells (Krasinska et al., 2007, Vazquez-Novelle et al., 2010, Tumurbaatar et al., 2011). A survey of phosphatase contribution to cell cycle progression in Drosophila failed to identify a specific candidate for a mitotic exit phosphatase (Chen et al., 2007), suggesting that more than one phosphatase may act redundantly, or that its involvement in mitotic exit is not the only function of the phosphatase. 2. Mitotic phosphatases in mammalian cells Recent efforts into identifying phosphatases other than Cdc14 that drive mitotic exit have revealed the serine-threonine calcium- and calmodulin-activated phosphatase, calcineurin (CaN or PP2B) (Chircop et al., 2010a), the protein tyrosine phosphatase containing domain 1 (Ptpcd-1) (Zineldeen et al., 2009), PP1 (Wu et al., 2009), PP2A (Mochida et al., 2009, Schmitz et al., 2010) and oculocerebrorenal syndrome of Lowe 1 (OCRL1) (Ben El et al., 2011) as being required for mitotic exit in mammalian cells (Table 1). 2.1 Cdc14A and Cdc14B Although the roles of human Cdc14A and Cdc14B are poorly understood, Cdc14A has been linked to centrosome separation and cytokinesis (Kaiser et al., 2002, Yuan et al., 2007), while Cdc14B participates in centrosome duplication and microtubule stabilization (Cho et al., 2005). 2.1.1 Cdc14A The role of Cdc14A in cytokinesis has been linked to the membrane abscission stage. Ectopically expressed Xenopus Cdc14A localizes to the midbody of cytokinetic cells. Xenopus oocytes overexpressing wild-type or phosphatase-dead Cdc14A arrests cells in late stage cytokinesis, whereby the nascent daughter cells are connected by a thin intracellular bridge. Neither central spindle formation, nor the re-localization of passenger proteins and centralspindlin complexes to the midbody are affected. Instead targeting of the essential www.intechopen.com 218 Protein Kinases midbody abscission components, exocyst and SNARE complexes to the midbody, are disrupted in these cells (Krasinska et al., 2007), indicating that Cdc14 midbody localization and more specifically its phosphatase activity is required for abscission. Phosphatase Substrates Function(s) References Cdc14A Unidentified Cdk substrates Centrosome separation, chromosome segregation and cytokinesis (Kaiser et al., 2002, Mailand et al., 2002, Yuan et al., 2007) Cdc25 Inhibition of Cdk1 (Krasinska et al., 2007) Cdc14B N.D. Stabilisation and bundling of MTs (Cho et al., 2005) SIRT2 Downregulation of SIRT2 deacetylase activity by promoting its degradation (Dryden et al., 2003) Ptpcd-1 Unidentified Cdk substrates Cytokinesis (Zineldeen et al., 2009) PP1 I2 Chromosome segregation (Wu et al., 2009) Moesin Cell shape changes for anaphase elongation (Kunda et al., 2011) AIB1 Relocate AIB1 to chromatin for transcription (Ferrero et al., 2011) PNUTS Chromosome decondensation (Landsverk et al., 2005) B-type lamins Targeting ER to chromatin and nuclear envelop reformation (Steen et al., 2000, Ito et al., 2007) Histone H3 Chromsomal reorganisation and nuclear envelope reformation (Vagnarelli et al., 2011) PP2A Unidentified Cdk substrates Mitotic exit (Schmitz et al., 2010, Burgess et al., 2010) CaN (PP2B) Dynamin II Membrane abscission (Chircop et al., 2010a, Chircop et al., 2010b) OCRL PI(4,5)P2 Cleavage furrow formation and membrane ingression (Ben El et al., 2011) Table 1. The substrates and function of mitotic phosphatases required for mitotic exit in mammalian cells. N.D. not determined. Table 1. The substrates and function of mitotic phosphatases required for mitotic exit in mammalian cells. N.D. not determined. Table 1. The substrates and function of mitotic phosphatases required for mitotic exit in mammalian cells. N.D. not determined. www.intechopen.com 219 Protein Phosphatases Drive Mitotic Exit Biochemical studies in human HeLa cells suggests that Plk1 regulates the phosphatase activity of Cdc14A during mitosis (Yuan et al., 2007). Plk1 interacts with and phosphorylates Cdc14A resulting in release of Cdc14 auto-inhibited phosphatase activity in vitro. This is likely to occur during anaphase. 2.2 Ptpcd-1 Of all the phosphatases implicated in mammalian cell mitotic exit to date, the dual- specificity phosphatase, Ptpcd-1, is structurally the most related to Cdc14 (Zineldeen et al., 2009). It is suggested to be a functional isozyme of mammalian Cdc14A (Zineldeen et al., 2009). Like Cdc14A, Ptpcd-1 associates with and co-localises with Plk1 at the midbody of cells in cytokinesis. Both overexpression of Ptpcd-1 and Plk1 cause cytokinesis failure and multinucleate cell formation. No Ptpcd-1 substrates have yet been identified, however like Cdc14B its function is most likely regulated by Plk1. Ptpcd-1 possesses four Plk1 consensus phosphorylation sites and its overexpression could not rescue cytokinesis failure induced by Plk1 depletion (Zineldeen et al., 2009), suggesting that it lies downstream of Plk1. In support of this idea, the yeast homolog of Plk1, Cdc5, regulates Cdc14 phosphorylation and its subcellular localization for mitotic exit (Visintin et al., 2003, Visintin et al., 2008). Based on their midbody co-localization, it is possible that this Plk1/Ptpcd-1 signalling pathway contributes to membrane abscission. 2.1.2 Cdc14B Although Cdc14B is not required for mitotic exit in mammalian cells, it does appear to play a role in mitosis during the latter stages. The SIRT2 protein is a NAD-dependent deacetylase (NDAC) that is a member of the SIR2 gene family with roles in chromatin structure, transcriptional silencing, DNA repair, and control of cellular life span. SIRT2 abundance and phosphorylation status increase upon mitotic entry. During late stages of mitosis, Cdc14B, but not Cdc14A, mediates SIRT2 dephosphorylation, which in turn targets it for degradation by the 26S proteasome (Dryden et al., 2003). Cells stably overexpressing wildtype SIRT2 but not missense mutants lacking NDAC activity have a prolonged mitotic phase (Dryden et al., 2003). Thus, Cdc14B may contribute to chromatin changes during mitotic exit such as chromosome decondensation by targeting SIRT2 for destruction. 2.1.1 Cdc14A Indeed, overexpression of a phospho-mimetic mutant of Cdc14A in HeLa cells results in aberrant chromosome alignment with delay in prometaphase (Yuan et al., 2007). This suggests that Cdc14A activity is associated with metaphase-anaphase progression and chromosome segregation. 2.3.1 PP1 PP1 is activated at the metaphase-anaphase transition by a mechanism involving both inactivation of Cdk1 and proteasome-dependent degradation of an unknown protein (Mochida and Hunt, 2007, Skoufias et al., 2007). During early stages of mitosis, PP1 activity is suppressed and this supression is maintained through dual inhibition by Cdk1 phosphorylation and the binding of inhibitor-1 (I1) (Wu et al., 2009). Protein kinase A phosphorylates I1, mediating its binding to PP1. Partial PP1 activation is achieved during anaphase following a drop in Cdk1 levels due to cyclin B degradation. This shifts the Cdk1/PP1 ratio in favour of PP1 allowing auto-dephosphorylation of PP1 at its Cdk1- mediated phosphorylation site. PP1 subsequently mediates the dephosphorylation of I2 at the I2 activating site, resulting in dissociation of the PP1-I2 inhibitor complex. This results in full activation of PP1 and initiation of mitotic exit. During anaphase, when the outer kinetochore is dis-assembled, I2 levels drop and this may also contribute to the up- regulation of PP1 (Li et al., 2007, Wang et al., 2008). PP1 itself participates in outer kinetochore dis-assembly and chromosome segregation and this may be due to its ability to dephosphorylate Aurora B substrates (Emanuele et al., 2008). Thus, several feedback loops exist and involve protein kinases to initiate and maintain PP1 activity for mitotic exit. PP1 plays roles in several mitotic events that need to occur in a sequential order and include cell elongation, chromosome segregation, chromosome decondensation and nuclear envelope re-formation. At the onset of mitosis, the cell rounds up and forms a stiff, rounded metaphase cortex. Moesin, the sole Drosophila Ezrin-Radixin-Moesin (ERM)-family protein which functions to regulate actin dynamics and cytoskeleton organization (Fehon et al., 2010), plays a critical role in this process and is dependent on the phospho-form of moesin (Kunda and Baum, 2009, Roch et al., 2010, Roubinet et al., 2011). Consequently, dephosphorylation of moesin at the cell poles is required to dismantle this rigid cortex to allow for anaphase elongation and cytokinesis. An RNAi screen for phosphatases involved in the temporal and spatial control of moesin identified PP1 as the responsible phosphatase (Kunda et al., 2011). Overexpression of phosphomimetic-moesin and PP1 depletion blocks proper anaphase elongation of the cell (Kunda et al., 2011). PP1 is involved in the first step of nuclear envelope re-formation by stimulating the targeting of endoplasmic reticulum to chromatin (Ito et al., 2007). 2.3 PP1 and PP2A The phosphatase inhibitor, okadaic acid, can induce mitotic entry in interphase cells (Yamashita et al., 1990) and this mitotic state can be maintained if Cdk1 activity is inhibited (Skoufias et al., 2007). Okadaic acid inhibits the activity of the protein phosphatase (PP)1 and PP2A. Consequently, both phosphatases have been implicated in reversing mitotic phosphorylation events in Xenopus egg extracts (Wu et al., 2009, Mochida et al., 2009). Not surprisingly, both phosphatases are inactivated during mitosis and their reactivation is important for mitotic exit (Wu et al., 2009, Mochida et al., 2009). However, both phosphatases have distinct substrates and are regulated via different mechanisms, which is in line with these enzymes being structurally diverse (Virshup and Shenolikar, 2009). www.intechopen.com 220 Protein Kinases www.intechopen.com 2.3.2 PP2A PP2A forms a complex with B-type regulatory subunits and these subunits contribute to PP2A localisation and substrate specificity. As such, PP2A-B55α and PP2A-B55ǅ were considered strong mitotic exit phosphatase candidates since these B55 regulatory subunits are substrate specifiers for Cdk substrates (Janssens et al., 2008). Indeed, both have been shown to be regulators of mitotic exit in human cells (Mochida et al., 2009, Schmitz et al., 2010). In Xenopus egg extracts, PP2A-B55ǅ is negatively regulated by the kinase greatwall (MASTL in humans) during early mitotic stages to allow the accumulation of mitotically phosphorylated proteins. This is achieved by greatwall-mediated phosphorylation of the small protein ARPP-19, which converts it into a potent PP2A inhibitor (Burgess et al., 2010). PP2A activation induced by MASTL knockdown leads to premature mitotic exit in human cells (Burgess et al., 2010). How PP2A is reactivated once Cdk1 activity decreases to drive mitotic exit remains unclear. Presumably greatwall needs to be inactivated and this is likely to involve an as yet unidentified phosphatase. Alternatively or in addition to this, PP2A may be activated via auto-phosphorylation in a similar manner to PP1 (Wu et al., 2009). Moreover, the identification of PP2A substrates and the role of PP2A for mitotic exit remain key questions for future investigation. 2.3.1 PP1 The assembly of the nuclear lamina depends on the dephosphorylation of B-type lamins, which is catalyzed by a PP1/AKAP149 complex that is associated with the nuclear envelope (Steen et al., 2000). The role of PP1 in chromosome decondensation involves its association with Repo-Man and the PP1 nuclear targeting subunit (PNUTS). During anaphase, a Repo-Man/PP1 complex forms following Repo-Man dephosphorylation. Repo-Man targets the complex to chromosomes to allow PP1 to mediate the dephosphorylation of histone H3 (Vagnarelli et al., 2011). This contributes to the loss of chromosome architecture (Vagnarelli et al., 2006, Trinkle-Mulcahy and Lamond, 2006, Trinkle-Mulcahy et al., 2006). During telophase, PP1 targets PNUTS to the reforming nuclei following the assembly of nuclear membranes concomitant with chromatin decondensation. Here, PNUTS enhances in vitro chromosome decondensation in a PP1-dependent manner (Landsverk et al., 2005). Thus, targeting of PNUTS to the reforming nuclei in telophase may be part of a signalling event promoting chromatin decondensation as cells re-enter interphase. www.intechopen.com 221 Protein Phosphatases Drive Mitotic Exit Finally, PP1 appears to play a role in the initiation of transcription upon entry into the next cell cycle, as it is responsible for reversing the inhibitory Cdk1-mediated phosphorylation events of the transcription factor, AIB1 (Ferrero et al., 2011). AIB1 phosphorylation does not appear to affect its transcriptional activity but instead excludes it from condensed chromatin during mitosis to prevent its access to the promoters of AIB1-dependent genes. Its dephosphorylation by PP1 would presumably allow AIB1 to relocate to decondensed chromatin upon entry into the next cell cycle to re-initiate gene transcription. www.intechopen.com 2.5 Oculocerebrorenal syndrome of Lowe 1 (OCRL), an inositol 5-phosphatase Generation of the cleavage furrow during the membrane ingression stage of cytokinesis involves an actin-myosin II contractile ring. At the cleavage furrow, the phosphoinositide phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) plays an important role in this process by recruiting and regulating essential proteins of the cytokinesis machinery (Janetopoulos and Devreotes, 2006). PI(4,5)P2 mis-regulation blocks cleavage furrow formation leading to generation of a multinucleated cell (Emoto et al., 2005, Field et al., 2005, Wong et al., 2005). In Drosophila, the localization of PI(4,5)P2 is restricted at the cleavage furrow by the Drosophila ortholog of human oculocerebrorenal syndrome of Lowe 1 (OCRL1) (Ben El et al., 2011), an inositol 5-phosphatase mutated in the X-linked disorder oculocerebrorenal Lowe syndrome. Depletion of this phosphatase results in cytokinesis failure due to mis- localization of several essential cleavage furrow components to giant cytoplasmic vacuoles that are rich in PI(4,5)P2 and endocytic markers (Ben El et al., 2011). dOCRL is associated with endosomes and mediates PI(4,5)P2 dephosphorylation on internal membranes to restrict this phosphoinositide at the plasma membrane and thereby regulate cleavage furrow formation and ingression. 2.4 CaN (PP2B) At the FMRs, a calcium influx activates CaN resulting in dephosphorylation of dynII. This is one of the last molecular events known to occur prior to abscission. Thus, it is possible that CaN-mediated dynII dephosphorylation may be the trigger for cellular abscission to complete cytokinesis. In the brain, for clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE), CaN not only targets dynI but also -adaptin, epsin and eps15 (Cousin and Robinson, 2001). Like dynII, epsin and -adaptin are mitotically phosphorylated (Chen et al., 1999, Kariya et al., 2000, Dephoure et al., 2008). Thus, they represent additional potential CaN substrates during cytokinesis. Once dephosphorylated they may contribute to the recruitment of the dephosphorylated form of dynII to the abscission site or directly in CME within the intracellular bridge. 2.4 CaN (PP2B) Endocytosis is thought to shut down during mitosis then resume during the final stage, cytokinesis (Schweitzer et al., 2005). Endocytosis is required for cytokinesis (Feng et al., 2002) and thought to contribute to the pool of recycling endosomes that are eventually delivered to the site of abscission. Here, they are proposed to (i) provide extra total cell surface area, an increase of at least 25% is required to complete division (Boucrot and Kirchhausen, 2007), (ii) deliver critical cytokinetic proteins to the abscission site (Low et al., 2003), and/or (iii) be directly involved in compound fusion, whereby numerous vesicles fuse with the plasma membrane during abscission to separate the daughter cells (Low et al., 2003, Gromley et al., 2005, Goss and Toomre, 2008, Prekeris and Gould, 2008). The calcium- and calmodulin-dependent phosphatase, calcineurin (CaN) is an excellent candidate phosphatase for restarting endocytosis during cytokinesis, since it initiates endocytosis in neurons (Liu et al., 1994). In support of this idea, the fission yeast CaN gene is required for cytokinesis and the CaN inhibitors cyclosporin A (CsA) and FK506 block yeast cytokinesis (Yoshida et al., 1994). CaN is required for calcium-induced mitotic exit in cytostatic factor-arrested Xenopus oocytes (Mochida and Hunt, 2007, Nishiyama et al., 2007). CaN is upregulated in Xenopus oocytes from metaphase of meiosis II. An increase in cytoplasmic calcium upon fertilisation triggers meiosis II exit in these oocytes, which involves calmodulin-activating kinase-dependent activation of the APC. APC-mediated www.intechopen.com 222 Protein Kinases inactivation of Cdk is not sufficient to drive Cdk substrate dephosphorylation and meiotic exit in these oocytes and thus activation of CaN is likely to occur in parrallel to drive this process. A recent report has indicated that CaN is also required for completion of abscission in human cells (Chircop et al., 2010a). During cytokinesis CaN locates to two 1.1 m diameter flanking midbody rings (FMRs) that reside on either side of the 1.6 m diameter Ǆ- tubulin midbody ring (MR) within the centre of the intracellular bridge. The endocytic protein, dynamin II (dynII), is mitotically phosphorylated by Cdk1/cyclin B1 upon mitotic entry and co-localises with CaN at the FMRs during cytokinesis (Chircop et al., 2010a, Chircop et al., 2010b). CaN inhibition by CsA, dynII depletion, phospho-mimetic dynII phosphopeptides and small molecule dynamin inhibitors lead to aborted cytokinesis and multinucleation (Joshi et al., 2010, Chircop et al., 2010a, Chircop et al., 2010b, Chircop et al., 2011). 3. Conclusion Here, the role of Cdc14A and Cdc14B, PP1, PP2A-B55, CaN, Ptpcd-1 and OCRL in regulating and driving mitotic exit in mammalian cells was reviewed. It is clear that we have only scraped the surface in our investigations into understanding the role and regulation of www.intechopen.com 223 Protein Phosphatases Drive Mitotic Exit protein phophatases in mitosis. The identification of all protein phosphatases involved in driving mitotic exit in mammalian cells, their relevant substrates and function as well as how their action is spatio- and temporal regulated in vivo remain key questions for future investigation. protein phophatases in mitosis. The identification of all protein phosphatases involved in driving mitotic exit in mammalian cells, their relevant substrates and function as well as how their action is spatio- and temporal regulated in vivo remain key questions for future investigation. Understanding how protein dephosphorylation regulates mitotic exit and how the responsible protein phosphatases are regulated will provide an improved understanding to how two independent daughter cells are generated. Mitotic exit failure results in aneuploidy, which leads to genomic instability and thus contributes to the initiation and progression of tumourigenesis. Thus, an understanding of the molecular pathways that drive mitotic exit may highlight molecular targets for the development of new anti-cancer chemotherapeutic agents. In line with this idea, a recent publication has identified the CaN substrate, dynII, as a molecular target for the treatment of cancer (Chircop et al., 2010a, Chircop et al., 2010b). Inhibitors of dynII possess anti-cancer properties due to their ability to cause cytokinesis failure and subsequent cell growth arrest or apoptotic cell death (Joshi et al., 2010). It will be interesting to pursue the development of other targeted inhibitors to determine if they also possess anti-cancer properties as well as being useful molecular tools to unravel the signalling pathways required for mitotic exit in mammalian cells. 4. 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Ptpcd-1 is a novel cell cycle related phosphatase that regulates centriole duplication and cytokinesis. Biochem Biophys Res Commun 2009;380:460-466. www.intechopen.com Yoshida T, Toda T, Yanagida M. A Calcineurin-Like Gene Ppb1(+) in Fission Yeast - Mutant Defects in Cytokinesis, Cell Polarity, Mating and Spindle Pole Body Positioning. J Cell Sci 1994;107:1725-1735. Protein Kinases Protein Kinases Edited by Dr. Gabriela Da Silva Xavier Edited by Dr. Gabriela Da Silva Xavier ISBN 978-953-51-0640-1 Hard cover, 484 pages Publisher InTech Published online 05, June, 2012 Published in print edition June, 2012 Proteins are the work horses of the cell. As regulators of protein function, protein kinases are involved in the control of cellular functions via intricate signalling pathways, allowing for fine tuning of physiological functions. This book is a collaborative effort, with contribution from experts in their respective fields, reflecting the spirit of collaboration - across disciplines and borders - that exists in modern science. Here, we review the existing literature and, on occasions, provide novel data on the function of protein kinases in various systems. We also discuss the implications of these findings in the context of disease, treatment, and drug development. Biophys Res Commun 2009;380:460-466. www.intechopen.com www.intechopen.com InTech Europe University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com InTech China Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following: Megan Chircop (2012). Protein Phosphatases Drive Mitotic Exit, Protein Kinases, Dr. Gabriela Da Silva Xavier (Ed.), ISBN: 978-953-51-0640-1, InTech, Available from: http://www.intechopen.com/books/protein- kinases/protein-phosphatases-drives-mitotic-exit In order to correctly reference this scholarly work, feel free to copy and paste the following: Megan Chircop (2012). Protein Phosphatases Drive Mitotic Exit, Protein Kinases, Dr. Gabriela Da Silva Xavier (Ed.), ISBN: 978-953-51-0640-1, InTech, Available from: http://www.intechopen.com/books/protein- kinases/protein-phosphatases-drives-mitotic-exit kinases/protein-phosphatases-drives-mitotic-exit InTech China Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 InTech Europe University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com © 2012 The Author(s). Licensee IntechOpen. This is an open access artic stributed under the terms of the Creative Commons Attribution 3.0 cense, which permits unrestricted use, distribution, and reproduction in ny medium, provided the original work is properly cited. © 2012 The Author(s). Licensee IntechOpen. This is an open access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
https://openalex.org/W3010443888	https://journals.umcs.pl/lrp/article/download/6888/7383	Polish	null	Spotkania dziecka ze sztuką – wychowanie przez książkę obrazkową	Lubelski Rocznik Pedagogiczny	2,019	cc-by	4,713	Joanna S. Ludwiczak Uniwersytet Łódzki ORCID – 0000-0002-2072-4685 Joanna S. Ludwiczak Uniwersytet Łódzki ORCID – 0000-0002-2072-4685 SPOTKANIA DZIECKA ZE SZTUKĄ – WYCHOWANIE PRZEZ KSIĄŻKĘ OBRAZKOWĄ Streszczenie: Przez lata liczne koncepcje pedagogiczne dowodziły, że sztuka dla dzieci posiada niezwykły potencjał jako medium w wychowaniu. Można ją uznać za znakomite narzędzie pośredniczące m.in. w kształtowaniu percepcji wzrokowej dziecka, co we współczesnej kulturze przesyconej wizualnością nabiera szczególnej wagi. Przykładem ogólnodostępnej, a jedno- cześnie wartościowej sztuki dla dzieci (jak również dorosłego) jest książka obrazkowa (ang. picturebook). Potencjał tego gatunku kryje się nie tylko w przekazie wizualnym i werbalnym, ale także całej „architekturze” książki. Wszystkie te elementy oraz zależności między nimi mogą stanowić inspirację do działań w obszarze edukacji plastycznej. Jednak kontakt dziecka z książką obrazkową przy współuczestnictwie dorosłego otwiera także przestrzeń do własnych poszukiwań, przemyśleń, interpretacji i stawiania pytań. Tym samym wywołuje refleksję na temat treści ukrytej w obrazach. Celem artykułu jest próba rozpoznania istotnych wycho- wawczo elementów formy i treści książek obrazkowych na przykładzie twórczości Iwony Chmielewskiej. Wskazane cechy wizualne i przytoczone fragmenty tekstu książek odniesiono do sformułowanych przez Herberta Reada zadań wychowania przez sztukę. Ta fundamentalna dla teorii wychowania estetycznego koncepcja w kontekście przeobrażeń kultury XXI wieku wydaje się nabierać nowych znaczeń i nadal inspiruje licznych badaczy. Słowa kluczowe: książka obrazkowa, sztuka dla dzieci, wychowanie przez sztukę, edukacja estetyczna, percepcja sztuki, Iwona Chmielewska WPROWADZENIE Trudno wyobrazić sobie współczesną kulturę bez przekazów wizualnych, które wypełniają naszą codzienność już od najmłodszych lat. Powszechność komuni- katów, których nośnikiem jest obraz, niesie wiele pozytywnych i pożytecznych aspektów, w postaci łatwego dostępu do zasobów kultury, lecz pociąga za sobą 98 Joanna S. Ludwiczak także szereg zagrożeń. Przytłaczający ogrom różnorodnych jakości wizualnych powoduje trudność w selekcji i właściwym odbiorze obrazów, a przede wszyst- kim w ich umiejętnym wartościowaniu i krytycznym osądzie. Często zdarza się, że tempo i styl życia nie sprzyjają refleksji dorosłego nad jakością obrazów (np. w książkach, czasopismach, kolorowankach, na opakowaniach, gadżetach, zabaw- kach itd.), z którymi styka się dziecko, zarówno w środowisku rodzinnym, jak i przedszkolnym lub szkolnym. Zdaniem Grzegorza Leszczyńskiego kultura ma- sowa wnosi do dziecięcego otoczenia wytwory przynależące najczęściej do „sztuki niskiej, płytkiej, niewymagającej refleksji i wysiłku intelektualnego, nierozwijającej wyobraźni, epatującej feerią powierzchownych wrażeń” (Leszczyński 2003, s. 79). Obcowanie dziecka z infantylnymi, słabymi, zakrawającymi o kicz przedsta- wieniami obrazowymi oraz brak dobrych i różnorodnych wzorców wizualnych utrwala postawę odtwórczą i hamuje rozwój wrażliwości, kreatywności i ciekawości świata (zob. Cackowska 2014, s. 275; Mazepa-Domagała, Wilk 2015, s. 89–91). p g Zagadnienie kształtowania kompetencji wizualnych dziecka coraz częściej pojawia się w opracowaniach podejmujących tematykę łączącą sztukę i edukację. Proces stymulowania tych kompetencji określany jako alfabetyzacja wizualna (Dylak 2012, s. 11) obejmuje rozwój umiejętności rozróżniania, trafnej interpretacji i wartościowania treści wizualnych oraz zdolności twórczego korzystania z tych kompetencji do komunikowania się z innymi (Pater-Ejgierd 2010, s. 159). W świe- cie, w którym język wizualny jest obecny niemal we wszystkich sferach życia, inicjowanie odpowiednich działań edukacyjnych w obszarze sztuk plastycznych lub szerzej wizualnych staje się uzasadnione i szczególnie istotne. Beata Mazepa- -Domagała i Teresa Wilk (2015, s. 100–101) podkreślają, że projektowanie tego rodzaju działań powinno uwzględniać następujące fazy: inspirację, wizualizację, kreację i wartościowanie. Te wzajemnie dopełniające się ogniwa zdaniem autorek posiadają wymiar idei nauczania całościowego. Jak zauważa Krystyna Pankowska, wraz z nowymi zjawiskami zmieniającymi kulturę XXI wieku powraca dylemat „jak wychowywać, by ocalić człowieczeństwo człowieka” (Pankowska 2010, s. 10). Pytanie to dotyczy nie tylko poszukiwania właściwej drogi nauczania, ale i ko- nieczności wkraczania w nowe przestrzenie i technologie. W dobie przeobrażeń otaczającej nas rzeczywistości pewne wartości pozo- stają jednak niezmienne i uniwersalne. Tytuł artykułu stanowi odwołanie do sformułowanej przez Herberta Reada koncepcji wychowania przez sztukę. WPROWADZENIE Ten swoisty manifest opublikowany w dobie II wojny światowej bez wątpienia jest jedną z teorii fundamentalnych dla rozwoju pedagogicznej refleksji i praktyki uwzględniających potencjał sztuki w edukacji. Koncepcja Reada nadal inspiruje wielu autorów odczytujących ją na nowo w kontekście współczesnych problemów edukacji estetycznej (zob. Pankowska 2010; Zalewska-Pawlak 2017). 99 SPOTKANIA DZIECKA ZE SZTUKĄ – WYCHOWANIE PRZEZ KSIĄŻKĘ OBRAZKOWĄ Twórca koncepcji wychowania przez sztukę zakładał, że „sztuka w najszerszym znaczeniu tego słowa powinna stać się zasadniczą podstawą wychowania. Tylko sztuka może sprawić, że w świadomości dziecka wyobrażenie i pojęcie, wrażenie i myśl wzajemnie sobie odpowiadają i łączą się. Równocześnie tylko ona może wyrobić w dziecku instynktowne rozumienie praw rządzących światem [...]” (Read 1976, s. 82). Pierwszorzędną rolę w całym tym procesie Read przypisywał wychowaniu estetycznemu, przed którym stawiał następujące zadania: „– zachowanie naturalnej intensywności wszystkich rodzajów percepcji i wrażeń; j k d j d jó ji i ż ń hż ś d „– zachowanie naturalnej intensywności wszystkich rodzajów percepcji i wrażeń; – wzajemna koordynacja rodzajów percepcji i wrażeń oraz tychże ze środo- „– zachowanie naturalnej intensywności wszystkich rodzajów percepcji i wrażeń; – wzajemna koordynacja rodzajów percepcji i wrażeń oraz tychże ze środo- wiskiem; – wzajemna koordynacja rodzajów percepcji i wrażeń oraz tychże ze środo- wiskiem; – ekspresja uczucia w formie komunikatywnej; – komunikatywna ekspresja różnych rodzajów psychicznego doświadczenia, które bez takiego uzewnętrzniania pozostałoby częściowo lub całkowicie nie- uświadomione; – kształcenie zdolności wyrażania myśli w trafnej formie” (Read 1976, s. 15) Z jednej strony sztuka jawi się jako instrument do pobudzania wrażliwości i przeżyć, do rozwijania zdolności postrzegania, z drugiej jako pretekst do wy- rażania własnych emocji i komunikowania się z innymi. Jak pisze Irena Wojnar, może ona „zmieniać mechanizmy oddziaływań wychowawczych, […] przyczy- nić się do ukształtowania człowieka […] nie tyle zrównoważonego, zharmoni- zowanego wewnętrznie, ale takiego, który funkcjonuje w pewnej różnorodno- ści swoich dyspozycji psychicznych i nieustannie wzbogacanych możliwości” (Wojnar 2010, s. 18). Tytułowe sformułowanie „wychowanie przez książkę obrazkową” należy od- czytywać jako pewną propozycję włączenia konkretnych przykładów sztuki do działań edukacyjnych. Sztuka oferuje niewyczerpane bogactwo form, z którego można dowolnie czerpać pod warunkiem odpowiedniej selekcji fragmentów/ eksponatów i przemyślanego ich uprzystępnienia. Jakość doświadczenia estetycz- nego dziecka w dużej mierze zależy od dostosowania treści i formy wybranych przykładów sztuki do możliwości percepcyjnych odbiorcy. Szczególnym obszarem sztuki jest twórczość adresowana do dziecięcego odbiorcy. Uwzględnia ona do- świadczenia, poziom wrażliwości i potrzeby rozwojowe dziecka. Zdaniem Marii Tyszkowej (1984, s. WPROWADZENIE 29) nie może jednak ograniczać się do aktualnych zainteresowań małego odbiorcy i naśladować jego przestrzeń życiową, ale powinna sprzyjać we- wnętrznemu wzbogacaniu się, poszerzaniu horyzontów i pozyskiwaniu nowychm doświadczeń. 100 Joanna S. Ludwiczak KSIĄŻKA OBRAZKOWA – ZAKRES POJĘCIOWY Rodzajem sztuki, z którym dziecko obcuje już od najmłodszych lat, jest literatura dziecięca, a wraz z nią ilustracja książkowa. Według Stefana Szumana „Ilustracja książek dla dzieci – tak samo jak literatura dla dzieci – nie jest sztuką dorosłych, która się zniża do poziomu dzieci, lecz jest odrębnym, społecznie doniosłym rodzajem sztuki” (Szuman, 1951, s. 92). Tematykę ilustracji książkowej dla dzieci w różnych kontekstach naukowych podejmowali w swoich opracowaniach m.in. Janusz Dunin, Irena Słońska, Stefan Szuman, Janina Wiercińska, a współcześnie Anna Boguszewska, Beata Mazepa-Domagała. Znamienną formą literatury, której przekaz tworzony jest przede wszystkim przez ilustrację (obraz), ale wymykającą się poza definicję książki ilustrowanej, jest książka obrazkowa (ang. picturebook). W Polsce gatunek ten nie posiada długiej tradycji, choć jej geneza sięga połowy ubiegłego wieku. Rozkwit tego zjawiska przypada na drugą dekadę XXI wieku, głównie za sprawą zaangażowania niewielkich wydawnictw, dzięki którym wielu polskich autorów zdobyło uznanie na prestiżowych międzynarodowych imprezach poświęconych książce dla dzieci. Definicje książki obrazkowej zaczerpnięte z literatury obcojęzycznej zgodnie podkreślają znaczące oddziaływanie ilustracji i tekstu na odbiorcę, a tym samym dostarczanie mu jednoczesnych doświadczeń wizualnych i werbalnych (Kiefer 1988). Badacze zajmujący się tematyką książki obrazkowej twierdzą, że cechą wyróżnia- jącą ten gatunek są szczególne zależności między warstwą obrazową i słowną, co otwiera przed odbiorcą wiele możliwości poszukiwań i eksploracji. Jak zauważają Maria Nikolajewa i Carole Scott (2001, s. 12), tekst i ilustracje mogą występować w różnych relacjach, tj. 1) symetrycznej wagi słowa i obrazu, 2) wzajemnego uzu- pełniania się, 3) wzmacniania wzajemnych znaczeń, 4) opowiadania odmiennych historii, 5) przedstawiania przeciwieństw. Każda z tych więzi potwierdza niezwykle głęboką współzależność między tekstem i obrazem. Nigdy nie opowiadają one dokładnie tej samej historii, wspólnie budują natomiast narrację, którą niemoż- liwym byłoby ukazać jedynie przy użyciu samego słowa. Jak pisze Carol Driggs Wolfenbarger i Lawrence Sipe (2007, s. 274), to właśnie ten dysonans przyciąga uwagę odbiorcy, ponieważ dziecko, aby rozwiązać konflikt między tym, co widzi i czyta (lub słyszy), musi podjąć wysiłek i odnaleźć własną drogę łączącą obrazy i słowa w jedną historię. Wartościowa książka obrazkowa tworzy przestrzeń dla gry, w której czytelnik odkrywa, eksperymentuje z relacjami pomiędzy obrazami i słowami, tworząc własną interpretację. Książki obrazkowe można nazwać wielopo- ziomowymi, ponieważ ich odbiorcy mogą być zróżnicowani w zależności od wieku lub doświadczeń. Ponadto zapraszają one do różnych form odczytywania, zapew- niając przy każdym kolejnym odnajdywanie nowych znaczeń (Becket 2012, s. 16). KSIĄŻKA OBRAZKOWA – ZAKRES POJĘCIOWY 101 SPOTKANIA DZIECKA ZE SZTUKĄ – WYCHOWANIE PRZEZ KSIĄŻKĘ OBRAZKOWĄ Pojęcie książki obrazkowej w polskiej literaturze zostało sformułowane w latach 70. ubiegłego wieku przez Janinę Wiercińską, jednak fakt ten nie korespondował wówczas z rozwojem tego rodzaju sztuki dla dzieci, w związku z czym książka obrazkowa przez kolejne dziesięciolecia pozostawała nieobecna na polskim rynku wydawniczym. Wiercińska prymarną rolę w książce obrazkowej przypisuje war- stwie wizualnej, która zdaniem autorki, „jeśli nie stanowi celu głównego, wiąże się z tekstem na zasadach równouprawnienia, gdzie pisarz i rysownik (jeśli nie jest to jedna i ta sama osoba) tak ściśle ze sobą współpracują, że istnienie tekstu jest uwarunkowane obrazem i na odwrót”(Wiercińska 1986, s. 76). Współcześnie próbę usystematyzowania problematyki związanej z polską terminologią w ob- szarze książki obrazkowej podejmuje Małgorzata Cackowska. Autorka zwraca uwagę na brak zaplecza teoretycznego precyzującego termin „książka obrazkowa”, wyznaczającego jego ramy definicyjne oraz określającego kryteria wyodrębnienia książki obrazkowej jako osobnego gatunku. Badaczka podkreśla nieocenione możliwości tego medium w alfabetyzacji wizualnej dziecka, akcentując także silny wpływ pośredników w tym procesie. Cackowska postrzega książkę obrazkową jako artefakt kulturowy, nazywając pełnowartościowym gatunkiem artystycznym (Cackowska i in. 2017, s. 11–48). Znamienną wersją książki obrazkowej jest jej autorska odmiana, która po- wstaje jako utwór kompleksowo stworzony przez jednego autora. Wszystkie detale opracowania graficznego korespondują z tematem i treścią książki, zachowując spójność ogólnej koncepcji. Przekaz kształtowany jest niemal przez każdy z ele- mentów publikacji (np. format, oprawę, układ stron, ilustracje, typografię, kolor itd.). Ponadto wszystkie elementy zespolone w swoiste sekwencje i wytwarzające wzajemne relacje mają kluczowe znaczenie dla zrozumienia książki (Sipe 2001, s. 24). Nośnikiem treści staje się więc nie tylko obraz i tekst, ale i inne detale, które mają swój udział np. w budowaniu nastroju lub uzupełnieniu prowadzonej przez ilustrację i słowo narracji. KSIĄŻKA OBRAZKOWA W ŚWIETLE ZADAŃ EDUKACJI ESTETYCZNEJ W wielu krajach świata walory książki obrazkowej są powszechnie doceniane i od kilku dekad mają ugruntowaną pozycję w praktyce pedagogicznej oraz w bada- niach naukowych. Jako wartości tego gatunku wskazuje się szeroko rozumiane aspekty poznawcze i estetyczne, wymieniając chociażby stymulację procesów percepcyjnych, rozwój różnych rodzajów myślenia, kształtowanie sfery emocji, pobudzanie twórczej wyobraźni czy kształtowanie postawy estetycznej. Książka 102 Joanna S. Ludwiczak obrazkowa, w tym autorska, postrzegana jest także jako narzędzie w procesie socjalizacji najmłodszych oraz wprowadzania w świat współczesnej kultury (Ca- ckowska 2017, s. 11–48). obrazkowa, w tym autorska, postrzegana jest także jako narzędzie w procesie socjalizacji najmłodszych oraz wprowadzania w świat współczesnej kultury (Ca- ckowska 2017, s. 11–48). Przedstawiona praca ma charakter badań wizualnych (zob. Kubinowski 2013, s. 183–184) przeprowadzonych na podstawie książki obrazkowej rozpatrywanej jako obiekt sztuki. Celem rozważań jest analiza formy i treści zastanych danych wizualnych oraz próba wskazania ich wychowawczych i estetycznych aspektów. Niniejszego artykułu nie można traktować jako całościowej interpretacji, ponieważ zjawisko książki obrazkowej jest bardzo złożone. Zagadnienie to daje możliwość interdyscyplinarnych analiz w różnych ujęciach i kontekstach. Materiał źródłowy stanowią wybrane książki obrazkowe autorstwa Iwony Chmielewskiej wydane na rynku polskim w latach 2011–2016. Autorka ta jest jedną z najbardziej utytułowa- nych polskich artystek w dziedzinie książek obrazkowych. Urodziła się w 1960 roku w Pabianicach. Mieszka i tworzy w Toruniu, gdzie ukończyła grafikę na Wydziale Artystycznym Uniwersytetu im. Mikołaja Kopernika. Jej utwory wydano w wielu krajach Europy, a także m.in. w Chinach, Japonii, Meksyku, Tajwanie oraz w Korei Południowej, gdzie cieszą się szczególnym uznaniem. Utwory Chmielewskiej zostały uhonorowane wieloma międzynarodowymi wyróżnieniami, w tym dwukrotnie główną nagrodą w kategorii „non fiction” podczas „Bologna Ragazzi Award” (2011: ilustracje do koreańskiego wydania Dom duszy: maum Kim-HeeKyung; 2013: koreańskie wydanie książki autorskiej Oczy). Adresatami książek Chmie- lewskiej są przede wszystkim dzieci, choć wypowiedzi autorki (wywiad z 2017 roku – w posiadaniu J.L.) wskazują, że odrzuca ona kryterium wieku i odwołuje się raczej do poziomu wrażliwości odbiorcy. Kryteriami poniższej analizy stały się kategorie sformułowane przez Reada i sparafrazowane przez Irenę Wojnar (1976, s. xlix). Odniesienia do „utrzymania naturalnej intensywności wszystkich rodzajów percepcji i wrażenia” (Wojnar 1976, s. xlix) w przypadku książek Chmielewskiej obecne są zarówno w ich treści, jak i elementach formy. Motyw znaczenia wrażeń zmysłowych w życiu człowieka po- jawia się np. w książce Oczy, która rozpoczyna się od słów: „Ten, kto widzi nawet nie wie jak cenny skarb dostaje w prezencie” (Chmielewska 2014b, b.p.). KSIĄŻKA OBRAZKOWA W ŚWIETLE ZADAŃ EDUKACJI ESTETYCZNEJ W utwo- rze tym autorka nie tylko przedstawia różne sposoby patrzenia i akcentuje wagę umiejętności widzenia w życiu, ale i ukazuje możliwości doświadczania świata z perspektywy innych zmysłów – słuchu, węchu, dotyku i smaku. Jakość odbieranych doznań wzrokowych w dużej mierze zdeterminowana jest przez cechy formalne tego, na co patrzymy. Pierwszy kontakt z książką jako obiektem estetycznym dostarcza więc przede wszystkim doświadczenia wzrokowe i dotykowe. Patrząc, odbieramy wrażenia określające informację dotyczącą np. formatu książki, proporcji, kolorystyki, kompozycji obrazów i tekstu itd. Cechą SPOTKANIA DZIECKA ZE SZTUKĄ – WYCHOWANIE PRZEZ KSIĄŻKĘ OBRAZKOWĄ 103 wspólną autorskich książek Chmielewskiej jest twarda oprawa i klasyczna budowa, obejmująca m.in. wyklejki, strony tytułowe, niekiedy również przedtytułowe. Format i styl oprawy wyklucza traktowanie książki jako zabawki, a jednocześnie stanowi wyraz szacunku dla dziecka jako pełnowartościowego odbiorcy. Odbiór utworu warunkuje także jego kolorystyka, którą w przypadku twórczości Chmielewskiej wyraża paleta subtelnych, niekiedy zgaszonych barw, od bieli, beży, szarości, przez ugry, umbry, błękity, po akcenty czerwieni, rzadziej zieleni. Nie bez znaczenia jest także dobór papieru. Jego matowa faktura eksponuje szlachetność barw, brak połysku nie rozprasza niepotrzebnie uwagi odbiorcy, a odcień kości słoniowej lub oliwkowo-szary sprawia, że książka emanuje swoistym ciepłem i wywołuje skojarzenia z przeszłością. Forma każdej książki dla dziecka, poza wzrokowymi, może stymulować także wrażenia dotykowe, np. poprzez zastosowanie uszla- chetnień druku. W książkach Chmielewskiej interesujące pod tym względem wydają się tłoczenia na okładkach Kłopotu i Czterech zwykłych misek, aksamitna faktura okładki Pamiętnika Blumki czy też owalne kształty wycięte w co drugiej stronie publikacji Oczy. Prócz doznań wzrokowych i dotykowych lektura książek aktywizuje także percepcję słuchową podczas czytania na głos lub – w przypadku dzieci w wieku przedczytelniczym – pośrednictwa w odbiorze warstwy słownej. Zgodnie z drugim zadaniem postawionym przed wychowaniem estetycznym sztuka powinna „koordynować różne rodzaje percepcji i wrażenia wzajemnie po- między sobą i w stosunku do otoczenia” (Wojnar 1976, s. xlix). W analizowanych publikacjach cel ten pośrednio wiąże się z poprzednim, bowiem kontakt z książką najczęściej dostarcza jednocześnie kilka typów wrażeń. Książka autorska to ze- tknięcie się dziecka z interpretacją świata częściowo narzuconą przez twórcę, lecz pozostawiającą także przestrzeń na własne interpretacje odbiorcy. Kompozycja rozkładówek w wielu utworach Iwony Chmielewskiej opiera się na współistnieniu warstwy słownej i wizualnej (np. Dwoje ludzi, Pamiętnik Blumki), które wspólnie stanowią konkretyzację pojęć i znaczeń. Inny układ kompozycyjny stosowany przez autorkę to umieszczenie tekstu i obrazu naprzemiennie na oddzielnych stronach (np. KSIĄŻKA OBRAZKOWA W ŚWIETLE ZADAŃ EDUKACJI ESTETYCZNEJ W kieszonce, Oczy, Kłopot), co wywołuje u odbiorcy ciekawość i zaskoczenie związane z przewracaniem kolejnych kart. Cechą znamienną dla pewnej części twórczości Chmielewskiej są puste powierzchnie – „powietrze” otaczające krótkie fragmenty tekstu oraz obrazy. Można odnieść wrażenie, że ta niezadrukowana przestrzeń odsłaniająca fakturę papieru to zaproszenie pozo- stawione przez autorkę do podjęcia dialogu ze słowem i obrazem oraz miejsce na własne refleksje i przemyślenia. Istotną rolę w „czytaniu” ilustracji odgrywa także technika wykonania. Chmielewska najczęściej sięga w swej twórczości do kolażu, rysunku kredką i ołówkiem, ale także do akwareli, haftu czy szycia. Au- torka chętnie wykorzystuje materiały odnalezione w starociach (np. ścinki tkanin, 104 Joanna S. Ludwiczak włóczki, suszone rośliny, stare papiery i druki itp.), które, jak sama pisze, „nie były już nikomu potrzebne i nikt nie podejrzewał, że kiedykolwiek się tak rozwiną” (O tych, którzy się rozwijali, b.p.), co więcej „teraz mogą żyć nowym życiem” (Cztery zwykłe miski, b.p.) i można by dodać, że ich nowa odsłona z pewnością wzbudza zaciekawienie dziecka i sentyment dorosłego odbiorcy. Charakterystyczna dla stylu Chmielewskiej sylwetowość kształtów i synteza form pozytywnie oddzia- łują na wyobraźnię czytelnika. Kształty, które wyraźnie wyodrębniają się z tła (np. O tych, którzy się rozwijali) lub cechują się linearyzmem (np. W kieszonce), pozwalają nawet najmłodszym dzieciom na łatwe wyszukiwanie, rozpoznawanie i nazywanie poszczególnych elementów widzianych na obrazie. Brak paginacji z kolei sugeruje możliwość nielinearnego odczytu treści i zachęca czytelnika do tworzenia kolejnych historii, rozpoczynających się w różnych miejscach książki. j y p y ją y ę y j ą Kolejnym celem wychowania estetycznego jest spowodowanie „wyrażania uczuć w formie komunikatywnej” (Wojnar 1976, s. xlix). Książki obrazkowe również w tym aspekcie mogą okazać się niezastąpione. Jak stwierdza sama autorka: „Picturebooki powinny być [...] tak tworzone, żeby odbiorca stał się ich współtwórcą, aby miał szansę przeżyć bardzo intymne emocje, aby mógł skonfrontować się ze swoimi problemami czy obawami” (Dzieci kończą naukę...). Niekiedy poruszają one trudne tematy i przedstawiają rozmaite, często złożone sytuacje z życia ludzi dorosłych i dzieci. W niezwykle poruszającym Pamiętniku Blumki tytułowa dziewczynka opowiada o epizodach z życia dzieci i wychowawców w prowadzonym przed II wojną światową przez Janusza Korczaka warszawskim Domu Sierot. Utwór po- kazuje dziecięce spojrzenie na świat i towarzyszące mu troski, radości, nadzieje, a także wielkie zaufanie do dorosłego – Pana Doktora. KSIĄŻKA OBRAZKOWA W ŚWIETLE ZADAŃ EDUKACJI ESTETYCZNEJ Zrealizowany w zupełnie innej konwencji Kłopot dotyka problemu wewnętrznych dylematów moralnych, przed którymi staje bohaterka – dziewczyna, która w zamyśleniu wypala żelazkiem ślad na pamiątkowym obrusie po babci. Cztery zwykłe miski z kolei skłaniają do zastanowienia nad tym, że „[...] jedni mają za dużo. A inni mają za mało” (Cztery zwykłe miski, b.p.). Realizacje tematów podejmowanych przez Chmielewską ukazują wielostronne możliwości doświadczania świata, poszukiwania piękna i dobra, nie brakuje w nich także wysmakowanego dowcipu. Przedstawione za pośrednictwem obrazów i słów reprezentacje uczuć i relacji międzyludzkich stanowią cenną pomoc w budowaniu kompetencji emocjonalnych dziecka i tworzeniu jego własnego syste- mu moralnego, uczą wrażliwości, empatii oraz szacunku do drugiego człowieka. Wyzwalanie konstruktywnych sił wyobraźni (Wojnar 1976, s. xlix) to cel, którego możliwość realizacji inicjują działania uwzględniające aktywną percepcję oraz ekspresję plastyczną. Warsztat Chmielewskiej charakteryzujący się lapidar- nością środków wyrazowych stanowi bogate źródło inspiracji. Cztery zwykłe miski czy Kłopot niemalże od pierwszych chwil podpowiadają gotowe rozwiązania do SPOTKANIA DZIECKA ZE SZTUKĄ – WYCHOWANIE PRZEZ KSIĄŻKĘ OBRAZKOWĄ 105 działań plastycznych z dziećmi, młodzieżą, a także dorosłymi. Na wyobraźnię odbiorcy oddziaływać może także tematyka książek, która dotyka zagadnień z po- zoru błahych, niekiedy kontrowersyjnych, trudnych, niejednokrotnie ukazanych w niebanalny sposób. Autorka pokazuje, że przy bacznej obserwacji wiele inspiracji możemy odnaleźć na co dzień w najbliższym otoczeniu, np. niepotrzebne przed- mioty, stare druki, faktury na powierzchniach, pozostawione ślady itd. Wybrane utwory zostały wzbogacone o kontynuację w postaci książki do uzupełniania (Kłopot, Moc kłopotów. Wytwórnik) lub dodatkowych materiałów do wydruku i tworzenia własnych wersji ilustracji (W kieszonce). Ostatnie z zadań stawianych sztuce przez Reada dotyczy nauki „właściwego wyrażania własnych myśli” (Wojnar 1976, s. xlix). Twórczość Chmielewskiej to utwory wymagające wysiłku i poświęcenia czasu, zachęcające do podjęcia dialogu z dzieckiem oraz poddania się wspólnym doświadczeniom czytelniczym. Znaczenie obecności dorosłego, który potrafi wysłuchać dziecko i podjąć z nim rozmowę ma tutaj kluczowe znaczenie. W jednym z wywiadów autorka wyjaśnia: „Zwykle tak konstruuję książki, by miały różne «stopnie wtajemniczenia», najprostszy, który interesuje małe dzieci, ale też wyższy: zawierający metafory, symbole, które można odczytać dopiero, gdy ma się już pewną wiedzę, a przede wszystkim doświadczenia. Ostatnie z zadań stawianych sztuce przez Reada dotyczy nauki „właściwego wyrażania własnych myśli” (Wojnar 1976, s. xlix). Twórczość Chmielewskiej to utwory wymagające wysiłku i poświęcenia czasu, zachęcające do podjęcia dialogu z dzieckiem oraz poddania się wspólnym doświadczeniom czytelniczym. Znaczenie obecności dorosłego, który potrafi wysłuchać dziecko i podjąć z nim rozmowę ma tutaj kluczowe znaczenie. KSIĄŻKA OBRAZKOWA W ŚWIETLE ZADAŃ EDUKACJI ESTETYCZNEJ W jednym z wywiadów autorka wyjaśnia: „Zwykle tak konstruuję książki, by miały różne «stopnie wtajemniczenia», najprostszy, który interesuje małe dzieci, ale też wyższy: zawierający metafory, symbole, które można odczytać dopiero, gdy ma się już pewną wiedzę, a przede wszystkim doświadczenia. Wtedy taka książka łączy pokolenia, można przy niej rozmawiać, drążyć tematy, zaspokajać swoje potrzeby duchowe. Babcia zobaczy w niej coś innego niż wnuczek, który znajdzie coś dla siebie. Mogą się od siebie nawzajem uczyć, poznawać się i szanować swoje punkty widzenia” (Dzieci kończą naukę...). Książki obrazkowe dają możliwość odczytywania znaczeń na wielu poziomach. Dziecko z pomocą dorosłego w kontakcie z książką nie tylko jest wprowadzane w świat pojęć, ale i ćwiczy procesy myślowe. Już najmłodsze dzieci poprzez utwór W kieszonce mogą kształtować zdolności w zakresie kategoryzowania i poszukiwania analogii, na- tomiast starszych z pewnością zainteresują bogate w metafory i symbole obrazy z książki Dwoje ludzi. Założeniem Chmielewskiej jest ofiarowanie czytelnikowi swoistej gry słowno-wizualnej, w której „trzeba połączyć różne fragmenty obra- zów, dopowiedzieć to, co tylko zasugerowane […], czasem poszukać rozwiązania między słowami albo domyślić się sensu ukrytego gdzieś za narysowaną ścianą lub oknem” (Dzieci kończą naukę...). LITERATURA PODMIOTU Chmielewska I., 2011, Pamiętnik Blumki. Poznań, Media Rodzina. Chmielewska I., 2012, Kłopot. Warszawa, Wytwórnia. Chmielewska I., 2013a, Cztery zwykłe miski. Wrocław, Format. Chmielewska I., 2013b, O tych, którzy się rozwijali. Poznań, Media Rodzina. Chmielewska I., 2014a, Dwoje ludzi. Poznań, Media Rodzina. Chmielewska I., 2014b, Oczy. Wrocław, Warstwy. Chmielewska I., 2015, W kieszonce. Poznań, Media Rodzina. Chmielewska I., 2016, Moc Kłopotów. Wytwórnik. Warszawa, Wytwórnia. Chmielewska I., 2011, Pamiętnik Blumki. Poznań, Media Rodzina. Chmielewska I., 2012, Kłopot. Warszawa, Wytwórnia. Chmielewska I., 2013a, Cztery zwykłe miski. Wrocław, Format. Chmielewska I., 2013b, O tych, którzy się rozwijali. Poznań, Media Rodzina Chmielewska I., 2014a, Dwoje ludzi. Poznań, Media Rodzina. Chmielewska I., 2014b, Oczy. Wrocław, Warstwy. Chmielewska I., 2014b, Oczy. Wrocław, Warstwy. Chmielewska I., 2015, W kieszonce. Poznań, Media Rodzina. Chmielewska I., 2016, Moc Kłopotów. Wytwórnik. Warszawa, Wytwórnia. PODSUMOWANIE Autorska twórczość Iwony Chmielewskiej to przykład sztuki dla dziecka two- rzonej na najwyższym poziomie artystycznym. Książki te mogą z powodzeniem realizować cele wychowania przez sztukę, będąc doskonałym instrumentem 106 Joanna S. Ludwiczak uwrażliwiania na piękno i nośnikiem humanistycznych wartości. Ich treść odsła- nia bogactwo metafor i symboli ujętych w poetyckie formy wizualne i lapidarne teksty literackie. Wiele utworów Chmielewskiej ukazuje relacje międzyludzkie, wprowadzając dziecko w sferę uczuć i emocji. Odwołania do świata realnego, na których autorka buduje przekaz w całej swojej twórczości, wpływają na rozwijanie u czytelnika zdolności obiektywnego i subiektywnego postrzegania świata oraz uważnej obserwacji otoczenia. Wspólne obcowanie dorosłego i dziecka z książką obrazkową stwarza przestrzeń do zadawania pytań, do odnajdywania sensu ukry- tego w obrazie i słowie, do wytyczania nowych wątków i poszukiwania odniesień do własnych przeżyć. Traktowanie książki obrazkowej jako medium kulturowego w przestrzeni socjalizacyjnej wyznacza jeszcze jedną płaszczyznę – osobliwej relacji między pośrednikiem i odbiorcą (Cackowska 2017, s. 43). Konfrontacja założeń wychowania przez sztukę z uwarunkowaniami współ- czesnej kultury sprawia, że zyskują one nowy wymiar. Nie ulega wątpliwości, że edukacja estetyczna posiada niezwykłą wagę w kształtowaniu pełnej osobowości człowieka, a w odniesieniu do współczesnego stylu życia może jednocześnie chronić wartości zagrożone i stłumione przez konsumpcyjny, technokratyczny i wirtualny charakter dzisiejszej rzeczywistości. Ponadto sztuka może stanowić instrument do pobudzania myślenia wizualnego. W nauczaniu sztuka niestety często bywa lekceważona, podobnie jak percepcja, gdyż zdaniem Rudolfa Arnheima „panuje przekonanie, że nie zawiera ona myśli”, a „młodzi ludzie uczą się widzieć w sztuce jedynie trening przyjemnych umiejętności, rozrywkę i odpoczynek od aktywności umysłowej” (Arnheim 2011, s. 11). Twórczość Chmielewskiej i, jak sądzę, także wiele picturebooków innych autorów posiada niezwykły potencjał w pobudzaniu procesów myślowych opartych na odczytywaniu warstwy obrazowej. Jak przekaz książki obrazkowej bazuje na ścisłej relacji słowo – obraz, tak jej odbiór opiera się na więzi spojrzenia z myśleniem. W Polsce książki obrazkowe nie wpisały się jeszcze na stałe do katalogów przedszkolnych i szkolnych bibliotek, ale coraz chętniej sięgają po nie poszukujący nauczyciele. Bywa, że są one przedmiotem różnych form edukacji pozaszkolnej, realizowanych między innymi przez samych artystów. Warto zaznaczyć, że ambitna oferta wydawnicza dedykowana najmłodszemu odbiorcy stale i systematycznie powiększa zakres swojego audytorium, co może być oznaką rosnącej potrzeby przeciwstawienia się masowej produkcji książek niedbale wydanych, o marnych tekstach i ilustracjach kopiujących telewizyjną estetykę. SPOTKANIA DZIECKA ZE SZTUKĄ – WYCHOWANIE PRZEZ KSIĄŻKĘ OBRAZKOWĄ 107 LITERATURA PRZEDMIOTU Becket S.L., 2012, Crossover Picturebooks. A Genre for All Ages. New York-London, Taylor & Francis. Cackowska M., 2014, Wzrastać radośnie w przedszkolnym i szkolnym kiczu. W: D. Klus-Stańska (red.), (Anty)edukacja wczesnoszkolna. Kraków, Impuls. Cackowska M., 2017, Współczesna książka obrazkowa – pojęcie, typologia, bada- nia, teorie, konteksty, dyskursy. W: M. Cackowska, H. Dymel-Trzebiatowska, J. Szyłak (red.), Książka obrazkowa. Wprowadzenie. Poznań, Instytut Kultury Popularnej, 11–48. Dylak S., 2012, Alfabetyzacja wizualna jako kompetencja współczesnego człowieka. W: W. Skrzydlewski, S. Dylak (red.), Media, edukacja, kultura: W stronę edu- kacji medialnej. Rzeszów, PTTiM. Kiefer B., 1988, Picturebooks as context for literacy, aesthetic, and real word under- stantings. „Language Arts”, vol. 65, 260–271. Leszczyński G., 2003, Literatura i książka dziecięca. Słowo – obiegi – konteksty. Warszawa, CEBID. Kubinowski D., 2013, Idiomatyczność, synergia, emergencje. Rozwój badań jakościowych w pedagogice polskiej na przełomie XX i XXI wieku. Lublin, Makmed. Mazepa-Domagała B., Wilk T. 2015, Edukacja w zakresie sztuk wizualnych, czyli o przygotowaniu dzieci w młodszym wieku szkolnym do odbioru i kreowania otaczającej je ikonosfery. „Chowanna”, t. 2(45), Katowice, Wydawnictwo Uni- wersytetu Śląskiego, 89–104. Nikolajeva M., Scott C., 2001, How picturebooks work. New York-London, Routledge Pankowska K. 2010, Wstęp. Wychowanie estetyczne dziś – nowa rzeczywistość, nowe zadania. W: K. Pankowska (red.), Sztuka i wychowanie. Współczesne problemy edukacji estetycznej. Warszawa, Wydawnictwo Akademickie ŻAK. 108 Joanna S. Ludwiczak Pater-Ejgierd N., 2010, Kultura wizualna a edukacja. Poznań, Fundacja Tranzy Pater-Ejgierd N., 2010, Kultura wizualna a edukacja. Poznań, Fundacja Tranzyt. Read H., 1976, Wychowanie przez sztukę. A. Trojanowska-Kaczmarska (przekł.). Wrocław-Warszawa-Kraków-Gdańsk, Ossolineum. Read H., 1976, Wychowanie przez sztukę. A. Trojanowska-Kaczmarska (przekł.). Wrocław-Warszawa-Kraków-Gdańsk, Ossolineum. Sipe L., 2001, Picturebooks as Aesthetic Objects. „Literacy and Learning”, vol. 6, nr 1, 23–42. Sipe L., Wolfenbarger C.D., 2007, A Unique Visual and Literacy Art Form: Recent Research on Picturebooks. „Language Arts”, vol. 83, Issue 3, 273–280. Szuman S., 1951, Ilustracja w książkach dla dzieci i młodzieży. Kraków, Wiedza – Zawód – Kultura. Tyszkowa M., 1984, Kultura symboliczna, wartości i rozwój jednostki. W: M. Tysz- kowa, B. Żurakowski (red.), Wartości w świecie dziecka i sztuki dla dziecka. Warszawa-Poznań, PWN. Wiercińska J., 1986, Sztuka i książka. Warszawa, PWN. Wojnar I., 1976, Wstęp. Wizja człowieka uskrzydlonego. W: H. Read, Wychowanie przez sztukę. Wrocław-Warszawa-Kraków-Gdańsk, Ossolineum. Wojnar I., 2010, Wychowanie estetyczne dziś. W czterdziestą rocznicę śmierci Herberta Reada. W: K. Pankowska (red.), Sztuka i wychowanie. Współczesne problemy edukacji estetycznej. Warszawa, Wydawnictwo Akademickie ŻAK, 15–22. Zalewska-Pawlak M., 2017, Sztuka i wychowanie w XXI wieku. LITERATURA PRZEDMIOTU W poszukiwaniu zagubionej teorii sztuki życia i sztuki w wychowaniu. Łódź, Wydawnictwo Uniwersytetu Łódzkiego. Dzieci kończą naukę o północy. Wywiad Sebastiana Frąckiewicza z Iwoną Chmielewską, opublikowano: http://www.clubmamy.pl/index.php?p=kultura&art=62&artic le=291 [dostęp: 5.01.2018]. Iwona Chmielewska: Dzieci mają na co dzień brutalny świat. Wywiad Sigutė Chlebinskaitė z Iwoną Chmielewską, opublikowano: http://zw.lt/kultura/iwona- chmielewska-dzieci-maja-na-co-dzien-brutalny-swiat/ [dostęp: 3.02.2018]. CHILD’S MEETINGS WITH ART – EDUCATION THROUGH PICTUREBOOK Abstract: For numerous years pedagogical conceptions have argued that art for children has the great potential as an educational medium. It can also be considered as a perfect tool that helps to develop child’s visual perception, which has a particular importance in contemporary culture saturated with visuality. The example of open access and valuable art for children (as well as adults) is a picturebook. The potential for this genre is hiding not just in visual and verbal massages but also throughout „architecture” of the book. All of these elements and relationships between them provide inspiration for activities in area of art education. However the contact which child at adult’s participation has with picturebook opens new space for own SPOTKANIA DZIECKA ZE SZTUKĄ – WYCHOWANIE PRZEZ KSIĄŻKĘ OBRAZKOWĄ 109 research, thoughts, interpretations and to ask questions. Hence it evokes a reflection on content hidden in images. There is an attempt in this article to recognise the important educational elements of the form and content of picturebooks on the example of the authorial work of Iwona Chmielewska. The visual characteristics and parts of the text has been presented in comarition to the basis education through art tasks by Herbert Read. This fundamental for theory of aesthetic education concept, in the context of transformations of culture in the 21st century, it acquires new meanings. Keywords: picturebook, art for children, education through art, aesthetic education, percepc- tion of art, Iwona Chmielewska
https://openalex.org/W2029053394	https://zenodo.org/records/578543/files/ZK_article_4134.pdf	English	null	Shallow-water zoantharians (Cnidaria, Hexacorallia) from the Central Indo-Pacific	ZooKeys	2,014	cc-by	26,675	Corresponding author: James D. Reimer (jreimer@sci.u–ryukyu.ac.jp) cademic editor: Leen van Ofwegen \| Received 18 March 2014 \| Accepted 27 August 2014 \| Published 7 October http://zoobank.org/FB83BDD3-958A-456D-BFEA-9C6C28D3E4D5 Citation: Reimer JD, Poliseno A, Hoeksema BW (2014) Shallow-water zoantharians (Cnidaria, Hexacorallia) from the Central Indo-Pacific. ZooKeys 444: 1–57. doi: 10.3897/zookeys.444.7537 Abstract Despite the Central Indo-Pacific (CIP) and the Indonesian Archipelago being a well-known region of coral reef biodiversity, particularly in the ‘Coral Triangle’, little published information is available on its zoantharians (Cnidaria: Hexacorallia: Zoantharia). In order to provide a basis for future research on the Indo-Pacific zoantharian fauna and facilitate comparisons between more well-studied regions such as Japan and the Great Barrier Reef, this report deals with CIP zoantharian specimens in the Naturalis collection in Leiden, the Netherlands; 106 specimens were placed into 24 morpho-species and were sup- plemented with 88 in situ photographic records from Indonesia, the Philippines, and Papua New Guinea. At least nine morpho-species are likely to be undescribed species, indicating that the region needs more research in order to properly understand zoantharian diversity within the CIP. The Naturalis’ zoanthar- ian specimens are listed by species, as well as all relevant collection information, and in situ images are provided to aid in future studies on zoantharians in the CIP. Shallow–water z ZooKeys 444: 1–57 (2014) doi: 10.3897/zookeys.444.7537 http://zookeys.pensoft.net Shallow–water z ZooKeys 444: 1–57 (2014) doi: 10.3897/zookeys.444.7537 http://zookeys.pensoft.net Shallow–water z ZooKeys 444: 1–57 (2014) doi: 10.3897/zookeys.444.7537 http://zookeys.pensoft.net nidaria, Hexacorallia RESEARCH ARTICLE Copyright James D. Reimer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Shallow-water zoantharians (Cnidaria, Hexacorallia) from the Central Indo-Pacific James D. Reimer1,2, Angelo Poliseno3, Bert W. Hoeksema2 1 Molecular Invertebrate Systematics and Ecology Laboratory, Faculty of Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903–0213, Japan 2 Department of Marine Zoology, Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands 3 Università Politecnica delle Marche, Via Brecce Bianche, 60131, Ancona, Italy Corresponding author: James D. Reimer (jreimer@sci.u–ryukyu.ac.jp) Introduction Zoantharians (Cnidaria: Anthozoa: Hexacorallia: Zoantharia) are a common compo- nent of benthos in subtropical and tropical coral reef systems, with many zooxanthel- late species found in shallow waters of both the Atlantic and Indo-Pacific Oceans. Nevertheless, common understanding of zoantharian species diversity is relatively poor when compared to the hard corals (Scleractinia). This lack of knowledge is due to a variety of reasons, including (1) high levels of intraspecific morphological variation hindering reliable identification (Burnett et al. 1997, Reimer et al. 2004), (2) problems in performing histological examinations owing to sand being incorporated in the body walls of many zoantharian species (Reimer et al. 2010), and (3) a confused taxonomic history as different researchers tried to properly classify and understand zoantharian diversity (Burnett et al. 1997, Reimer et al. 2004, Sinniger et al. 2005). Despite these problems, an understanding of zoantharian diversity and their cor- responding taxonomy have slowly become clearer as molecular techniques have been implemented into zoantharian research. The first molecular works of Burnett and co- workers (Burnett et al. 1994, 1995, 1997) combined with the ecological and descrip- tive works of Ryland (Ryland and Lancaster 2003, 2004) have led to more recent pa- pers dealing with the molecular phylogeny of zoantharians (Reimer et al. 2004, 2012b, Sinniger et al. 2005, Swain 2010), resulting in a reassessment of zoantharian taxonomy (Fujii and Reimer 2011, 2013, Sinniger et al. 2013). Consequently, zoantharians are now perhaps the hexacorallian order for which the taxonomy most accurately reflects molecular phylogenetic understanding. However, whereas zoantharian supraspecific taxonomy and diversity is increasingly well understood, many problems remain at the species level (Reimer et al. 2007b), and total species diversity of zoantharians is still poorly known (Appeltans et al. 2012).i p y pp Recent work on zoantharians has focused on many regions of the Indo-Pacific, including Japan (Reimer 2010), Singapore (Reimer and Todd 2009), New Caledonia (Sinniger 2006), the Great Barrier Reef (Burnett et al. 1997, Reimer et al. 2011a) and Palau (Reimer et al. 2014a). In the center between these regions lies the central Indo-Pacific “Coral Triangle” (Hoeksema 2007), including parts or all of Malaysia, Indonesia, Brunei, the Philippines, and Papua New Guinea, the Solomons, and Timor Leste. This region is believed to harbor the highest species diversity in hard corals of the order Scleractinia (Hoeksema 2007, Veron et al. Keywords Zoantharians, Indonesia, Indo-Pacific, biodiversity, coral reef, benthos Copyright James D. Reimer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 2 Introduction 2009, 2011), and it is believed that other coral reef organisms likely have similar diversity patterns (Roberts et al. 2002). Despite this, shallow-water zoantharian species within the Coral Triangle have only briefly been reported on in scientific literature and only a few publications exist (e.g. Den Hartog 1997, Sinniger et al. 2005, Di Camillo et al. 2010), and most informa- tion is made up of photographs in aquarium handbooks (Fosså and Nilsen 1998) and field guides (Colin and Arneson 1995, Gosliner et al. 1996, Erhardt and Knop 2005). Therefore, efforts to compare the regional zoantharian fauna of the Indo-Pacific are hampered by this almost complete lack of published scientific distribution informa- tion. Basic data on zoantharians from the Coral Triangle, such as species lists and Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 3 3 distribution records, are critical to achieve a comprehensive understanding of Indo- Pacific zoantharian diversity. i The present study addresses this lack of Central Indo-Pacific (CIP) zoantharian data via examinations of specimen collections housed in Naturalis Biodiversity Center, Leiden, the Netherlands: RMNH (the former Rijksmuseum van Natuurlijke Histo- rie) and ZMA (the former Zoologisch Museum van Amsterdam). These zoantharian collections are partly based on specimens from numerous surveys in Indonesia dating from the Snellius Expedition (1929–1930) to a recent Marine Biodiversity Workshop in Lembeh Strait (2012), with the large majority of these specimens collected from coral reef environments. Despite the presence of these large and scientifically valuable collections, no previous effort has been made to comprehensively catalogue or examine these historical collections for over 80 years, which could also serve as base-line mate- rial for studies on biotic change (Hoeksema et al. 2011). Here, for the first time, we report on the zoantharian specimens from Indonesia housed at Naturalis, and list shal- low water species of the CIP, including specimen collection information. Our records are further enhanced by numerous in situ images from more recent fieldwork in Indo- nesia taken by the last author starting with the Snellius–II Expedition (1984–1985). Finally, we discuss the shallow water zoantharian diversity of CIP in relation to infor- mation from surrounding regions, and make recommendations for future zoantharian research in the region. Specimen collection Zoantharian specimens from the Naturalis collections in Leiden (RMNH + ZMA) were collected primarily from expeditions to the Indonesia region, starting with the Snellius Expedition (1929–1930). Our examinations showed 22 regions in which ei- ther specimens or photographic records were present. All specimen/record localities are shown in Figure 1 with location and reference details in Table 1. Regions (numbers also referred to in species notes and in distributional maps, with names used hereafter in bold, and with representative publications included): 1. West Sumatra, Indonesia. Fieldwork by B.W. Hoeksema in collaboration with Dr. A. Kunzmann, Bung Hatta University, Padang, West Sumatra, in 1996–1997. Sumatra, Indonesia. Fieldwork by B.W. Hoeksema in collaboration with . Kunzmann, Bung Hatta University, Padang, West Sumatra, in 1996–1997. 2. Southwest Java, Indonesia. Collections from Teluk Pelabuhan Ratu by Dr. P.H. van Doesburg, RMNH, in 1977. 3. Thousand Islands, off Jakarta, Java Sea, northwest Java, Indonesia. Expedition organized by the Research Center for Oceanography (RCO–LIPI) and Naturalis in 2005 (Tuti and Soemodihardjo 2006). 3. Thousand Islands, off Jakarta, Java Sea, northwest Java, Indonesia. Expedition organized by the Research Center for Oceanography (RCO–LIPI) and Naturalis in 2005 (Tuti and Soemodihardjo 2006). j 4. West Bali, Indonesia. Fieldwork by B.W. Hoeksema in collaboration with K.S. Putra of WWF Indonesia Marine Program in 1998 (Hoeksema and Putra 2000). j 4. West Bali, Indonesia. Fieldwork by B.W. Hoeksema in collaboration with K.S. Putra of WWF Indonesia Marine Program in 1998 (Hoeksema and Putra 2000). James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 4 5. East Bali (including southeast Bali, Nusa Lembongan, Nusa Penida in Lombok Strait), Indonesia. Fieldwork by B.W. Hoeksema in collaboration with K.S. Putra of WWF in 1997 and 1998 (Hoeksema and Putra 2000). Expedition organized by the Research Center for Oceanography (RCO–LIPI), WWF Bali Indonesia Marine Program, and Naturalis in 2001 (Hoeksema and Tuti 2001). 6. Northeast Sumba, Indonesia. Indonesian – Dutch Snellius–II Expedition in 1984 (Van der Land and Sukarno 1986, Best et al. 1989). . South Flores, Indonesia. Snellius Expedition in 1929–1930 (Boschma 1936) 8. Komodo Island, Indonesia. Indonesian – Dutch Snellius–II Expedition (Van der Land and Sukarno 1986, Best et al. 1989). 9. Spermonde Archipelago, South Sulawesi, Indonesia. Snellius Expedition in 1929–1930 (Boschma 1936). Indonesian – Dutch Snellius–II Expedition in 1984 (Van der Land and Sukarno 1986, Best et al. 1989). Fieldwork by Dr. H. Moll in 1980 (Moll 1983). Fieldwork by B.W. Specimen collection Hoeksema around reefs along onshore-off- shore gradients in 1984–1987 (Hoeksema 2012a) and in 1993–1998 (Hoeksema and Crowther 2011). 10. Salayer Island, South Sulawesi, Indonesia. Indonesian – Dutch Snellius–II Expe- dition in 1984 (Van der Land and Sukarno 1986, Best et al. 1989). 11. Taka Bone Rate (Tiger Islands), Flores Sea, Indonesia. Indonesian – Dutch Snel- lius–II Expedition in 1984 (Van der Land and Sukarno 1986, Best et al. 1989). 12. Tukang Besi Islands (Wakatobi), Southeast Sulawesi, Indonesia. Indonesian – Dutch Snellius–II Expedition in 1984 (Van der Land and Sukarno 1986, Best et al. 1989). Rapid Ecological Assessment (REA) Wakatobi National Park in 2003 (Pet–Soede and Erdmann 2004). 13. Maisel Islands, Banda Sea, Indonesia. Indonesian – Dutch Snellius–II Expedition in 1984 (Van der Land and Sukarno 1986, Best et al. 1989). 14. Ambon and Haruku, Moluccas, Indonesia. Snellius Expedition in 1929–1930 (Boschma 1936). Indonesian – Dutch Snellius–II Expedition in 1984 (Van der Land and Sukarno 1986, Best et al. 1989). Rumphius Biohistorical Expedition to Ambon in 1990 (Strack 1993). Fauna Malesiana Marine Maluku Expedition in 1996 (Van der Land 1996). 15. Bo Islands, Halmahera Sea, Indonesia. Snellius Expedition in 1929–1930 (Boschma 1936). 16. West Halmahera Sea, Indonesia. Ekspedisi Widya Nusantara (E–Win): Ternate Expedition in 2009, involving reefs on volcanic slopes and reefs around sand-cays (Hoeksema and Van der Meij 2010; Gittenberger et al. 2014). 17. Lembeh Strait, North Sulawesi, Indonesia. Fauna Malesiana Marine Sulawesi Expedition organized by Research Center for Oceanography (RCO–LIPI) and Naturalis in 1994. Marine Biodiversity Workshop North Sulawesi organized by Research Center for Oceanography (RCO–LIPI), Universitas Sam Ratulang and Naturalis in 2012. Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 5 Figure 1. Sampling regions in this study. Note that the region numbers correspond with numbers given in text. 1 West Sumatra 2 Southwest Java 3 Thousand Islands, northwest Java, Java Sea 4 West Bali 5 East Bali 6 Northeast Sumba 7 South Flores 8 Komodo Island 9 Spermonde Archipelago, Southwest Sulawesi 10 Salayer Island, Southwest Sulawesi 11 Taka Bone Rate, Flores Sea 12 Tukang Besi Islands (Wakatobi), Southeast Sulawesi 13 Maisel Islands, Banda Sea 14 Ambon and Haruku, Moluccas 15 Bo Islands, Halmahera Sea 16 West Halmahera 17 Lembeh Strait, North Sulawesi 18 Bunaken, North Sulawesi 19 Berau Islands, East Kali- mantan 20 Sulu Islands, Philippines 21 Cebu, Philippines 22 Madang, Papua New Guinea. Regions with no country names are in Indonesia. Oceanic names in italics. Specimen collection Dark grey: land masses. Light grey: continental flats. Figure 1. Sampling regions in this study. Note that the region numbers correspond with numbers given in text. 1 West Sumatra 2 Southwest Java 3 Thousand Islands, northwest Java, Java Sea 4 West Bali 5 East Bali 6 Northeast Sumba 7 South Flores 8 Komodo Island 9 Spermonde Archipelago, Southwest Sulawesi 10 Salayer Island, Southwest Sulawesi 11 Taka Bone Rate, Flores Sea 12 Tukang Besi Islands (Wakatobi), Southeast Sulawesi 13 Maisel Islands, Banda Sea 14 Ambon and Haruku, Moluccas 15 Bo Islands, Halmahera Sea 16 West Halmahera 17 Lembeh Strait, North Sulawesi 18 Bunaken, North Sulawesi 19 Berau Islands, East Kali- mantan 20 Sulu Islands, Philippines 21 Cebu, Philippines 22 Madang, Papua New Guinea. Regions with no country names are in Indonesia. Oceanic names in italics. Dark grey: land masses. Light grey: continental flats. 18. Bunaken, North Sulawesi, Indonesia. Fieldwork by B.W. Hoeksema in collabora- tion with Universitas Sam Ratulang, Manado, in 1994 and 1998. g 19. Berau Islands, East Kalimantan, Indonesia. East Kalimantan Program in 2003 (Hoeksema 2004). 20. Sulu Islands, Philippines. Snellius Expedition in 1929–1930 (Boschma 1936). 21. Cebu, Philippines. Cebu Fieldwork by M. L. Esmeno in 1976. Strait Expedition organized by San Carlos University and National Museum of Natural History, Leiden in 1999. 22. Madang, Bismarck Sea, north coast of Papua New Guinea. Fieldwork by B.W. Hoeksema with Christensen Research Institute in 1992 (Hoeksema 1993). James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 6 Table 1. Overview of field surveys from which order Zoantharia specimens examined in this study were collected. Area Year(s) References Remarks on locality and conditions of sample collecting 1 West Sumatra, Indonesia 1996 Jonker and Johan (1999: Figure 1) Reefs off Padang and Siberut. Coral reef survey in collaboration with Bung Hatta University, Padang. Most reefs damaged, possibly as a result of blast fishing and red tide. Observer/collector: B.W. Hoeksema. 2 Southwest Java, Indonesia 1977 NA Locality: Teluk Pelabuhan Ratu. Observer/collector: P.H. van Doesburg, RMNH. 3 Northwest Java, Indonesia 2005 Tuti and Soemodihardjo (2006): Annex 2 (157–161), Annex 5 (179) Thousand Islands Expedition, off Jakarta, Java Sea. In collaboration with RCO–LIPI. Zoantharians were observed during a coral survey along an onhsore-offshore gradient. 4 West Bali, Indonesia 1998 Hoeksema and Putra (2000: Figure 1) Coral biodiversity survey in collaboration with WWF Indonesia Marine Program. Specimen collection 5 Eastern Bali, Indonesia 1997, 1998 Hoeksema and Putra (2000: Figure 1) Includes southeast Bali and Lombok Strait. Coral biodiversity surveys in collabora- tion with RCO–LIPI and WWF Bali Indonesia Marine Program. 2001 Hoeksema and Tuti (2001: 12–15) Includes southeast Bali and Lombok Strait. Coral biodiversity surveys in collabora- tion with RCO–LIPI and WWF Bali Indonesia Marine Program. 6 Northeast Sumba, Indonesia 1984 Van der Land and Sukarno (1986: 2.4–2.6, 3.5), Best et al. (1989: 108) Indonesian – Dutch Snellius–II Expedition. 7 South Flores, Indonesia 1930 Boschma (1936: 24) Snellius Expedition. 8 Komodo Island, Indonesia 1984 Van der Land and Sukarno (1986: 2.6–2.9, 2.19–2.20, 3.6), Best et al. (1989: 108) Indonesian – Dutch Snellius–II Expedition. 9 Spermonde Archipelago, South Sulawesi. Indonesia 1980 Moll (1983: 26) Coral reef surveys on reefs along onshore-offshore gradients. Observer/collector: H. Moll 1984 Van der Land and Sukarno (1986: 2.22, 3.10), Best et al. (1989: 108) Indonesian – Dutch Snellius–II Expedition. 1984–1987 Hoeksema (2012a: Figure 1) Coral reef surveys on reefs along onshore-offshore gradients. 1993–1998 Hoeksema and Crowther (2011: Figure 1) Coral reef surveys on reefs along onshore-offshore gradients. 10 Salayer Island, S Sulawesi, Indonesia 1984 Van der Land and Sukarno (1986: 2.12–2.17, 3.9), Best et al. (1989: 108) Indonesian – Dutch Snellius–II Expedition. 11 Taka Bone Rate (Tiger Is.), Indonesia 1984 Van der Land and Sukarno (1986: 2.11– 2.12, 2.17–2.19, 3.8), Best et al. (1989: 108) Indonesian – Dutch Snellius–II Expedition. 12 Tukang Besi Is. (Wakatobi), SE Sulawesi, Indonesia 1984 Van der Land and Sukarno (1986: 2.2–2.4, 3.4), Best et al. (1989: 108) Indonesian – Dutch Snellius–II Expedition. 2003 Pet-Soede and Erdmann (2004: 57, 117, 143) Rapid Ecological Assessment (REA) Wakatobi National Park. 7 7 Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 13 Maisel Is., Banda Sea, Indonesia 1984 Van der Land and Sukarno (1986: 2.2, 3.3), Best et al. (1989: 108) Indonesian – Dutch Snellius–II Expedition. 14 Moluccas (Ambon, Haruku), Indonesia 1930 Boschma (1936: 19–20) Snellius Expedition. 1984 Van der Land and Sukarno (1986: 2.1–2.2), Best et al. (1989: 108) Indonesian – Dutch Snellius–II Expedition. 1990 Strack (1993: 16–42) Rumphius Biohistorical Expedition to Ambon. 1996 Van der Land (1996) Fauna Malesiana Marine Maluku Expedition. 15 Bo Is., Halmahera Sea, Indonesia 1930 Boschma (1936: 23) Snellius Expedition. 16 West Halmahera Sea, Indonesia 2009 Hoeksema and Van der Meij (2010: 80–85) Ekspedisi Widya Nusantara (E–Win): Ternate Expedition. Coral biodiversity survey B.W. Hoeksema. Specimen registration and identification Examination of the registered (n=52) and unregistered zoantharian specimens (n=570) of the Naturalis collection showed that of a total 622 specimens, 105 were from Indonesia, with an additional four from the Philippines. Of these 109 specimens, 106 form the basis of this research, as we excluded three specimens that could not be conclusively identified as zoantharians. 88 photographic records of zoantharians specimens were also examined. Although most species are from depths in the range of SCUBA (<40 m), we also included all Epizoanthus illoricatus Tischbierek, 1930 specimens, as although some specimens were from >40 m (and down to 190 m), the range of this species does extend into shallower (<40 m) depths. Additionally, three specimens of Parazoanthus collected by rectangular dredge from depths of 50–100 m were included in analyses. In this study, these 106 zoantharian specimens are collectively referred to as “shallow- water zoantharians”. All unregistered specimens were newly registered into the Naturalis collection in the course of our research. All specimens, newly registered or not, were re-identified by the first author. A list of specimens, their collection information, and Naturalis (RMNH Coel) registration numbers are given within each species’ section. Descrip- tions of each species are given to aid in field and specimen identification, and are not formal taxonomic redescriptions.i Most zoantharian specimens were easily identifiable to genus level without micro- scopic examination. Species determinations were made consulting previous literature (listed with each species). However, many specimens were only identified to “confers with” (cf.) or “affinity” (aff.) levels. Asides from a few species (e.g. Palythoa heliodiscus), very few records of zoantharians had previously been formally reported from the CIP/ Coral Triangle region. Given these reasons, we followed recent research (Burnett et al. 1994, 1995, 1997, Reimer et al. 2006a, 2007b, Sinniger 2006, Sinniger et al. 2010, Reimer and Todd 2009, Reimer 2010, Reimer et al. 2011a, Fujii and Reimer 2011) from neighboring regions and used Zoanthus and Palythoa species names for which nu- merous references, molecular data and/or accurate descriptions were available, unless specimens and/or images clearly did not match with previously published information. p g y p y p Sizes of specimens are averages taken from measurements of 10 polyps per specimen, unless the specimen contained less than 10 polyps, in which case all non-damaged polyps were examined. Specimen collection 2009 Gittenberger et al. (2014: Figure 1) Ekspedisi Widya Nusantara (E–Win): Ternate Expedition. Coral biodiversity survey B.W. Hoeksema. 17 Lembeh Strait, North Sulawesi, Indonesia 1994 Van der Land (1994: 7–9) Fauna Malesiana Marine Sulawesi Expedition in collaboration with RCO–LIPI. 2012 NA Marine Biodiversity Workshop North Sulawesi in collaboration with RCO–LIPI and Universitas Sam Ratulang. Observer/collector: B.W. Hoeksema. 18 Bunaken, North Sulawesi, Indonesia 1994, 1998 NA Fieldwork in collaboration with Universitas Sam Ratulang, Manado. Observer/col- lector: B.W. Hoeksema. 19 Berau Islands, East Kalimantan, Indonesia 2003 Hoeksema (2004: 57–60, Figure 1) East Kalimantan Program. Coral biodiversity survey B.W. Hoeksema. 20 Sulu Islands, Philippines 1929 Boschma (1936: 8–9) Snellius Expedition. 21 Cebu, Bohol Philippines 1976 NA Fieldwork by M.L. Esmeno. 1999 NA Coral biodiversity survey B.W. Hoeksema during Cebu Strait Expedition in collabo- ration with San Carlos University, Cebu City. 22 Madang, Bismarck Sea, Papua New Guinea 1992 Hoeksema (1993: Figures 1–2) Coral biodiversity survey in collaboration with Christensen Research Institute. James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 8 8 Specimen registration and identification For species’ dimensions, average dimensions were taken from the overall average of specimens, unless there were less than three specimens within a species. In such cases, dimensions are stated only as a range (minimum to maximum). Specimens and species Abbreviations: NA=not available. Abbreviations: NA=not available. Order Zoantharia Gray, 1832 Suborder Brachycnemina Haddon & Shackleton, 1891a Family Zoanthidae Rafinesque, 1815 Genus Acrozoanthus Saville–Kent, 1893 Results From specimen examination, the 106 Indonesian zoantharian specimens in the Natu- ralis collection supplemented with images were placed into 24 morphospecies, detailed below. Locations are in Indonesia unless otherwise noted, and all photographic images Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 9 9 were taken by B.W. Hoeksema unless otherwise noted. Duplicate photographic images of the same species from the same site are counted as one record. Latitude and longi- tude are given when available. were taken by B.W. Hoeksema unless otherwise noted. Duplicate photographic images of the same species from the same site are counted as one record. Latitude and longi- tude are given when available. 1. Acrozoanthus australiae Saville–Kent, 1893 Figures 2A, 3 Specimens examined (n=16). RMNH Coel 23405, Tg. Bengteng (=Galghoek), Am- bon, Moluccas, depth = 3 to 4 m, collected November 10, 1990 by J.C. den Hartog; RMNH Coel 23406, outer bay, Ruhmatiga, Hitu, Ambon, Moluccas, depth = ap- prox. 3 m, collected December 3, 1990 by J.C. den Hartog; RMNH Coel 23407, station 17, southeast side of Pombo Island, Ambon, Moluccas, depth = 6 m, collected November 17, 1994 by J.C. den Hartog; RMNH Coel 23408, west-northwest of Bar- rang Lompo, Spermonde Archipelago, South Sulawesi, depth = 1.5 to 4 m, collected December 23, 1994 by J.C. den Hartog; RMNH Coel 23409, entrance of harbor near light beacon, northwest of Gusung, Spermonde Archipelago, South Sulawesi, depth = 5 to 7 m, collected October 7, 1990 by J.C. den Hartog; RMNH Coel 23410, 7.5 km west of Makassar, Spermonde Archipelago, South Sulawesi (05°07'S, 119°20'E), depth = NA, collected May 31, 1994 by J.C. den Hartog; RMNH Coel 23411, west of Gusung (=Lae–Lae Keke) (=1 km northwest of Makassar), Spermonde Archipelago, South Sulawesi (05°07.5'S, 119°23'E), depth = NA, collected May 31, 1994 by J.C. den Hartog; RMNH Coel 24100, station MAL04, south coast northeast of Cape Ha- hurong, Ambon, Moluccas (03°47'S, 128°06'E), depth = 2 to 28 m, collected June 6, 1996 by J.C. den Hartog; RMNH Coel 40361, NNM–LIPI–WWF Expedition station BAL.16, southeast side of Pulau Serangan, Bali (08°44'48"S, 115°14'26"E), depth = to 10 m, collected April 6, 2001 by J. Goud; RMNH Coel 40549, Snellius–II Expedition station 4.011, reef edge west of Mai, Maisel Islands, Banda Sea (05°28'S, 127°31'E), depth = 1 to 30 m, collected September 7, 1984; RMNH Coel 40550, Snellius–II Ex- pedition station 4.001, near Tawiri, Ambon Bay, Moluccas (03°42'S, 128°07'E), depth = approx. 1.5 to 8 m, collected September 4, 1984; RMNH Coel 40554, Snellius–II Expedition station 4.006, near Eri, Ambon Bay, Moluccas (03°45'S, 128°8'E), depth = approx. 1.5 to 5 m, collected September 4, 1984; RMNH Coel 40556, Snellius–II Expedition station 4.006, near Eri, Ambon Bay, Moluccas (03°45'S, 128°8'E), depth James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 10 = approx. 1.5 to 5 m, collected August 29, 1984; RMNH Coel 40558, Snellius–II Expedition station 4.030, west coast of Binongko, Tukang Besi Islands, Banda Sea (05°55'S, 123°59'E), depth = approx. 3 to 4 m, collected September 10, 1984 by M. 1. Acrozoanthus australiae Saville–Kent, 1893 Figures 2A, 3 Slierings; RMNH Coel 40566, west side of Pulau Samalona, 7.5 km west of Makassar, Spermonde Archipelago, South Sulawesi (05°07'S, 119°20'E), depth = NA, collected February 18, 1994 by B.W. Hoeksema; RMNH Coel 40569, Fauna Malesiana Ma- rine Sulawesi Expedition station SUL.06, Pantai Parigi, Pulau Lembeh, Selat Lembeh, North Sulawesi (01°28'N, 125°14'E), depth = 0 to 6 m, collected October 15, 1994 by M. Slierings. Photographic records (n=6). West side of Pulau Lae–Lae (05°08'05"S, 119°23'15"E), South Sulawesi, May 24, 1997; station MAL.19 (03°43'S, 128°03'E), Tanjune Batu Dua, east of Hatu, north coast of Ambon Bay, Moluccas, Novem- ber 19, 1996; station MAL.22 (03°48'S, 128°06'E), southwest coast, east of Tun- jung Nusanive, Ambon Bay, Moluccas, November 21, 1996; Nusa Penida, Lombok Strait, east Bali, May 26, 1998 (08°40'56"S, 115°28'56"E); northwest Pulau Sama- lona, Spermonde Archipelago, South Sulawesi (05°07'25"S, 119°20'10"E), January 12, 1997; western slope of Bone Lola shoal, Spermonde Archipelago, South Sulawesi (05°03'15"S, 119°21'15"E), April 22, 1998. Description. Non-incrusted zooxanthellate zoantharian that inhabits the outside of eunicid worm tubes (Haddon 1895), with a unique asexual form of “budding” (Ryland 1997). Easily recognizable as it is an epibiont on outside surface of eunicid worm tube, and has a reduced stoloniferous coenenchyme, long pale yellow-green or pale purple tentacles (n=approx. 40–50) with occasional fluorescent green markings and black tips, and light brown/purple to white ectoderm with similarly colored oral disks (Figure 2A). Preserved specimens in this study had polyps of average 6.0 mm in height (range 2.5–14 mm), 3.2 mm in width (range 2–5 mm) (n=8 specimens exam- ined [RMNH Coel 40361, 40549, 40550, 40554, 40556, 40558, 40566, 40569], 10 polyps/specimen), and oral disks approximately 6 mm in diameter when expanded in situ (partially adapted from Reimer et al. 2011b). Specimen RMNH Coel 40566 had much larger polyps than other specimens (average height 10.6 mm, average width 4.3 mm), but this may be due to preservation in formalin as opposed to ethanol than to any phenotypic difference. f Distribution. Regions recorded in this study (Figure 3). East coast of Bali (5), Spermonde Archipelago (9), Tukang Besi Islands (12), Maisel Islands (13), Moluccas (14), Lembeh Strait (17). 2. Zoanthus sansibaricus Carlgren, 1900 Figures 2B, 3 Specimens examined (n=1). RMNH Coel 40476, Rumphius Biohistorical Expedi- tion station 27, Leitimur, south coast, Hutumuri, Ambon, Moluccas, depth = inter- tidal, collected November 26, 1990 by M.S.S. Lavaleye. y y Photographic records (n=9). Southeast Siberut, West Sumatra (01°44'S, 99°15'E), December 15, 1996; east Menjangan Island, West Bali (08°05'25"S, 114°31'40"E), May 21, 1998; west Pulau Lumu Lumu, Spermonde Archipelago, South Sulawesi (04°58'30"S, 119°12'30"E), October 8, 1997; west Pulau Kudingareng Keke, Spermonde Archipela- go, South Sulawesi (05°06'20"S, 119°17'03"E), May 29, 1997; northwest Pulau Barang Lompo, Spermonde Archipelago, South Sulawesi (05°02'35"S, 119°19'10"E), July 21, 1998; south Pulau Samalona, Spermonde Archipelago, South Sulawesi (05°07'45"S, 119°20'25"E), October 27, 1997; west Pulau Lae Lae Besar, Spermonde Archipelago, South Sulawesi (05°08'15"S, 119°23'10"E), November 12, 1997; northwest Pulau Lae Lae Keke, Spermonde Archipelago, South Sulawesi (05°07'10"S, 119°23'25"E), Octo- ber 11, 1997; Station BER.26, northeast Buliulin (south of Samama Island), Berau Is- lands, East Kalimantan, (02°07'07"N, 118°20'32"E), October 15, 2003. Description. Can form colonies of up to 1 m2, but often forming much smaller colonies in cracks and small overhangs in intertidal and shallow waters (<5 m). with polyps well clear and free of the coenenchyme (“liberae”) (Pax 1910, Reimer et al. 2006b). Adult polyps 2–12 mm in diameter when open, up to 20 mm in length but usually shorter, particularly in locations with strong currents or waves. The sole speci- men (RMNH Coel 40476) in this study has small polyps (height average 2.8 mm, range 2–5 mm; width average 2.4 mm, range 1.5–4 mm) within the reported range of this species. External polyp surface generally uniform, light to dark gray-blue with no significant markings or patterns. Tentacles 40–58, mesenteries 48–54. Wide variation in oral disk colors, patterns, and in colors of tentacles (Figure 2B) (Reimer et al. 2004, 2006a) (partially adapted from Reimer and Hickman 2009). Distribution. Regions recorded in this study (Figure 3). West Sumatra (1), West Bali (4), Spermonde Archipelago (9), Moluccas (14), Berau Islands (19). Previous records. This species has previously been reported from Zanzibar (type locality), Singapore (Reimer and Todd 2009), Taiwan (Reimer et al. 2011c, 2013a), Palau (Reimer et al. 2014a), southern Japan (Reimer et al. 2004, 2006a, Kamezaki et al. 2013), and is considered to have a very wide Indo-Pacific distribution.i i Remarks. Based on its wide Indo-Pacific distribution, it is very likely that this zooxanthellate species is much more common within the CIP than reported here. (14), Lembeh Strait (17). Previous records. Originally described from Australia, where it has been reported from both the coast of northern Queensland, and the region around Darwin in the Northern Territory. Subsequent records reported from North Sulawesi, Indonesia (Sin- niger et al. 2005, Reimer et al. 2011b), and photographic records from Mactan Island, Philippines (Reimer et al. 2011b). Also reported from southern Taiwan (Reimer et al. 2011b) and at Ningaloo, Western Australia (Y. Irei and J.D. Reimer, unpubl. data). g Remarks. This genus is positioned within the genus Zoanthus based on phyloge- netic analyses (Reimer et al. 2011b). Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 11 2. Zoanthus sansibaricus Carlgren, 1900 Figures 2B, 3 One possible reason for the lack of records from the CIP is that this species is most com- monly found in the intertidal zone, which is under-sampled during SCUBA surveys. However, this species is also found to depths of 52 m (Kamezaki et al. 2013), although below the shallow littoral zone it rarely forms colonies >100 polyps. 12 James D. Reimer et al. / ZooKeys 444: 1–57 (2014) Additionally, as preserved specimens of Zoanthus are notoriously hard to identify to species level, the large number of unidentified Zoanthus specimens in this study undoubtedly include some Z. sansibaricus colonies. This is also likely one important reason explaining the presence of comparatively more photographic records of this species in this study, as in situ identification of colonies with expanded oral polyps is easier than preserved specimen identification. i This species may be the same as Zoanthus coppingeri Haddon & Shackleton, 1891b from the Great Barrier Reef, Australia, based on molecular data (Reimer, data not shown), which has been reported to be a senior synonym of Z. jukesii Haddon & Shackleton, 1891b, Z. macgillivrayi Haddon & Shackleton, 1891b, Z. annae Carlgren, 1937, Z. mantoni Carlgren, 1937, Z. fraseri Carlgren, 1937, all described from the Great Barrier Reef based on nematocyst data (Burnett et al. 1997). 3. Zoanthus sp. Figures 2C, D, 3 Specimens examined (n=10). RMNH Coel 40360, NNM–LIPI–WWF Expedition station BAL.03, south of tidal channel, Palung Semawang, off Kesumasari Beach, Sa- nur, Bali (08°42'39"S, 115°16'09"E), depth to 5 m, collected by L. P. van Ofwegen and M. Slierings on March 31, 2001; RMNH Coel 40457, piers of harbor, Cebu City, Cebu, Philippines by M. L. Esmeno in 1976 (original label “specimen 196”); RMNH Coel 40516, Snellius–II Expedition station 27, west side of Bone Tambung, South Sulawesi (05°03'00"S, 119°15'45"E), depth = 1 m, collected October 23, 1980 by H. Moll; RMNH Coel 40537, Snellius–II Expedition station 4.139, reef flat edge south of Tarupa Kecil, northeast Taka Bone Rate (06°30'S, 121°08'E), depth = 30 m, col- lected September 25, 1984; RMNH Coel 40539, Snellius–II Expedition station 4.011, reef edge west of Mai, Maisel Islands, Banda Sea (05°28'S, 127°31'E), depth 1 to 30 m, collected September 7, 1984; RMNH Coel 40542, Snellius–II Expedition station 4.084, Selat Linta, east of Komodo I. (08°35'S, 119°34'E), depth = approx. Photographic records (n=3). West side of Pulau Lae–Lae, Spermonde Archipela- go, South Sulawesi (05°08'05"S, 119°23'15"E), September 16, 1997; west side of Pu- 2. Zoanthus sansibaricus Carlgren, 1900 Figures 2B, 3 3 m, col- lected September 18, 1984; RMNH Coel 40551, Snellius–II Expedition station 4.079, Selat Linta, east of Komodo I. (08°35'S, 119°34.2'E), collected September 10, 1984; RMNH Coel 40560, Snellius–II Expedition station 4.096, northeast cape of Komodo I. (08°29'S, 119°34.1'E), from “shallow water”, collected September 20, 1984; RMNH Coel 40564, Fauna Malesiana Marine Sulawesi Expedition station SUL.08, channels between lava outflows, south of Tanjung Batuangus, Selat Lembeh, North Sulawesi (01°30'N, 125°15'E), depth 5 to 10 m, collected by M. Slierings on October 16 or 25, 1994; RMNH Coel 40565, Fauna Malesiana Marine Sulawesi Expedition station SUL.08, channels between lava outflows, south of Tanjung Batuangus, Selat Lembeh, North Sulawesi (01°30'N, 125°15'E), depth to 10 m, collected on October 16 or 25, 1994. Specimens examined (n=10). RMNH Coel 40360, NNM–LIPI–WWF Expedition station BAL.03, south of tidal channel, Palung Semawang, off Kesumasari Beach, Sa- nur, Bali (08°42'39"S, 115°16'09"E), depth to 5 m, collected by L. P. van Ofwegen and M. Slierings on March 31, 2001; RMNH Coel 40457, piers of harbor, Cebu City, Cebu, Philippines by M. L. Esmeno in 1976 (original label “specimen 196”); RMNH Coel 40516, Snellius–II Expedition station 27, west side of Bone Tambung, South Sulawesi (05°03'00"S, 119°15'45"E), depth = 1 m, collected October 23, 1980 by H. l Specimens examined (n=10). RMNH Coel 40360, NNM–LIPI–WWF Expedition station BAL.03, south of tidal channel, Palung Semawang, off Kesumasari Beach, Sa- nur, Bali (08°42'39"S, 115°16'09"E), depth to 5 m, collected by L. P. van Ofwegen and M. Slierings on March 31, 2001; RMNH Coel 40457, piers of harbor, Cebu City, Cebu, Philippines by M. L. Esmeno in 1976 (original label “specimen 196”); RMNH Coel 40516, Snellius–II Expedition station 27, west side of Bone Tambung, South Sulawesi (05°03'00"S, 119°15'45"E), depth = 1 m, collected October 23, 1980 by H. l Photographic records (n=3). West side of Pulau Lae–Lae, Spermonde Archipela- go, South Sulawesi (05°08'05"S, 119°23'15"E), September 16, 1997; west side of Pu- Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 13 Figure 2. Images of Acrozoanthus and Zoanthus species from photographic records in this study. A Acrozoanthus australiae at Nusa Penida, Lombok Strait, east Bali, May 26, 1998 B Zoanthus san- sibaricus at Station BER.26, northeast Buliulin (south of Samama Island), East Kalimantan, Berau Islands, October 15, 2003 C Zoanthus sp. at west side of Pulau Samalona, Spermonde Archipelago, South Sulawesi, September 16, 1997; and D Zoanthus sp. 2. Zoanthus sansibaricus Carlgren, 1900 Figures 2B, 3 west of Gusung (=Pulau Lae–Lae Keke), Spermonde Archipelago, South Sulawesi, October 11 1997. Figure 2. Images of Acrozoanthus and Zoanthus species from photographic records in this study. A Acrozoanthus australiae at Nusa Penida, Lombok Strait, east Bali, May 26, 1998 B Zoanthus san- sibaricus at Station BER.26, northeast Buliulin (south of Samama Island), East Kalimantan, Berau Islands, October 15, 2003 C Zoanthus sp. at west side of Pulau Samalona, Spermonde Archipelago, South Sulawesi, September 16, 1997; and D Zoanthus sp. west of Gusung (=Pulau Lae–Lae Keke), Spermonde Archipelago, South Sulawesi, October 11 1997. lau Samalona, Spermonde Archipelago, South Sulawesi (05°07'25"S, 119°20'10"E), September 16, 1997; west of Gusung (=Pulau Lae–Lae Keke), Spermonde Archipela- go, South Sulawesi (05°07.5'S, 119°23'E), October 11, 1997. Description. This group includes all Zoanthus spp. specimens that could not be identified to species level (n=10). Almost all of these specimens are ‘liberae’ or ‘inter- mediae’, with polyps rising out from the coenenchyme (see Pax 1910) (Figure 2C). One specimen, RMNH Coel 40560, is more ‘immersae’ with polyps only slightly protruding from the coenenchyme (average height 2.25 mm, width 2.0 mm, n=10). Overall, polyps for all specimens fit within the range of several described species, with an average for all specimens of a height of 6.7 mm (range 2–17 mm), and width of 3.3 mm (range 1.5–6 mm) (n=10 specimens). Thus, given the high variation within Zoanthus species, particularly polyp height based on microenvironment (Ong et al. 2013), and the lack of other diagnostic characteristics, for now these species cannot be identified to species level. Based solely on sizes, two specimens, RMNH Coel 40542 and 40565, have much larger polyps compared to the other specimens (height 10.3 mm, width 4.3 mm; height 10.8 mm, width 4.4 mm), but whether these are a sepa- rate species from other specimens or the size difference is due to fixation method in formalin is unknown. James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 14 Figure 3. Distribution of Acrozoanthus and Zoanthus species from specimens and photographic records from this study. Acrozoanthus australiae specimens in red, Z. sansibaricus in green, and Zoanthus sp. in blue. Region numbers correspond to locations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. Figure 3. Distribution of Acrozoanthus and Zoanthus species from specimens and photographic records from this study. 2. Zoanthus sansibaricus Carlgren, 1900 Figures 2B, 3 Acrozoanthus australiae specimens in red, Z. sansibaricus in green, and Zoanthus sp. in blue. Region numbers correspond to locations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. Distribution. Regions recorded in this study (Figure 3). Eastern Bali (5), Ko- modo (8), Spermonde Archipelago (9), Taka Bone Rate (11), Maisel Is. (13), Lembeh Strait. (17). Distribution. Regions recorded in this study (Figure 3). Eastern Bali (5), Ko- modo (8), Spermonde Archipelago (9), Taka Bone Rate (11), Maisel Is. (13), Lembeh Strait. (17). y Photographic records. NA. Description. Species in this genus are zooxanthellate, not incrusted, with a simple mesogleal sphincter muscle, and have non-erect, recumbent polyps that do not have lacunae or mesogleal canals, unlike Zoanthus species. Isaurus tuberculatus has tubercles on the exterior surface of polyps (=endodermal invagination) (Figures 4A, B). For detailed discussion of I. tuberculatus, refer to Muirhead and Ryland (1985), with phy- logenetic analyses in Reimer et al. (2008b). Specimens examined in this study varied greatly in size from relatively large RMNH Coel 40567 (height 28–31 mm, width = 6–7 mm, n=2 polyps) to relatively small RMNH Coel 40473 (height average 10.6 mm, width average 2.9 mm, n=7 pol- yps). However, Isaurus polyps are known to vary greatly in size both between different colonies and within large colonies (Larson and Larson 1982; Muirhead and Ryland 1985; Reimer et al. 2008b). Furthermore, the two other valid Pacific Isaurus spp. asides from I. tuberculatus are both very distinct from these specimens, and found in Fiji and southwestern Australia, respectively. Thus, the identity of these specimens as I. tuberculatus is largely certain. Distribution. Regions recorded in this study (Figure 5). Moluccas (14), Lembeh Strait (17). Distribution. Regions recorded in this study (Figure 5). Moluccas (14), Lembeh Strait (17). Distribution. Regions recorded in this study (Figure 5). Moluccas (14), Lembeh Strait (17). Previous records. Originally described from the West Indies, this species is dis- tributed throughout the subtropical and tropical Atlantic and Indo-Pacific (e.g. Muir- head and Ryland 1985), although populations in each ocean basin likely constitute different species (Reimer et al. 2008a). In the Indo-Pacific, it has previously been reported from the Great Barrier Reef, Fiji, Hawaii (summarized in Muirhead and Ry- land 1985), and also from Indonesia (Sinniger et al. 2005), New Caledonia (Laboute and Richer de Forges 2004), and Japan (Reimer et al. 2008b). Previous records. Originally described from the West Indies, this species is dis- tributed throughout the subtropical and tropical Atlantic and Indo-Pacific (e.g. Muir- head and Ryland 1985), although populations in each ocean basin likely constitute different species (Reimer et al. 2008a). In the Indo-Pacific, it has previously been reported from the Great Barrier Reef, Fiji, Hawaii (summarized in Muirhead and Ry- land 1985), and also from Indonesia (Sinniger et al. 2005), New Caledonia (Laboute and Richer de Forges 2004), and Japan (Reimer et al. 2008b). Remarks. As seen in previous studies (Reimer et al. 4. Isaurus tuberculatus Gray, 1828 Figures 4A, B, 5 Specimens examined (n=3). RMNH Coel 40472, Rumphius Biohistorical Expedi- tion station 27, Leitimur, south coast, Hutumuri, Ambon Bay, Moluccas (03°41'50"S, 128°17'00"E), intertidal under stones, collected on November 27, 1990 by J.C. den Hartog; RMNH Coel 40473, Rumphius Biohistorical Expedition station 27, Leit- imur, south coast, Hutumuri, Ambon Bay, Moluccas (03°41'50"S, 128°17'00"E), in- tertidal under stones, collected on November 27, 1990 by J.C. den Hartog; RMNH Coel 40567, Fauna Malesiana Marine Sulawesi Expedition station SUL.04, bay south of Pulau Putus, Lembeh Strait, North Sulawesi (01°31'N, 125°16'E), depth approx. 1 to 2 m, on October 27, 1994 by J.C. den Hartog. Previous records. NA.h Previous records. NA.h Remarks. This designation simply consists of all Zoanthus spp. specimens that could not be identified to species-level. It is likely this designation includes more than one species based on depths of specimens sampled. However, as preserved speci- mens were contracted (polyps closed) and many described Zoanthus spp. present no readily diagnostic external characters, identification to species level is not potentially possible without detailed molecular examination. Attempts at molecular identifica- tion also failed for these (and most other specimens), perhaps due to initial preserva- tion in 10% seawater formalin for older specimens, or in ethanol with additives for newer specimens. Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 15 Genus Isaurus Gray, 1828 4. Isaurus tuberculatus Gray, 1828 Figures 4A, B, 5 y Photographic records. NA. 2008b), it appears from the low numbers of specimens here that Isaurus is either somewhat rare throughout its range, or cryptic in nature (e.g. well-camouflaged), resulting in few reports of this species. 16 James D. Reimer et al. / ZooKeys 444: 1–57 (2014) Figure 4. Images of Isaurus and Neozoanthus species from specimens and photographic records in this study. A Isaurus tuberculatus specimen RMNH Coel 40567 from Fauna Malesiana Marine Sulawesi Expedition sta- tion SUL.04, bay south of Pulau Putus, Lembeh Strait, North Sulawesi, depth approx. 1 to 2 m, on October 27, 1994 by J.C. den Hartog B I. tuberculatus specimen RMNH Coel 40472 from Rumphius Biohistorical Expedition station 27, Leitimur, south coast, Hutumuri, Ambon Bay, Moluccas, intertidal under stones, col- lected on November 27, 1990 by J.C. den Hartog C Neozoanthus sp. at station WAK.13, southwest tip of To- landono Island, REA Wakatobi National Park, Wakatobi, Southeast Sulawesi, on May 9, 2003; and D Neozo- anthus sp. at Lembongan Bay, Nusa Lembongan, Lombok Strait, on May 19, 1998. Scales in A and B 1 cm. Figure 4. Images of Isaurus and Neozoanthus species from specimens and photographic records in this study. A Isaurus tuberculatus specimen RMNH Coel 40567 from Fauna Malesiana Marine Sulawesi Expedition sta- tion SUL.04, bay south of Pulau Putus, Lembeh Strait, North Sulawesi, depth approx. 1 to 2 m, on October 27, 1994 by J.C. den Hartog B I. tuberculatus specimen RMNH Coel 40472 from Rumphius Biohistorical Expedition station 27, Leitimur, south coast, Hutumuri, Ambon Bay, Moluccas, intertidal under stones, col- l d N b b J C d H C N h WAK h f T Figure 4. Images of Isaurus and Neozoanthus species from specimens and photographic records in this study. A Isaurus tuberculatus specimen RMNH Coel 40567 from Fauna Malesiana Marine Sulawesi Expedition sta- tion SUL.04, bay south of Pulau Putus, Lembeh Strait, North Sulawesi, depth approx. 1 to 2 m, on October 27, 1994 by J.C. den Hartog B I. tuberculatus specimen RMNH Coel 40472 from Rumphius Biohistorical Expedition station 27, Leitimur, south coast, Hutumuri, Ambon Bay, Moluccas, intertidal under stones, col- lected on November 27, 1990 by J.C. den Hartog C Neozoanthus sp. at station WAK.13, southwest tip of To- landono Island, REA Wakatobi National Park, Wakatobi, Southeast Sulawesi, on May 9, 2003; and D Neozo- anthus sp. at Lembongan Bay, Nusa Lembongan, Lombok Strait, on May 19, 1998. y Photographic records. NA. Scales in A and B 1 cm. Family Neozoanthidae Herberts, 1972 Genus Neozoanthus Herberts, 1972 5. Neozoanthus sp. Figures 4C, D, 5 Specimen regions. NA. Specimen regions. NA. / ZooKeys 444: 1–57 (2014) 18 Remarks. This genus was originally described from Madagascar with the type spe- cies N. tulearensis Herberts, 1972. Subsequently, two species have been reported from Australia and Japan (Reimer et al. 2012a). As no specimens exist, it is impossible to determine if the Indonesian photographs constitute one or both of the species reported in Reimer et al. (2012a), or an as of yet undescribed species. Specimen regions. NA. p g Specimens examined. NA. Photographic records (n=8). Gili Selang, eastern Bali (08°23'55"S, 115°42'30"E), on June 3, 1998; Lembongan Bay, Nusa Lembongan, Lombok Strait (08°40'25"S, Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 17 Figure 5. Distribution of Isaurus and Neozoanthus species from specimens and photographic records from this study. Isaurus tuberculatus specimens in red, and Neozoanthus sp. in green. Region numbers cor- respond to locations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. Figure 5. Distribution of Isaurus and Neozoanthus species from specimens and photographic records from this study. Isaurus tuberculatus specimens in red, and Neozoanthus sp. in green. Region numbers cor- respond to locations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. 115°26'18"E), on May 19, 26, 27, 29, 1998 (4 records); Tanjung Taal, Nusa Lembon- gan, Lombok Strait (08°39'33"S, 115°26'37"E), on May 25, 1998; station WAK.22, north channel pass of Karang Koromaha, REA Wakatobi National Park, Wakatobi, Southeast Sulawesi (05°42'54"S, 124°10'53"E), on May 12, 2003; station WAK.13, southwest tip of Tolandono Island, REA Wakatobi National Park, Wakatobi, South- east Sulawesi (05°46'35"S, 123°53'38"E), on May 9, 2003. 115°26'18"E), on May 19, 26, 27, 29, 1998 (4 records); Tanjung Taal, Nusa Lembon- gan, Lombok Strait (08°39'33"S, 115°26'37"E), on May 25, 1998; station WAK.22, north channel pass of Karang Koromaha, REA Wakatobi National Park, Wakatobi, Southeast Sulawesi (05°42'54"S, 124°10'53"E), on May 12, 2003; station WAK.13, southwest tip of Tolandono Island, REA Wakatobi National Park, Wakatobi, South- east Sulawesi (05°46'35"S, 123°53'38"E), on May 9, 2003. y Description. Unique among zoantharians, species in this genus have an endoder- mal sphincter with brachycnemic mesentery arrangement. Polyps are only partially incrusted, with the oral end of polyps lacking incrustation (Figures 4C, D). Phyloge- netically, this genus is closely related to Isaurus (within family Zoanthidae). Zooxan- thellate. Adapted from Herberts (1972), Reimer et al. (2012a). Distribution. Regions recorded in this study (Figure 5). Eastern Bali (5), Sper- monde Archipelago (9), Tukang Besi Islands (12), Moluccas (14). Previous records. Species of this genus have been reported from Madagascar (Herberts 1972), southern Japan (Reimer et al. 2011a, 2012a, 2013b), and the south- ern Great Barrier Reef (Reimer et al. 2011a, 2012a). James D. Reimer et al. Photographic records (n=2). Main coast, West Bali (08°06'50"S, 114°30'40"E), May 22, 1998; west Pulau Bone Batang, South Sulawesi, Spermonde Archipelago (05°01'00"S, 119°19'15"E), October 22, 1997. 6. Palythoa cf. mutuki (Haddon & Shackleton, 1891b) Figures 6A, B, 7 Although all specimens in this grouping match with previously reported P. mu- tuki based on sizes (average polyp height 9.6 mm, range 3–31 mm, average width 4.8 mm, range 2–8 mm, n=12 specimens) and overall morphology (‘intermediae’ or ‘liberae’ [Pax 1910]; visible capitulary ridges on closed polyps [Ryland and Lan- caster 2003]) (Figure 6B), we have identified all specimens in this study as “cf.”. Recent work has shown the presence of more than two closely related species groups within P. mutuki (Reimer et al. 2006b, 2013a) that are exceedingly difficult to distinguish without molecular data. For this reason, we have preliminarily assigned “cf.” to these specimens. Distribution. Regions recorded in this study (Figure 7). West Bali (4), eastern Bali (5), Komodo Island (8), Spermonde Archipelago (9), Taka Bone Rate (11), Mai- sel Islands (13), Moluccas (14), Cebu (21). Distribution. Regions recorded in this study (Figure 7). West Bali (4), eastern Bali (5), Komodo Island (8), Spermonde Archipelago (9), Taka Bone Rate (11), Mai- sel Islands (13), Moluccas (14), Cebu (21). Previous records. Ryland and Lancaster (2003) in their treatment of P. mutu- ki also mentioned records from Fiji, and synonymized records of other species from Tuvalu (Gemmaria willeyi Hill & Whitelegge, 1898), eastern Australia (G. arenacea Wilsmore, 1909; P. yongei Carlgren, 1937; P. australiensis Carlgren, 1950) and Singa- pore (P. singaporensis Pax & Müller, 1956) with this species. However, asides from the specimens directly examined by Ryland and Lancaster, there is much confusion over the true identity of these species. For example, Ryland and Lancaster (2003) them- selves state that G. willeyi is likely a Zoanthus species based on the figures in the original description. Ryland and Lancaster state “Probably only the use of genetic methods, so successfully applied by Burnett et al. (1997), will settle identities over wide geographic areas”. However, in the Pacific, records of this species with phylogenetic confirmation have previously been reported from the Great Barrier Reef in Australia (Burnett et al. 1997), Singapore (Reimer and Todd 2009), to the south Pacific coast of Japan (e.g. Reimer et al. 2006b, 2007b), New Caledonia (Sinniger 2006), and across to the Galapagos (Reimer and Hickman 2009), and thus it is known that this species has a very wide Indo-Pacific distribution. i Remarks. This species is likely common in Indonesia as in other regions such as Okinawa (Irei et al. 2011) and Taiwan (Reimer et al. 2011c). 6. Palythoa cf. mutuki (Haddon & Shackleton, 1891b) Figures 6A, B, 7 Specimens examined (n=13): RMNH Coel 40458, harbor pier, Cebu City, Cebu, Philip- pines, collected in 1976 by M.L. Esmeno; RMNH Coel 40459, harbor pier, Cebu City, Cebu, Philippines, collected in 1976 by M.L. Esmeno; RMNH Coel. 40468, Rumphius Biohistorical Expedition station 29, Hitu, Ambon Bay, Ambon, Moluccas (03°38'05"S, 128°12'36"E), depth = intertidal, collected on November 28, 1990 by M.S.S. Lavaleye; RMNH Coel. 40470, Rumphius Biohistorical Expedition station 4, Leitimur, outer Am- bon Bay, Wainitu, Moluccas (03°42'10"S, 128°09'15"E), depth = littoral on old shipwreck, collected on November 7–8, 1990 by H. Strack; RMNH Coel. 40475, Rumphius Biohis- torical Expedition station 27, Leitimur, south coast, Hutumuri, Moluccas (03°41'50"S, 128°17'00"E), depth = intertidal, on November 26, 1990 by M.S.S. Lavaleye; RMNH Coel. 40514, Fauna Malesiana Maluku Expedition station MAL.15, Ambon Bay, south coast, cape west of Amahusu, Moluccas (03°44'S, 128°08'E), collected on November 16, 1996; RMNH Coel. 40528, Snellius–II Expedition station 4.096, northeast Komodo, Ko- modo (08°29'S, 119°34'E), depth = to 30 m, collected on October 26, 1984; RMNH Coel 40532, NNM–LIPI–WWF Bali–Lombok Strait 2001 Expedition station BAL.09, Loloan Batu Agung, Sanur, eastern Bali (08°43'31"S, 115°15'57"E), depth = 10 to 15 m, collected on April 3, 2001 by B.W. Hoeksema; RMNH Coel. 40540, Snellius–II Expedition station 4.010, near Tawiri, Ambon Bay, Moluccas (03°42'S, 128°07'E), depth = 1 to 5 m, collect- ed on September 5, 1984; RMNH Coel. 40559, Snellius–II Expedition sta 4.012, north Pulau Mai, Maisel Islands, Banda Sea (05°28'S, 127°31'E), depth = 0 to 1.5 m, collected on 07.09.1984; RMNH Coel. 40561, Snellius–II Expedition station 4.133, east Pulau Tarupa Kecil, Taka Bone Rate (06°29'S, 121°08'E), depth = 11 m, collected on September 26, 1984; RMNH Coel. 40562, Snellius–II Expedition station 4.096, northeast Komodo, Komodo (08°29'S, 119°34'E), depth = to 30 m, collected on September 20, 1984; RMNH Coel. 40741, Rumphius Biohistorical Expedition station 11, Leitimur, Tanjung Nasaniwe, Moluccas (03°47'10"S, 128°05'20"E), depth = littoral, collected on November 12, 1990; Photographic records (n=2). Main coast, West Bali (08°06'50"S, 114°30'40"E), Photographic records (n=2). Main coast, West Bali (08°06'50"S, 114°30'40"E), May 22, 1998; west Pulau Bone Batang, South Sulawesi, Spermonde Archipelago (05°01'00"S, 119°19'15"E), October 22, 1997. Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 19 Description. Originally described from the Torres Strait, Australia, this species was redescribed in detail in Ryland and Lancaster (2003). Description. Originally described from the Torres Strait, Australia, this species was redescribed in detail in Ryland and Lancaster (2003). 6. Palythoa cf. mutuki (Haddon & Shackleton, 1891b) Figures 6A, B, 7 However, species de- lineation in Palythoa is confused due to the close phylogenetic relationships between P. mutuki, P. tuberculosa, and some other undescribed species, and a potential reticu- late evolutionary history (Reimer et al. 2007b, Shiroma and Reimer 2010, M. Miz- uyama and J.D. Reimer unpubl. data). Furthermore, distinguishing P. mutuki, from other, more distantly related species such as P. heliodiscus based solely on morphol- ogy is often difficult (Ryland and Lancaster 2003). For this study, we have included all “P. mutuki-like” specimens as one species group for convenience, although it is likely the specimens will encompass more than one species once the taxonomy of this genus is clarified. James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 20 Figure 6. Images of Palythoa cf. mutuki from specimens and photographic records in this study. A P. cf. mutuki at west Pulau Bone Batang, South Sulawesi, Spermonde Archipelago, October 22, 1997 B P. cf. mutuki at main coast, West Bali, May 22, 1998 C Palythoa sp. specimen RMNH Coel 40508, Fauna Malesiana Maluku Expedition station MAL.13, west coast near Larike, Ambon, Moluccas, depth = 3 m, collected on November 15, 1996; and D Palythoa sp. specimen RMNH Coel 40512, Pelabuhan Ratu, Southwest Java, collected on October 13, 1977, by P.H. van Doesburg. Scales in C and D 1 cm. Figure 6. Images of Palythoa cf. mutuki from specimens and photographic records in this study. A P. cf. mutuki at west Pulau Bone Batang, South Sulawesi, Spermonde Archipelago, October 22, 1997 B P. cf. mutuki at main coast, West Bali, May 22, 1998 C Palythoa sp. specimen RMNH Coel 40508, Fauna Malesiana Maluku Expedition station MAL.13, west coast near Larike, Ambon, Moluccas, depth = 3 m, collected on November 15, 1996; and D Palythoa sp. specimen RMNH Coel 40512, Pelabuhan Ratu, Southwest Java, collected on October 13, 1977, by P.H. van Doesburg. Scales in C and D 1 cm. 7. Palythoa sp. Figures 6C, D, 7 Specimens examined (n=2): RMNH Coel 40508, Fauna Malesiana Maluku Expedi- tion station MAL.13, west coast near Larike, Ambon, Moluccas (03°43'S, 127°56'E), depth = 3 m, collected on November 15, 1996; RMNH Coel 40512, Pelabuhan Ratu, southwest Java (07°01'N, 106°34'E), collected on October 13, 1977, by P.H. van Doesburg. 6. Palythoa cf. mutuki (Haddon & Shackleton, 1891b) Figures 6A, B, 7 Specimens examined (n=2): RMNH Coel 40508, Fauna Malesiana Maluku Expedi- tion station MAL.13, west coast near Larike, Ambon, Moluccas (03°43'S, 127°56'E), depth = 3 m, collected on November 15, 1996; RMNH Coel 40512, Pelabuhan Ratu, southwest Java (07°01'N, 106°34'E), collected on October 13, 1977, by P.H. van Doesburg. Photographic records. NA.h Description. This group consists of two specimens that do not clearly fit with previously described Palythoa species. Both specimens have dimensions very different from other Palythoa specimens reported here; whether this is due to unusual fixation or relaxation methods, or to true phenotypic differences is unknown. RMNH Coel 40508 (Figure 6C) has very long ‘liberae’ polyps (average 23.6 mm height, n=4 polyps) that are more robust (average 5 mm, n=4 polyps) than seen in P. heliodiscus, but with almost no development of the coenenchyme, unlike as in P. RMNH Coel 40508 (Figure 6C) has very long ‘liberae’ polyps (average 23.6 mm height, n=4 polyps) that are more robust (average 5 mm, n=4 polyps) than seen in P. heliodiscus, but with almost no development of the coenenchyme, unlike as in P. Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 21 Figure 7. Distribution of Palythoa species from specimens and photographic records from this study. Palythoa cf. mutuki specimens in red, Palythoa sp. in green, P. cf. heliodiscus in blue, P. aff. tuberculosa in yellow, and P. tuberculosa in pink. Region numbers correspond to locations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. Figure 7. Distribution of Palythoa species from specimens and photographic records from this study. Palythoa cf. mutuki specimens in red, Palythoa sp. in green, P. cf. heliodiscus in blue, P. aff. tuberculosa in yellow, and P. tuberculosa in pink. Region numbers correspond to locations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. mutuki or other closely related species. As well, this specimen is from 3 meters depth, a shallower depth than usually seen for P. heliodiscus. 6. Palythoa cf. mutuki (Haddon & Shackleton, 1891b) Figures 6A, B, 7 RMNH Coel 40512 (Figure 6D) is a small ‘intermediae’ colony consisting of four polyps that are squat and robust (average width 8.3 mm, n=3 polyps, height approxi- mately same as width) with large oral discs (average 12 mm in diameter, n=3 polyps) with no tentacles visible and a large oral opening. Distribution. Regions recorded in this study (Figure 7). Southwest Java (2), Moluccas (14). Previous records. NA.h Previous records. NA.h Remarks. The morphology of these specimens do not clearly match any described species from the central Indo-Pacific. In particular, specimen RMNH Coel 40512 is different than any other zoantharian previously observed by the first author. However, it is unknown if fixation has resulted in degradation of fine scale structures (e.g. tenta- cles, which are absent), but the specimen is clearly a zoantharian due to sand encrusta- tion in body wall. James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 22 8. Palythoa cf. heliodiscus (Ryland & Lancaster, 2003) Figures 7, 8 Specimens examined (n=2). RMNH Coel 40504, Fauna Malesiana Maluku Expedi- tion station MAL.12, north coast near Morela, Ambon, Moluccas (03°33'S, 128°12'E), depth = 35 m, collected on November 13, 1996; RMNH Coel. 40513, Rumphius Biohistorical Expedition station 24, south Seri Bay, Ambon, Moluccas (03°34'50"S, 128°09'45"E), depth = 12 m, November 22, 1990. p Photographic records (n=13). Pulau Ular, off Padang, West Sumatra (01°07'05"S, 100°20'02"E), December 16, 1996; Pemuteran, West Bali (08°11'20"S, 114°50'30"E), May 23, 1998; Tulamben, eastern Bali (08°16'26"S, 115°35'28"E), July 12, 1997; Nusa Lembongan, Lombok Strait (08°40'S, 115°26'E), May 29, 1998; west side Pulau Samalona, Spermonde Archipelago, South Sulawesi (05°07'25"S, 119°20'10"E), November, 1984; northwest side Pulau Samalona, Spermonde Ar- chipelago, South Sulawesi (05°07'25"S, 119°20'10"E), November 23, 1997; north- west Kudingareng Keke, Spermonde Archipelago, South Sulawesi (05°06'15"S, 119°17'10"E), August 6, 1997; west side Pulau Badi, Spermonde Archipelago, South Sulawesi (04°58'06"S, 119°16'57"E), November 1, 1994; REA Wakatobi National Park station WAK.18, southwest Pulau Binongko, Southeast Sulawesi, Wakatobi, Tukang Besi Islands (05°59'48"S, 124°02'55"E), May 10, 2003; REA Wakatobi Na- tional Park station WAK.22, north channel pass of Karang Koromaha, Southeast Sulawesi, Wakatobi, Tukang Besi Is. (05°42'54"S, 124°10'53"E), May 12, 2003; Fauna Malesiana Maluku Expedition station MAL.12, north coast near Morela, Am- bon (03°33'S, 128°12'E), November 13–14, 1996; East Kalimantan–Berau Expedi- tion station BER.03, south side of Pulau Derawan, East Kalimantan (02°17'03"N, 118°14'49"E), October 16, 2003; Christensen Research Institute, Madang, Papua New Guinea (05°09'30"S, 145°48'10"E), June 1992. Description. This zooxanthellate species was described in detail recently by Ry- land and Lancaster (2003). Superficially similar in appearance to P. mutuki, externally the species can be distinguished by its short tentacles (length <20% of oral disk) and subtidal distribution, compared to primarily intertidal P. mutuki, which also has long- er tentacles (~45% of oral disk) (Ryland and Lancaster 2003). Sizes of specimens agree well with specimens seen in other localities (average polyp heights 11.3 mm and 17.0 mm for each specimen, range 7–20 mm; average width 3.9 mm and 4.4 mm for each specimen, range 3.5–5.5 mm; n=2 specimens of 8 and 5 polyps, respectively). Depth of collected specimens (12 and 35 m) also fits well with the description of this species as primarily subtidal in the original description, and from data in Okinawa, Japan (e.g. Reimer 2010). p g Distribution. Regions recorded in this study (Figure 7). West Bali (4), eastern Bali (5), Spermonde Archipelago (9), Tukang Besi Islands (12), Moluccas (14). 8. Palythoa cf. heliodiscus (Ryland & Lancaster, 2003) Figures 7, 8 p g Distribution. Regions recorded in this study (Figure 7). West Bali (4), eastern Bali (5), Spermonde Archipelago (9), Tukang Besi Islands (12), Moluccas (14). Previous records. Palythoa heliodiscus has been reported from Australia (Ryland and Lancaster 2003) and is likely widespread in the Indo-Pacific (Ryland and Lancaster Previous records. Palythoa heliodiscus has been reported from Australia (Ryland and Lancaster 2003) and is likely widespread in the Indo-Pacific (Ryland and Lancaster Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 23 Figure 8. Images of Palythoa cf. heliodiscus from photographic records in this study. A P. cf. heliodiscus at the northwest side of Pulau Samalona, Spermonde Archipelago, South Sulawesi, November 23, 1997 B P. cf. heliodiscus at the south side of Pulau Derawan, East Kalimantan, October 16, 2003 C P. cf. heliodiscus at REA Wakatobi National Park station WAK.22, north channel pass of Karang Koromaha, Southeast Sulawesi, Wakatobi, Tukang Besi Is., May 12, 2003; and D P. cf. heliodiscus at REA Wakatobi National Park station WAK.18, Southwest Pulau Binongko, Southeast Sulawesi, Wakatobi, Tukang Besi Islands, May 10, 2003. Figure 8. Images of Palythoa cf. heliodiscus from photographic records in this study. A P. cf. heliodiscus at the northwest side of Pulau Samalona, Spermonde Archipelago, South Sulawesi, November 23, 1997 B P. cf. heliodiscus at the south side of Pulau Derawan, East Kalimantan, October 16, 2003 C P. cf. heliodiscus at REA Wakatobi National Park station WAK.22, north channel pass of Karang Koromaha, Southeast Sulawesi, Wakatobi, Tukang Besi Is., May 12, 2003; and D P. cf. heliodiscus at REA Wakatobi National Park station WAK.18, Southwest Pulau Binongko, Southeast Sulawesi, Wakatobi, Tukang Besi Islands, May 10, 2003. 2003 and references within), as well as Japan (Reimer et al. 2006b), Palau (Reimer et al. 2014a), while P. toxica Walsh & Bowers, 1971 has been reported from Hawai’i. Remarks. We have identified all specimens here as “cf.” as in situ images (Figure 8) there are two different morphotypes. One morphotype matches with Palythoa heliodiscus, with a brown oral disk with no patterns (Figures 8A, B), while the other morphotype’s polyps have either green or purple oral disks with various semi-irregular patterns, as well as blue/gray or light orange tentacles (Figures 8C, D). Based on data from Okinawa and Australia, both of these morphotypes are almost identical asides from the oral disk colora- tion and small but consistent differences in ITS–rDNA (T. Nishimura and J.D. g Previous records: NA.h Remarks. This specimen is unlike any other previous specimen observed in the field or museums by the first author. Unfortunately, as it was collected in 1930, at- tempts to acquire utilizable DNA sequences able to distinguish this specimen’s affinity were unsuccessful, and identification was made on gross morphology alone. 8. Palythoa cf. heliodiscus (Ryland & Lancaster, 2003) Figures 7, 8 Reimer, unpubl. data) that may be either intraspecific or interspecific. Thus, it is still uncertain if the green/purple morphotype is an undescribed species or not (Reimer et al. 2014a). Furthermore, the overall morphology of the green/purple morphotype closely re- sembles P. toxica from Hawai’i, and whether these Indonesian specimens are P. toxica or P. heliodiscus, and if these two species are synonyms needs to be ascertained before any formal description occurs. In situ images and further DNA sequences are therefore needed from future specimens. James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 24 9. Palythoa aff. tuberculosa (Esper, 1805) Figures 7, 9A Specimens examined (n=1). RMNH Coel 40521, Snellius Expedition, Pulau Haroe- koe, east of Ambon, Ambon, Moluccas, collected on May 03–07, 1930. Description. This specimen superficially resembles zooxanthellate Palythoa sp. yoron sensu Shiroma and Reimer (2010) with its very well developed coenenchyme and ‘intermediae–immersae’ morphology (Figure 9B). However, there are some differ- ences between this specimen and P. sp. yoron from Okinawa. The current specimen consists of two large portions of colonies consisting of >50 polyps, while P. sp. yoron usually is found in very small colonies of <10 polyps. As well, P. sp. yoron consists of a very well developed coenenchyme from which all individual polyps partially emerge, while the current specimen appears to consist more of large robust polyps that have merged together at many locations, but not at others, giving the specimen the ap- pearance of P. tuberculosa from the top, and often of P. mutuki from side angles. On the other hand, P. sp. yoron has an appearance, although intermediate between P. tuberculosa and P. mutuki, unique to and of itself. Polyps’ height (when not merged) is approximately 7.0 mm, and average width is 7.3 mm (n=10 polyps). Thus, for now, this specimen is identified as P. aff. tuberculosa. For details on P. tuberculosa, refer to the relevant species section below. p Distribution. Regions recorded in this study (Figure 7): Moluccas (14). Previous records: NA.h p Distribution. Regions recorded in this study (Figure 7): Moluccas (14). Previous records: NA. p Distribution. Regions recorded in this study (Figure 7): Moluccas (14). 10. Palythoa tuberculosa Esper, 1805 Figures 7, 9B 8 m, September 10, 1984; RMNH Coel 40530, Snellius–II Expedi- tion station 4.071, Slawi Bay, east Komodo, Komodo (08°34'30"S, 119°31'18"E), depth sublittoral, collected on September 17, 1984; RMNH Coel 40531, Snellius–II Expedition station 4.030, west coast of Pulau Binongko, Southeast Sulawesi, Tukang Besi Islands, Wakatobi (05°55'S, 123°59'E), depth approx. 3 to 4 m, September 10, 1984; RMNH Coel 40534, Snellius–II Expedition station 4.169, reef north of Pulau Bahuluang, Southwest Salayer, Salayer Island, South Sulawesi (06°27'S, 120°26'E), collected on September 30, 1984; RMNH Coel 40535, Snellius–II Expedition station 4.059, off Melolo, northeast Sumba (09°52'30"S, 120°40'18"E), collected on Sep- tember 14, 1984; RMNH Coel 40541, Snellius–II Expedition station 4.006, Ambon Bay near Eri, Ambon, Moluccas (03°45'S, 128°08'E), depth approx. 3 m, collected on August 29, 1984; RMNH Coel 40543, Snellius–II Expedition station 4.006, Am- bon Bay near Eri, Ambon, Moluccas (03°45'S, 128°08'E), depth = 0 to 10 m, col- lected on August 29, 1984; RMNH Coel 40548, Snellius–II Expedition station 4.052, east of Melolo, northeast Sumba (09°55'S, 120°45'E), depth approx. 3 m, collected on September 13, 1984; RMNH Coel 40552, Snellius–II Expedition station 4.048, east of Melolo, northeast Sumba (09°54'00"S, 120°43'30"E), depth = 12 m, collected on September 14, 1984; RMNH Coel 40553, Snellius–II Expedition station 4.096, northeast cape, Komodo (08°29'S, 119°34'E), depth to 30 m, collected on Septem- ber 20, 1984; RMNH Coel 40555, Snellius–II Expedition station 4.096, northeast cape, Komodo (08°29'S, 119°34'E), depth to 30 m, collected on September 20, 1984; RMNH Coel 40557 Snellius–II Expedition station 4 096 northeast cape Komo- tion station 27, Leitimur, south coast, Hutumuri, Ambon, Moluccas (03°41'50"S, 128°17'00"E), depth = 1 to 3 m, collected on November 27, 1990 by J.C. den Hartog; RMNH Coel 40505, south side of Barang Lompo, Spermonde Archipelago, South Sulawesi (05°03'23"S, 119°19'45"E), depth = 18 m, collected on October 18, 1980, by H. Moll; RMNH Coel 40511, west side of Pulau Samalona, Spermonde Archi- pelago, South Sulawesi (05°07'25"S, 119°20'10"E), depth = 2.5 m, collected on Sep- tember 4, 1980 by H. Moll; RMNH Coel. 40517, west side of Pulau Samalona, Sper- monde Archipelago, South Sulawesi (05°07'25"S, 119°20'10"E), depth = 2.5 m, col- lected on September 4, 1980 by H. 10. Palythoa tuberculosa Esper, 1805 Figures 7, 9B Specimens examined (n=31). RMNH Coel 40465, Rumphius Biohistorical Ex- pedition station 11, Leitimur, Tanjung Nasaniwe, Ambon, Moluccas (03°47'10"S, 128°05'20"E), depth = 2–5 m, collected on November 12, 1990; RMNH Coel 40466, Rumphius Biohistorical Expedition station 30, Hitu, Baguala Bay, Suli, Ambon, Mo- luccas (03°37'40"S, 128°17'50"E), collected on November 29, 1990; RMNH Coel 40467, Rumphius Biohistorical Expedition station 15, Hitu, Baguala Bay, 0.5 km west of Tial, Ambon, Moluccas (03°38'20"S, 128°19'40"E), depth = 2 m, collected on November 13–14, 1990; RMNH Coel 40471, Rumphius Biohistorical Expedition station 4, Leitimur, Ambon Bay, outer bay, Wainitu (near Ambon City), Ambon, Moluccas (03°42'10"S, 128°09'15"E), littoral on old shipwreck, collected on Novem- ber 7–8, 1990 by H. Strack; RMNH Coel 40474, Rumphius Biohistorical Expedi- Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 25 ( ) f fi tion station 27, Leitimur, south coast, Hutumuri, Ambon, Moluccas (03°41'50"S, 128°17'00"E), depth = 1 to 3 m, collected on November 27, 1990 by J.C. den Hartog; RMNH Coel 40505, south side of Barang Lompo, Spermonde Archipelago, South Sulawesi (05°03'23"S, 119°19'45"E), depth = 18 m, collected on October 18, 1980, by H. Moll; RMNH Coel 40511, west side of Pulau Samalona, Spermonde Archi- pelago, South Sulawesi (05°07'25"S, 119°20'10"E), depth = 2.5 m, collected on Sep- tember 4, 1980 by H. Moll; RMNH Coel. 40517, west side of Pulau Samalona, Sper- monde Archipelago, South Sulawesi (05°07'25"S, 119°20'10"E), depth = 2.5 m, col- lected on September 4, 1980 by H. Moll; RMNH Coel 40519, Snellius Expedition, Rumah Fija, Bo Islands, Halmahera Sea, collected on October 7, 1930; RMNH Coel 40522, Snellius Expedition, Sulu Islands, Philippines, collected on September 11–17, 1930; RMNH Coel 40523, Snellius Expedition, probably Indonesia, no locality data; RMNH Coel 40524, Snellius–II Expedition station 4.011, reef edge west of Mai, Maisel Islands, Banda Sea (05°28'S, 127°31'E), depth = 1–30 m, collected on Sep- tember 7, 1984; RMNH Coel 40526, Snellius–II Expedition station 4.030, west coast of Pulau Binongko, Southeast Sulawesi, Tukang Besi Islands, Wakatobi (05°55'S, 123°59'E), depth approx. 2 m, September 10, 1984; RMNH Coel 40527, Snellius–II Expedition station 4.030, west coast of Pulau Binongko, Southeast Sulawesi, Tukang Besi Islands, Wakatobi (05°55'S, 123°59'E), depth approx. 0.5 m, September 10, 1984; RMNH Coel 40529, Snellius–II Expedition station 4.030, west coast of Pulau Binongko, Southeast Sulawesi, Tukang Besi Islands, Wakatobi (05°55'S, 123°59'E), depth approx. 10. Palythoa tuberculosa Esper, 1805 Figures 7, 9B / ZooKeys 444: 1–57 (2014) 26 do (08°29'S, 119°34'E), depth = “shallow water”, collected on September 20, 1984; RMNH Coel 40568, northwest of Pulau Kapoposang, Spermonde Archipelago, South Sulawesi (04°41'40"S, 118°54'55"E), collected on May 2, 1998 by B.W. Hoeksema; RMNH Coel 40769, Snellius Expedition, Eude, South Flores, collected on March 6–8, 1930; RMNH Coel 40770, Snellius Expedition, Maratua, Berau Islands, East Kalimantan, collected on October 14–17, 1930; RMNH Coel 40771, Snellius Expe- dition, Maratua, Berau Islands, East Kalimantan, collected on October 14–17, 1930; RMNH Coel 40772, Snellius–II Expedition station 4.006, Ambon Bay near Eri, Am- bon, Moluccas (03°45'S, 128°08'E), depth = 0 to 10 m, collected on August 29, 1984. do (08°29'S, 119°34'E), depth = “shallow water”, collected on September 20, 1984; RMNH Coel 40568, northwest of Pulau Kapoposang, Spermonde Archipelago, South Sulawesi (04°41'40"S, 118°54'55"E), collected on May 2, 1998 by B.W. Hoeksema; RMNH Coel 40769, Snellius Expedition, Eude, South Flores, collected on March 6–8, 1930; RMNH Coel 40770, Snellius Expedition, Maratua, Berau Islands, East Kalimantan, collected on October 14–17, 1930; RMNH Coel 40771, Snellius Expe- dition, Maratua, Berau Islands, East Kalimantan, collected on October 14–17, 1930; RMNH Coel 40772, Snellius–II Expedition station 4.006, Ambon Bay near Eri, Am- bon, Moluccas (03°45'S, 128°08'E), depth = 0 to 10 m, collected on August 29, 1984. Photographic records (n=12). Pemuteran, West Bali (08°08'S, 114°41'E), May 20, 1998; Pemuteran, West Bali (08°08'S, 114°41'E), May 23, 1998; Napoleon Reef, West Bali (08°08'S, 114°41'E), May 20, 1998; Nusa Lembongan, Lombok Strait, East Bali, July 13, 1997; Nusa Lembongan, Lombok Strait, east Bali, July 19, 1997; Nusa Lembongan, Lombok Strait, east Bali, May 26, 1998; south of Pulau Samalona, Sper- monde Archipelago, South Sulawesi (05°07'45"S, 119°20'25"E), October 27, 1997; northwest Pulau Samalona, Spermonde Archipelago, South Sulawesi (05°07'25"S, 119°20'10"E), November 25, 1997; Fauna Malesiana Maluku Expedition station MAL.12, north coast near Morela, Ambon, Moluccas November 13, 1996; North Sulawesi, Bunaken, (01°36'N, 124°47'E), April 9, 1996; Cebu, Philippines, Novem- ber 21, 1998; Madang, Papua New Guinea, June 1992. Photographic records (n=12). 10. Palythoa tuberculosa Esper, 1805 Figures 7, 9B Moll; RMNH Coel 40519, Snellius Expedition, Rumah Fija, Bo Islands, Halmahera Sea, collected on October 7, 1930; RMNH Coel 40522, Snellius Expedition, Sulu Islands, Philippines, collected on September 11–17, 1930; RMNH Coel 40523, Snellius Expedition, probably Indonesia, no locality data; RMNH Coel 40524, Snellius–II Expedition station 4.011, reef edge west of Mai, Maisel Islands, Banda Sea (05°28'S, 127°31'E), depth = 1–30 m, collected on Sep- tember 7, 1984; RMNH Coel 40526, Snellius–II Expedition station 4.030, west coast of Pulau Binongko, Southeast Sulawesi, Tukang Besi Islands, Wakatobi (05°55'S, 123°59'E), depth approx. 2 m, September 10, 1984; RMNH Coel 40527, Snellius–II Expedition station 4.030, west coast of Pulau Binongko, Southeast Sulawesi, Tukang Besi Islands, Wakatobi (05°55'S, 123°59'E), depth approx. 0.5 m, September 10, 1984; RMNH Coel 40529, Snellius–II Expedition station 4.030, west coast of Pulau Binongko, Southeast Sulawesi, Tukang Besi Islands, Wakatobi (05°55'S, 123°59'E), depth approx. 8 m, September 10, 1984; RMNH Coel 40530, Snellius–II Expedi- tion station 4.071, Slawi Bay, east Komodo, Komodo (08°34'30"S, 119°31'18"E), depth sublittoral, collected on September 17, 1984; RMNH Coel 40531, Snellius–II Expedition station 4.030, west coast of Pulau Binongko, Southeast Sulawesi, Tukang Besi Islands, Wakatobi (05°55'S, 123°59'E), depth approx. 3 to 4 m, September 10, 1984; RMNH Coel 40534, Snellius–II Expedition station 4.169, reef north of Pulau Bahuluang, Southwest Salayer, Salayer Island, South Sulawesi (06°27'S, 120°26'E), collected on September 30, 1984; RMNH Coel 40535, Snellius–II Expedition station 4.059, off Melolo, northeast Sumba (09°52'30"S, 120°40'18"E), collected on Sep- tember 14, 1984; RMNH Coel 40541, Snellius–II Expedition station 4.006, Ambon Bay near Eri, Ambon, Moluccas (03°45'S, 128°08'E), depth approx. 3 m, collected on August 29, 1984; RMNH Coel 40543, Snellius–II Expedition station 4.006, Am- bon Bay near Eri, Ambon, Moluccas (03°45'S, 128°08'E), depth = 0 to 10 m, col- lected on August 29, 1984; RMNH Coel 40548, Snellius–II Expedition station 4.052, east of Melolo, northeast Sumba (09°55'S, 120°45'E), depth approx. 3 m, collected on September 13, 1984; RMNH Coel 40552, Snellius–II Expedition station 4.048, east of Melolo, northeast Sumba (09°54'00"S, 120°43'30"E), depth = 12 m, collected on September 14, 1984; RMNH Coel 40553, Snellius–II Expedition station 4.096, northeast cape, Komodo (08°29'S, 119°34'E), depth to 30 m, collected on Septem- ber 20, 1984; RMNH Coel 40555, Snellius–II Expedition station 4.096, northeast cape, Komodo (08°29'S, 119°34'E), depth to 30 m, collected on September 20, 1984; James D. Reimer et al. 10. Palythoa tuberculosa Esper, 1805 Figures 7, 9B Pemuteran, West Bali (08°08'S, 114°41'E), May 20, 1998; Pemuteran, West Bali (08°08'S, 114°41'E), May 23, 1998; Napoleon Reef, West Bali (08°08'S, 114°41'E), May 20, 1998; Nusa Lembongan, Lombok Strait, East Bali, July 13, 1997; Nusa Lembongan, Lombok Strait, east Bali, July 19, 1997; Nusa Lembongan, Lombok Strait, east Bali, May 26, 1998; south of Pulau Samalona, Sper- monde Archipelago, South Sulawesi (05°07'45"S, 119°20'25"E), October 27, 1997; northwest Pulau Samalona, Spermonde Archipelago, South Sulawesi (05°07'25"S, 119°20'10"E), November 25, 1997; Fauna Malesiana Maluku Expedition station MAL.12, north coast near Morela, Ambon, Moluccas November 13, 1996; North Sulawesi, Bunaken, (01°36'N, 124°47'E), April 9, 1996; Cebu, Philippines, Novem- ber 21, 1998; Madang, Papua New Guinea, June 1992. , ; g, p , J Description. This zooxanthellate species was originally described from India (Es- per 1805), and subsequently redescribed utilizing specimens from the Red Sea (Klun- zinger 1877). Recent work by Hibino et al. (2013) indicates the species may include some junior synonyms, and has a wide distribution across the subtropical and tropical Indo-Pacific. Polyps are embedded within a well-developed coenenchyme (‘immersae’, Pax 1910), and colonies vary in color from fluorescent green-yellow to dark brown or even ochre (Figure 9A). Specimens in this study averaged 4.7 mm in polyp diameter (n=29 specimens), ranging from 2 to 8 mm. One specimen, RMNH Coel 40553, was notable for its very small polyps (average diameter 2.4 mm, n=10 polyps). Other colonies ranged from 3.1 to 6.5 mm in average diameter, similar to previous reported sizes. All speci- mens were ‘immersae’. Generally, morphology fit well within the accepted range of P. tuberculosa (see Table 1 in Hibino et al. 2013), although some specimens’ polyps were somewhat smaller than previously observed. These smaller sizes may also be partly due to preservation methods. Description. This zooxanthellate species was originally described from India (Es- per 1805), and subsequently redescribed utilizing specimens from the Red Sea (Klun- zinger 1877). Recent work by Hibino et al. (2013) indicates the species may include some junior synonyms, and has a wide distribution across the subtropical and tropical Indo-Pacific. Polyps are embedded within a well-developed coenenchyme (‘immersae’, Pax 1910), and colonies vary in color from fluorescent green-yellow to dark brown or even ochre (Figure 9A). g Specimens in this study averaged 4.7 mm in polyp diameter (n=29 specimens), ranging from 2 to 8 mm. 10. Palythoa tuberculosa Esper, 1805 Figures 7, 9B One specimen, RMNH Coel 40553, was notable for its very small polyps (average diameter 2.4 mm, n=10 polyps). Other colonies ranged from 3.1 to 6.5 mm in average diameter, similar to previous reported sizes. All speci- mens were ‘immersae’. Generally, morphology fit well within the accepted range of P. tuberculosa (see Table 1 in Hibino et al. 2013), although some specimens’ polyps were somewhat smaller than previously observed. These smaller sizes may also be partly due to preservation methods. Distribution. Regions recorded in this study (Figure 7). West Bali (4), east Bali (5), northeast Sumba (6), south Flores (7), Komodo (8), Spermonde Archipelago (9), Salayer Island (10), Tukang Besi Islands (12), Maisel Islands (13), Moluccas (14), Bo Is- lands (15), Bunaken (18), Berau Islands (19), Sulu Islands (20), Cebu (21), Madang (22). Distribution. Regions recorded in this study (Figure 7). West Bali (4), east Bali (5), northeast Sumba (6), south Flores (7), Komodo (8), Spermonde Archipelago (9), Salayer Island (10), Tukang Besi Islands (12), Maisel Islands (13), Moluccas (14), Bo Is- lands (15), Bunaken (18), Berau Islands (19), Sulu Islands (20), Cebu (21), Madang (22).hi Previous records. This species has been phylogenetically confirmed as distributed over the entire subtropical and tropical Indo-Pacific, from at least the Red Sea to Sin- gapore (Reimer and Todd 2009), Taiwan (Reimer et al. 2011c), Japan (e.g. Reimer et al. 2006a), New Caledonia (Sinniger 2006), and the Galapagos Islands (Reimer and Hickman 2009). Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 27 ( , ) f fi 7 Figure 9. Images of Palythoa tuberculosa and P. aff. tuberculosa from specimens and photographic records in this study. A P. aff. tuberculosa specimen RMNH Coel 40521, Snellius Expedition, Sulu Islands, Philip- pines, collected on September 11–17, 1930; and B P. tuberculosa at Madang, Papua New Guinea, June 1992. Scale in A 1 cm. Figure 9. Images of Palythoa tuberculosa and P. aff. tuberculosa from specimens and photographic records in this study. A P. aff. tuberculosa specimen RMNH Coel 40521, Snellius Expedition, Sulu Islands, Philip- pines, collected on September 11–17, 1930; and B P. tuberculosa at Madang, Papua New Guinea, June 1992. Scale in A 1 cm. Figure 9. Images of Palythoa tuberculosa and P. aff. tuberculosa from specimens and photographic records in this study. A P. aff. tuberculosa specimen RMNH Coel 40521, Snellius Expedition, Sulu Islands, Philip- pines, collected on September 11–17, 1930; and B P. 11. Sphenopus marsupialis (Gmelin, 1791) Figures 10A, B, 11 Specimens examined (n=2). RMNH Coel 40506, East Kalimantan–Berau Expedition station BER.14, lighthouse northeast side of Pulau Panjang, Berau Islands, East Ka- limantan (02°23'14"N, 118°12'34"E), depth = 12 m, collected on October 09, 2003 by B.W. Hoeksema; RMNH Coel 40509, East Kalimantan–Berau Expedition station BER.01, east side of Pulau Derawan, Berau Islands, East Kalimantan (02°17'32"N, 118°15'43"E), depth = 14 m, collected on October 11, 2003 by B.W. Hoeksema. p y Photographic records (n=7). west Pulau Barang Caddi, Spermonde Archi- pelago, South Sulawesi (05°05'08"S, 119°18'55"E), October 06, 1997; east Bone Lola shoal, Spermonde Archipelago, South Sulawesi (05°03'15"S, 119°21'30"E), October 27, 1997; east Pulau Kudingareng Keke, Spermonde Archipelago, South Sulawesi (05°06'15"S, 119°17'35"E), September 17, 1997; north Pulau Kudingareng Keke, Spermonde Archipelago, South Sulawesi (05°06'07"S, 119°17'15"E), Octo- ber 1, 1997; station BER.01, east Pulau Derawan, East Kalimantan, Berau Islands (02°17'32"N, 118°15'43"E), October 11, 2003; station BER.14, lighthouse northeast Pulau Panjang Island, East Kalimantan, Berau Islands (02°23'14"N, 118°12'34"E), October 9, 2003; station BER.24, southeast Pulau Samama, East Kalimantan, Berau Islands (02°07'51"N, 118°20'23"E), October 15, 2003. Photographic records (n=7). west Pulau Barang Caddi, Spermonde Archi- pelago, South Sulawesi (05°05'08"S, 119°18'55"E), October 06, 1997; east Bone Lola shoal, Spermonde Archipelago, South Sulawesi (05°03'15"S, 119°21'30"E), October 27, 1997; east Pulau Kudingareng Keke, Spermonde Archipelago, South Sulawesi (05°06'15"S, 119°17'35"E), September 17, 1997; north Pulau Kudingareng Keke, Spermonde Archipelago, South Sulawesi (05°06'07"S, 119°17'15"E), Octo- ber 1, 1997; station BER.01, east Pulau Derawan, East Kalimantan, Berau Islands (02°17'32"N, 118°15'43"E), October 11, 2003; station BER.14, lighthouse northeast Pulau Panjang Island, East Kalimantan, Berau Islands (02°23'14"N, 118°12'34"E), October 9, 2003; station BER.24, southeast Pulau Samama, East Kalimantan, Berau Islands (02°07'51"N, 118°20'23"E), October 15, 2003. Description. The type species of the azooxanthellate genus Sphenopus, this species has an Indo-West Pacific distribution (Reimer et al. 2012b). Uniquely for the order, species in this genus are unitary (not colonial), and usually free-living, as they are not attached to substrate, and instead embedded in sand or loose gravel/substrate (Figures 10A, B). Individuals can often grow to large sizes (for zoantharians); up to several cm in both length and polyp diameter. Taxonomic examination of this genus is quite limited, with only two recent studies (Soong et al. 1999, Reimer et al. 2012b), both of which clearly state that further research is needed to more clearly understand this group. 10. Palythoa tuberculosa Esper, 1805 Figures 7, 9B tuberculosa at Madang, Papua New Guinea, June 1992. Scale in A 1 cm. Figure 9. Images of Palythoa tuberculosa and P. aff. tuberculosa from specimens and photographic records in this study. A P. aff. tuberculosa specimen RMNH Coel 40521, Snellius Expedition, Sulu Islands, Philip- pines, collected on September 11–17, 1930; and B P. tuberculosa at Madang, Papua New Guinea, June 1992. Scale in A 1 cm. James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 28 Remarks. It is highly likely this species is the senior synonym of P. caesia Dana, 1846 (Hibino et al. 2013), described from Fiji and commonly reported from Australia (Burnett et al. 1997). This species is also part of the P. tuberculosa–P. mutuki species complex (Reimer et al. 2007b, M. Mizuyama and J.D. Reimer unpubl. data). Distribution. Regions recorded in this study (Figure 11). Spermonde Archi- pelago (9), Berau Islands (19).h Previous records. This species has been reported from many locations in the Indo- West Pacific, including Taiwan (Soong et al. 1999) and Brunei Darussalam (Reimer et al. 2012b). Previous records. This species has been reported from many locations in the Indo- West Pacific, including Taiwan (Soong et al. 1999) and Brunei Darussalam (Reimer et al. 2012b). Remarks. Specimen RMNH Coel 40506 may be similar to a putative undescribed Sphenopus species mentioned in Soong et al. (1999) from Taiwan based on its smaller size. 11. Sphenopus marsupialis (Gmelin, 1791) Figures 10A, B, 11 Specimen RMNH Coel 40506 consists of seven polyps, with an average height of 24.4 mm (range 18.5 to 30 mm), and an average width of 8.4 mm (range 6 to 11 mm). The non-peduncle portions of the polyps are 15–20 mm in height, with the remainder made up of peduncle. Specimen RMNH Coel 40506 has some polyps (five of seven) somewhat different in morphology from RMNH Coel 40509 and other Naturalis S. marsupialis specimens from the Indian Ocean. These polyps have regularly spaced small round “tubercles” Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 29 (approx. 1 mm in diameter) on the upper half of their scapus arranged in vertical lines (n=8–14 vertical tubercle lines on each polyp, with 6–10 tubercles per line), making this portion of the polyp appear furrowed. As well, polyps have a small, stubby “pe- duncle” (2 to 5 mm in width) that is not attached to any hard substrate, intermediate between S. marsupialis with its completely rounded bottom end and S. pedunculatus with its long, attached peduncle. For now, we identify these specimens as S. marsupia- lis as their peduncles were not attached to the substrate, but it is clear more examina- tion of these specimens is needed. p Specimen RMNH Coel 40509 consists of two polyps of different sizes, with the smaller one being 16 by 5 mm, and the larger one 24 by 15 mm. Both polyps have no peduncle and are tapered. Both polyps are somewhat rugged on their outer sur- face, with no discernable tubercles, and have intermittent (=not one clear stripe) small darker vertical patterns in between the capitulary ridges only on the top 3–5 mm of the oral end of polyps. p Specimen RMNH Coel 40509 consists of two polyps of different sizes, with the smaller one being 16 by 5 mm, and the larger one 24 by 15 mm. Both polyps have no peduncle and are tapered. Both polyps are somewhat rugged on their outer sur- face, with no discernable tubercles, and have intermittent (=not one clear stripe) small darker vertical patterns in between the capitulary ridges only on the top 3–5 mm of the oral end of polyps. Distribution. Regions recorded in this study (Figure 11). Spermonde Archi- pelago (9), Berau Islands (19).h Distribution. Regions recorded in this study (Figure 11). Spermonde Archi- pelago (9), Berau Islands (19).h 12. Sphenopus pedunculatus Hertwig, 1888 Figures 10C, D, 11 Specimens examined (n=2). RMNH Coel 40507, Kepulauan Seribu Expedition sta- tion SER.29, north side of Pulau Tikus, Thousand Islands off Jakarta, northwest Java (05°51'13"S, 106°34'43"E), depth = 30 m, collected on September 18, 2005 by B.W. Hoeksema; RMNH Coel 40510, East Kalimantan–Berau Expedition station BER.03, south side of Pulau Derawan, East Kalimantan (02°17'03"N, 118°14'49"E), depth = 15 m, collected on October 21, 2003 by B.W. Hoeksema. y Photographic records (n=2). Images of RMNH Coel. 40507 and RMNH Coel 40510 as above. Description. This azooxanthellate species was originally described from the Philip- pines, and has not been reported in the literature for over 80 years, excepting two brief mentions in Reimer et al. (2012b). Easily discernable from other Sphenopus species by the presence of a ‘foot’ (=peduncle) that is attached to substrate (e.g. small rocks). The two specimens here varied in length from 33 to 62 mm in polyp length, and had a width between 9 to 11 mm (polyp head). The “swollen”, non-peduncle part of the polyp was between 15 to 20 mm in height, with the remainder of the length made up of the peduncle which was between 0 5 to 3 mm in width RMNH Coel 40507 Description. This azooxanthellate species was originally described from the Philip- pines, and has not been reported in the literature for over 80 years, excepting two brief mentions in Reimer et al. (2012b). Easily discernable from other Sphenopus species by the presence of a ‘foot’ (=peduncle) that is attached to substrate (e.g. small rocks). y p p g The two specimens here varied in length from 33 to 62 mm in polyp length, and had a width between 9 to 11 mm (polyp head). The “swollen”, non-peduncle part of the polyp was between 15 to 20 mm in height, with the remainder of the length made up of the peduncle, which was between 0.5 to 3 mm in width. RMNH Coel 40507 polyps were generally smooth in appearance, while the upper portions of polyps of James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 30 Figure 10. Images of Sphenopus species from photographic records in this study. A S. marsupialus at east Bone Lola shoal, Spermonde Archipelago, South Sulawesi, October 27, 1997 B S. marsupialus at station BER.14, lighthouse northeast Pulau Panjang Island, East Kalimantan, Berau Islands, October 9, 2003 C S. 12. Sphenopus pedunculatus Hertwig, 1888 Figures 10C, D, 11 pedunculatus specimen RMNH Coel 40507, Kepulauan Seribu Expedition station SER.29, north side of Pulau Tikus, Thousand Islands off Jakarta, northwest Java, depth = 30 m, collected on September 18, 2005 by B.W. Hoeksema; and D S. pedunculatus specimen RMNH Coel 40510, East Kalimantan– Berau Expedition station BER.03, south side of Pulau Derawan, East Kalimantan, depth = 15 m, col- lected on October 21, 2003 by B.W. Hoeksema. Figure 10. Images of Sphenopus species from photographic records in this study. A S. marsupialus at east Bone Lola shoal, Spermonde Archipelago, South Sulawesi, October 27, 1997 B S. marsupialus at station BER.14, lighthouse northeast Pulau Panjang Island, East Kalimantan, Berau Islands, October 9, 2003 C S. pedunculatus specimen RMNH Coel 40507, Kepulauan Seribu Expedition station SER.29, north side of Pulau Tikus, Thousand Islands off Jakarta, northwest Java, depth = 30 m, collected on September 18, 2005 by B.W. Hoeksema; and D S. pedunculatus specimen RMNH Coel 40510, East Kalimantan– Berau Expedition station BER.03, south side of Pulau Derawan, East Kalimantan, depth = 15 m, col- lected on October 21, 2003 by B.W. Hoeksema. RMNH Coel 40510 were somewhat rugged, with small round tubercules 0.5 mm in diameter roughly arranged in vertical lines. The spaces between these small tubercules were colored a much darker color than the remainder of the polyps’ outer surfaces. The peduncle of specimens and images (Figure 10C) are much thinner and longer than the sketch in Hertwig (1888). However, so few data are available for this (and other Sphenopus species) that currently nothing is known about intraspecific variation, and for now, we group these two specimens within this species. Distribution. Regions recorded in this study (Figure 11). Northwest Java (3), Berau Islands (19). Distribution. Regions recorded in this study (Figure 11). Northwest Java (3), Berau Islands (19). Previous records. This species was originally described from the Philippines, but has not been mentioned in recent literature (except for Reimer et al. 2012b), and hence very little is known on its distribution or ecology. Previous records. This species was originally described from the Philippines, but has not been mentioned in recent literature (except for Reimer et al. 2012b), and hence very little is known on its distribution or ecology. Remarks. 12. Sphenopus pedunculatus Hertwig, 1888 Figures 10C, D, 11 It is unknown as to whether the peduncle is a morphological charac- teristic that forms only when there is a hard substrate available, and this needs to be investigated to confirm this is truly a different species from S. marsupialis. Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 31 Figure 11. Distribution of Sphenopus species from specimens and photographic records from this study. Sphenopus marsupialus specimens in red, and S. pedunculatus in green. Region numbers correspond to lo- cations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. Figure 11. Distribution of Sphenopus species from specimens and photographic records from this study. Sphenopus marsupialus specimens in red, and S. pedunculatus in green. Region numbers correspond to lo- cations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. Figure 11. Distribution of Sphenopus species from specimens and photographic records from this study. Sphenopus marsupialus specimens in red, and S. pedunculatus in green. Region numbers correspond to lo- cations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. Photographic records (n=5). Southwest Nusa Penida, eastern Bali (08°49'S, 115°34"E), May 25, 1998; Desa Ped, Nusa Penida, Lombok Strait, east Bali Suborder Macrocnemina Haddon & Shackleton, 1891a , Family Hydrozoanthidae Sinniger, Reimer & Pawlowski, 2010 Genus Hydrozoanthus Sinniger, Reimer & Pawlowski, 2010 Family Hydrozoanthidae Sinniger, Reimer & Pawlowski, 2010 Genus Hydrozoanthus Sinniger, Reimer & Pawlowski, 2010 Regions recorded in this study (Figure 13). East Bali (5), Komodo Island (8), Maisel Islands (13), Moluccas (14), Berau Islands (19), Sulu Islands (20). Previous records. Originally reported from Sagami Bay, Japan (Lwowsky 1913), and subsequently reported from Taiwan (Reimer et al. 2011c), New Caledonia (Sin- niger 2006), and Indonesia (Sinniger et al. 2005, Di Camillo et al. 2010). It appears this species has an Indo-West Pacific distribution.hf i Remarks. This morphotype differs from the other known morphotype of the spe- cies (sensu Carlgren 1934) associated with this binomen, which is yellow in coloration. The original description of the species from Sagami Bay, Japan by Lwowsky (1913) was of a “gray, sandy” morphotype, but this was preserved in formalin, and thus could be either morphotype discussed here, or even a different one altogether. Phylogenetic analyses have shown subtle differences of sequences of specimens within this species (Sinniger et al. 2008), indicating that taxonomic revision may be needed in the future for this species group. 14. Hydrozoanthus sp. 1 Figures 12C, 13 Family Hydrozoanthidae Sinniger, Reimer & Pawlowski, 2010 Genus Hydrozoanthus Sinniger, Reimer & Pawlowski, 2010 Genus Hydrozoanthus Sinniger, Reimer & Pawlowski, 2010 13. Hydrozoanthus gracilis (Lwowsky, 1913) sensu Di Camillo et al. (2010) Figures 12A, B, 13 Specimens examined (n=3). RMNH Coel 40692, Snellius–II Expedition station 4.098, East Komodo, Komodo (08°29'54"S, 119°38'06"E), depth = 75 m, collected on September 19, 1984 by rectangular dredge; RMNH Coel. 40518, Snellius–II Expe- dition station 4.022, north Pulau Mai, Maisel Islands, Banda Sea (05°29'S, 127°32'E), depth = 0 to 1.5 m, collected on September 7, 1984; RMNH Coel 3816, Snellius Expedition, Sipankat Island, near Siburu Island, Sulu Islands, Philippines, collected on September 10–14, 1929. Specimens examined (n=3). RMNH Coel 40692, Snellius–II Expedition station 4.098, East Komodo, Komodo (08°29'54"S, 119°38'06"E), depth = 75 m, collected on September 19, 1984 by rectangular dredge; RMNH Coel. 40518, Snellius–II Expe- dition station 4.022, north Pulau Mai, Maisel Islands, Banda Sea (05°29'S, 127°32'E), depth = 0 to 1.5 m, collected on September 7, 1984; RMNH Coel 3816, Snellius Expedition, Sipankat Island, near Siburu Island, Sulu Islands, Philippines, collected on September 10–14, 1929. Photographic records (n=5). Southwest Nusa Penida, eastern Bali (08°49'S, 115°34"E), May 25, 1998; Desa Ped, Nusa Penida, Lombok Strait, east Bali Photographic records (n=5). Southwest Nusa Penida, eastern Bali (08°49'S, 115°34"E), May 25, 1998; Desa Ped, Nusa Penida, Lombok Strait, east Bali James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 32 (08°40'28"S, 115°30'50"E), May 25, 1998; east Tanjung Taal, Nusa Lembongan, Lombok Strait, east Bali (08°39'33"S, 115°26'37"E), May 24, 1998; Fauna Male- siana Maluku Expedition station MAL.21, west of Lilibooi, north coast Ambon Bay, Ambon, Moluccas (03°44'S, 128°02'E), November 20, 1996; East Kalimantan Program station BER.16, northeast Pulau Maratua, East Kalimantan, Berau Islands (02°17'29"N, 118°35'29"E), October 10, 2003. Description. As originally and previously described (Di Camillo et al. 2010), this azooxanthellate, colonial species is found as an epibiont on hydrozoans, particularly Plumularia habereri Stechow, 1909. In this study, this species consists of only one morphotype, with a gray to brown scapus, and reddish-brown oral disk and tentacles (Figure 12B). The appearance matches well with the morphotype of the species ob- served by Di Camillo et al. (2010). In this study, measurements are only available for two specimens, with polyps averaging 2.4 mm in height and 2.1 mm in width. These data also fit well with Di Camillo et al. (2010), who mention polyp heights of 2–5 mm, widths of 1.6 to 3 mm, with 32 tentacles and mesenteries. Distribution. p Photographic records (n=1). Balicasag Island, Cebu Strait, Philippines (09°31'01"N 123°41'04”E), November 21, 1999. Specimens examined. NA. Photographic records (n 1) Balicasag Island Cebu Strait Philippines 15. Hydrozoanthus sp. 2 Figures 12D, 13 15. Hydrozoanthus sp. 2 Figures 12D, 13 Specimens examined. NA. Specimens examined. NA. Photographic records (n=1). Balicasag Island, Cebu Strait, Philippines (09°31'01"N 123°41'04”E), November 21, 1999. Description. Similar to H. gracilis above, this azooxanthellate, colonial species is found as an epibiont on Plumularia habereri. As described in Di Camillo et al. (2010; as Parazoanthus sp.), this species has much smaller polyps than H. gracilis, forming colonies only on the main branch(es) of Pl. habereri colonies. Polyps are much less in- Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 33 Figure 12. Images of Hydrozoanthus species from photographic records in this study. A H. gracilis from Fauna Malesiana Maluku Expedition, station MAL.21, west of Lilibooi, north coast Ambon Bay, Ambon, Moluccas, November 20, 1996 B H. gracilis at Southwest Nusa Penida, east Bali, May 25, 1998 C Hy- drozoanthus sp. 1 at Balicasag Island, Cebu Strait, Philippines, November 21, 1999; and D Hydrozoanthus sp. 2 at East Kalimantan Program station BER.20, Tanjung Pandan shoal, Southwest of Pulau Panjang, East Kalimantan, Berau Islands, October 22, 2003. Figure 12. Images of Hydrozoanthus species from photographic records in this study. A H. gracilis from Fauna Malesiana Maluku Expedition, station MAL.21, west of Lilibooi, north coast Ambon Bay, Ambon, Moluccas, November 20, 1996 B H. gracilis at Southwest Nusa Penida, east Bali, May 25, 1998 C Hy- drozoanthus sp. 1 at Balicasag Island, Cebu Strait, Philippines, November 21, 1999; and D Hydrozoanthus sp. 2 at East Kalimantan Program station BER.20, Tanjung Pandan shoal, Southwest of Pulau Panjang, East Kalimantan, Berau Islands, October 22, 2003. crusted than H. gracilis. The Pl. habereri colonies hosting this species are much bigger than those with H. gracilis, as shown by (Di Camillo et al. 2010). Red scapus with yel- low tentacles, 22 to 24 tentacles slightly longer than oral disk diameter (Figure 12C). Distribution. Regions recorded in this study (Figure 13). Cebu (21). g g Distribution. Regions recorded in this study (Figure 13). Cebu (21 g g Previous records. Reported from Bunaken, North Sulawesi, in Di Camillo et al. (2010). Remarks. This undescribed species was informally and well described by Di Camillo et al. (2010) as “Parazoanthus sp.”. Specimens and DNA sequences are need- ed to properly describe this species. p Photographic records (n=1). East Kalimantan Program station BER.20, Tan- jung Pandan shoal, southwest of Pulau Panjang, East Kalimantan, Berau Islands (02°19'15"N, 118°06'33"E), October 22, 2003. Specimens examined. NA. Photographic records (n=1). East Kalimantan Program station BER.20, Tan- jung Pandan shoal, southwest of Pulau Panjang, East Kalimantan, Berau Islands (02°19'15"N, 118°06'33"E), October 22, 2003. James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 34 Figure 13. Distribution of Hydrozoanthus species from specimens and photographic records from this study. Hydrozoanthus gracilis specimens in red, Hydrozoanthus sp. 1 in green, and Hydrozoanthus sp. 2 in blue. Region numbers correspond to locations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. Figure 13. Distribution of Hydrozoanthus species from specimens and photographic records from this study. Hydrozoanthus gracilis specimens in red, Hydrozoanthus sp. 1 in green, and Hydrozoanthus sp. 2 in blue. Region numbers correspond to locations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. Description. Similar to H. gracilis and Hydrozoanthus sp. 1 above, this azooxanthel- late, colonial species is found as an epibiont on Plumularia habereri. Similar to Hydro- zoanthus sp. 1, this completely white species has much smaller polyps than H. gracilis, forming colonies only on the main branch(es) of Pl. habereri colonies (Figure 12D). Polyps are much less incrusted than H. gracilis. yp g Regions recorded in this study (Figure 13). Berau Islands (19). Previous records. NA.h Remarks. This undescribed species may be a different colored morphotype of Hydrozoanthus sp. 1 (above) informally described by Di Camillo et al. (2010) as “Parazoanthus sp.”. Specimens and DNA sequences are needed to properly describe this species. Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 35 Genus Terrazoanthus Reimer & Fujii, 2010 Specimens examined (n=1). RMNH Coel 40469, Fauna Malesiana Maluku Expedi- tion station MAL.05, Leitimur, outer Ambon Bay, Tanjung Bentang, Ambon, Moluccas (03°35'S, 128°05'E), depth = NA, collected on November 7, 1996 by J.C. den Hartog. Photographic records (n=1). West Pulau Badi, Spermonde Archipelago, South Sulawesi (04°58'06"S, 119°16'57"E), September 29, 1997. Specimens examined (n=1). RMNH Coel 40469, Fauna Malesiana Maluku Expedi- tion station MAL.05, Leitimur, outer Ambon Bay, Tanjung Bentang, Ambon, Moluccas (03°35'S, 128°05'E), depth = NA, collected on November 7, 1996 by J.C. den Hartog. tion station MAL.05, Leitimur, outer Ambon Bay, Tanjung Bentang, Ambon, Moluccas (03°35'S, 128°05'E), depth = NA, collected on November 7, 1996 by J.C. den Hartog. Photographic records (n=1). West Pulau Badi, Spermonde Archipelago, South Sulawesi (04°58'06"S, 119°16'57"E), September 29, 1997. p y g Photographic records (n=1). West Pulau Badi, Spermonde Archipelago, South Sulawesi (04°58'06"S, 119°16'57"E), September 29, 1997. Description. Azooxanthellate. Polyps well free and clear of coenenchyme. Outer surface of polyps covered with dense incrustation of irregularly sized sand particles, reminiscent of Microzoanthus sp. Oral disk semi-translucent with dark, almost black coloration, except for oral opening, which is much lighter in color. 40 to 50 tenta- cles, at least as long as oral disk diameter, with same blackish coloration as oral disk, with terminal 1/4 whitish in coloration. Colonies attached to non-living substrate. Specimen RMNH Coel 40469 is apparently a fragment of a whole colony, while the photographic record shows a colony of approximately 50 polyps arising from a com- mon coenenchyme (Figure 14A). The single specimen had polyps averaging 6.8 mm in length (n=3) and 3.1 mm in width (n=6). Figure 14. Images of Terrazoanthus species from photographic records in this study. A and B Terrazo- anthus sp. 1 at the west side of Pulau Badi, Spermonde Archipelago, South Sulawesi, September 29, 1997; and C and D Terrazoanthus sp. 2 at the west side of Bone Lola shoal, Spermonde Archipelago, South Sulawesi, April 22, 1998. Figure 14. Images of Terrazoanthus species from photographic records in this study. A and B Terrazo- anthus sp. 1 at the west side of Pulau Badi, Spermonde Archipelago, South Sulawesi, September 29, 1997; and C and D Terrazoanthus sp. 2 at the west side of Bone Lola shoal, Spermonde Archipelago, South Sulawesi, April 22, 1998. James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 36 Figure 15. Genus Terrazoanthus Reimer & Fujii, 2010 Distribution of Terrazoanthus species from specimens and photographic records from this study. Terrazoanthus sp. 1 specimens in red, and Terrazoanthus sp. 2 in green. Region numbers correspond to lo- cations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. Figure 15. Distribution of Terrazoanthus species from specimens and photographic records from this study. Terrazoanthus sp. 1 specimens in red, and Terrazoanthus sp. 2 in green. Region numbers correspond to lo- cations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Overlapping symbols indicate the same region. 17. Terrazoanthus sp. 2 Figures 14C, D, 15 17. Terrazoanthus sp. 2 Figures 14C, D, 15 Distribution. Regions recorded in this study (Figure 15). Spermonde Archi- pelago (9), Moluccas (14). Distribution. Regions recorded in this study (Figure 15). Spermonde Archi- pelago (9), Moluccas (14). Previous records. None, although similar undescribed specimens have been pho- tographed in the Philippines (P. Poppe, pers. comm.), and collected from Okinawa, Japan (Reimer, unpubl. data), indicating a potential West Pacific distribution.hf i Remarks. This species is similar in appearance but different in coloration to T. onoi from the Galapagos and west coast of Central and South America. Specimens examined. NA. Photographic records (n=1). West Bone Lola shoal, Spermonde Archipelago, South Sulawesi (05°03'15"S, 119°21'15"E), April 22, 1998. Photographic records (n=1). West Bone Lola shoal, Spermonde Archipelago, South Sulawesi (05°03'15"S, 119°21'15"E), April 22, 1998. Description. With only a single photographic record available, even an informal description of this undescribed species is limited. Asides from yellow coloration, this Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 37 species is outwardly similar to Terrazoanthus sp. 1 above. Polyps appear to be more crowded than in Terrazoanthus sp. 1, with 40 to 54 yellow tentacles longer than oral disk diameter (Figure 14D). species is outwardly similar to Terrazoanthus sp. 1 above. Polyps appear to be more crowded than in Terrazoanthus sp. 1, with 40 to 54 yellow tentacles longer than oral disk diameter (Figure 14D). g Regions recorded in this study (Figure 15). Spermonde Archipelago (9). g Regions recorded in this study (Figure 15). Spermonde Archipelago (9) Overall distribution. Unknown, although similar specimens have been photo- graphed in the Philippines (P. Poppe, pers. comm.).h Remarks. This species has been traded in the aquarium trade as “yellow polyps”, and is thought to be distributed primarily in Indonesia, yet no museum specimens exist, preventing this species from being formally described. Colonies often appear to be intermixed with Zoanthus spp. colonies in shallow water (J.D. Reimer, pers. obs.). Although undescribed, this putative species has been placed with the genus Terrazo- anthus based on DNA sequences acquired from aquarium trade polyps (Sinniger et al. 2005, Reimer and Fujii 2010). Family Parazoanthidae Delage & Hérouard, 1901 Family Parazoanthidae Delage & Hérouard, 1901 18. Parazoanthidae sp. 1 Figures 16A, B, 17 18. Parazoanthidae sp. 1 Figures 16A, B, 17 18. Parazoanthidae sp. 1 Figures 16A, B, 17 Specimens examined (n=2). RMNH Coel 40766, Fauna Malesiana Maluku Ex- pedition station MAL.09, southwest coast, Ambon, Latuhalat, Moluccas (03°46'S, 128°06'E), depth = to 24 m, collected on November 11, 1996; RMNH Coel 40768, Snellius Expedition, Pulau Bo Islands, Halmahera Sea, collected on October 5, 1930. Specimens examined (n=2). RMNH Coel 40766, Fauna Malesiana Maluku Ex- pedition station MAL.09, southwest coast, Ambon, Latuhalat, Moluccas (03°46'S, 128°06'E), depth = to 24 m, collected on November 11, 1996; RMNH Coel 40768, Snellius Expedition, Pulau Bo Islands, Halmahera Sea, collected on October 5, 1930. Photographic records (n=1). Station BER.30, north of Lighthouse 1 Reef, south of Pulau Derawan, East Kalimantan, Berau Islands (02°16'02"N, 118°14'23"E), Oc- tober 22, 2003. Description. Azooxanthellate, epibiotic on Keroeides sp., polyps approximately the same height as width (approximately 1–3 mm), connected by coenenchyme vis- ible on the outer surface of the octocoral colony. Polyps numerous, placed between smaller octocoral polyps, pale yellow in coloration, with outer surface of polyps slightly reddish in color similar to host octocoral. Tentacles relatively short, ap- proximately half of the oral disk diameter, also pale yellow, and approximately 20 in number (Figure 16A). Description. Azooxanthellate, epibiotic on Keroeides sp., polyps approximately the same height as width (approximately 1–3 mm), connected by coenenchyme vis- ible on the outer surface of the octocoral colony. Polyps numerous, placed between smaller octocoral polyps, pale yellow in coloration, with outer surface of polyps slightly reddish in color similar to host octocoral. Tentacles relatively short, ap- proximately half of the oral disk diameter, also pale yellow, and approximately 20 in number (Figure 16A). Specimen RMNH Coel 40766 is larger than RMNH Coel 40768 (polyp average width 2.6 mm vs 1.6 mm, respectively). However, the latter specimen is quite old (from the original Snellius Expedition) and this difference may be due to fixation methods. Specimen RMNH Coel 40766 is larger than RMNH Coel 40768 (polyp average width 2.6 mm vs 1.6 mm, respectively). However, the latter specimen is quite old (from the original Snellius Expedition) and this difference may be due to fixation methods. fi Distribution. Regions recorded in this study (Figure 17). Moluccas (14), Bo Islands (15), Berau Islands (19). fi Distribution. Regions recorded in this study (Figure 17). Moluccas (14), Bo Islands (15), Berau Islands (19). Previous records. NA. Previous records. NA. Remarks. Only two specimens and one photographic record of this undescribed species exist. However, these records are each from different expeditions, and it is rea- sonable to expect that this species is at least distributed in the Banda and Celebes Seas. 38 James D. Reimer et al. / ZooKeys 444: 1–57 (2014) Figure 16. Images of Parazoanthidae sp. 1 and Parazoanthidae sp. 2 from specimens and photographic records in this study. A and B Parazoanthidae sp. 1 at station BER.30, north of Lighthouse 1 Reef, south of Pulau Derawan, East Kalimantan, Berau Islands, October 22, 2003. Note octocoral polyps on antipa- tharian on left side of image C and D Parazoanthidae sp. 2 specimen RMNH Coel 40762, Snellius–II Expedition, Station 4.227, west Pulau Tinanja, Taka Bone Rate, depth = 60 m, collected on October 15, 1984 by rectangular dredge. Scale in C 1 cm. Figure 16. Images of Parazoanthidae sp. 1 and Parazoanthidae sp. 2 from specimens and photographic records in this study. A and B Parazoanthidae sp. 1 at station BER.30, north of Lighthouse 1 Reef, south of Pulau Derawan, East Kalimantan, Berau Islands, October 22, 2003. Note octocoral polyps on antipa- tharian on left side of image C and D Parazoanthidae sp. 2 specimen RMNH Coel 40762, Snellius–II Expedition, Station 4.227, west Pulau Tinanja, Taka Bone Rate, depth = 60 m, collected on October 15, 1984 by rectangular dredge. Scale in C 1 cm. Recently, many different genera in the family Parazoanthidae have been described based on a combination of epizoitic relationships and phylogenetic analyses (e.g. Sin- niger et al. 2010, 2013). However, no parazoanthids have been reported in association with Keroeides, and therefore currently it is impossible to place these specimens and records into a genus without both further examination of specimens combined with DNA sequence data. 19. Parazoanthidae sp. 2 Figures 16C, D, 17 Specimens examined (n=1). RMNH Coel 40762, Snellius–II Expedition, Station 4.227, west Pulau Tinanja, Taka Bone Rate (06°32'48"S, 121°09'36"E), depth = 60 m, collected on October 15, 1984 by rectangular dredge. Specimens examined (n=1). RMNH Coel 40762, Snellius–II Expedition, Station 4.227, west Pulau Tinanja, Taka Bone Rate (06°32'48"S, 121°09'36"E), depth = 60 m, collected on October 15, 1984 by rectangular dredge. Photographic records. NA. Photographic records. NA. Description. Epibiotic on Cirripathes sp. (specimen RMNH Coel 24832). Polyps of this azooxanthellate zoantharian specimen are relatively small (average Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 39 Figure 17. Distribution of Parazoanthidae sp. 1 and Parazoanthidae sp. 2 from specimens and pho- tographic records from this study. Parazoanthidae sp. 1 specimens in red, and Parazoanthidae sp. 2 in green. Region numbers correspond to locations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. Figure 17. Distribution of Parazoanthidae sp. 1 and Parazoanthidae sp. 2 from specimens and pho- tographic records from this study. Parazoanthidae sp. 1 specimens in red, and Parazoanthidae sp. 2 in green. Region numbers correspond to locations given in species’ information. Boxes indicate presence of specimens (with or without photographic records), while circles indicate only photographic records. width 2.1 mm, n=8 polyps) and do not protrude much from the coenenchyme, with polyp height approximately same as width. Polyps and coenenchyme are heavily encrusted, and golden yellow-brown in color. Coenenchyme forms a thin sheath over the antipatharian surface. Capitulary ridges not clearly discernable. Polyps form semi-regular vertical rows over short distances of the antipatharian (e.g. approx. 5 cm), but with no observable pattern for the entire colony (Figure 16C). Colony encrusts the top approximately 1/2 of the Cirripathes specimen; starting approximately 15 cm from the bottom holdfast. The Cirripathes colony’s proximal tip appears to be broken off and missing. f Distribution. Regions recorded in this study (Figure 17). Taka Bone Rate (11). Past records. NA. Specimens examined. NA. Photographic records (n=3). West side of Pulau Kudengareng Keke, Spermon- de Archipelago, South Salawesi (05°06'20"S, 119°17'03"E), June 4, 1997; Cabilao Island, Cebu Strait, Philippines (09°52'35”N, 123°46'33”E), November 16, 1999; station WAK.24, Ndaa Atoll northwest outer slope, REA Wakatobi National Park, Tukang Besi Islands, Wakatobi, Southeast Sulawesi, (05°38'46"S, 124°02'42"E), May 12, 2003. Description. Very small (polyp diameter likely approximately 1 mm) azooxan- thellate polyps regularly spaced and embedded within encrusting sponge tissue (Fig- ure 18A). Polyps differentially colored from sponges; dark red (Cebu), yellow (Pu- lau Kudengareng Keke), white (Wakatobi). Tentacles up to 24 in number (in images here), as long as diameter of oral disk. g Distribution. Regions recorded in this study (Figure 19). Spermonde Archi- pelago (9), Tukang Besi Islands (12), Cebu (21). p g g Past records. Previously, similar specimens have been reported from Japan (Sin- niger et al. 2008) and the Red Sea (Reimer et al. 2014b). Past records. Previously, similar specimens have been reported from Japan (Sin- niger et al. 2008) and the Red Sea (Reimer et al. 2014b). Remarks. Based on phylogenetic data (J. Montenegro, F. Sinniger and J.D. Re- imer, unpubl. data) it appears that this group includes several undescribed species. The species has been found on cave ceilings (Figure 18A), which may explain why it is azooxanthellate as in some other hexacorals with white polyps (Hoeksema 2012b, Reimer et al. 2014, Irei et al. subm). Past records. NA.h Remarks. This species may belong to genus Antipathozoanthus, which was described recently by Sinniger et al. (2010) and includes species from both the Atlantic and Indo- Pacific, with reports of specimens also from the Red Sea (Reimer et al. 2014b). It is likely several undescribed Antipathozoanthus species are present in the Indo-Pacific, as only one Antipathozoanthus species from the Galapagos has been formally described. In situ images and DNA sequences are needed to formally describe this species. James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 40 Genus Parazoanthus Haddon & Shackleton, 1891a 20. Parazoanthus sp. 1 Figures 18A, 19 Past records. NA.h Remarks. The only sponge-associated Parazoanthus species formally described from the Indo-Pacific are P. elongatus McMurrich, 1904 from the west coast of South America and New Zealand (Sinniger and Haussermann 2009) and P. darwini Reimer & Fujii, 2010 from the Galapagos. Thus, no similar species have been reported from past or recent zoantharian work in surrounding CIP regions, and therefore it is likely that these specimens constitute an undescribed species. Although Parazoanthus has re- cently been taxonomically redescribed (Sinniger et al. 2010), and the species now only encompasses sponge-associated species, the genus is still paraphyletic and taxonomic revision is needed. DNA sequences from undescribed species are needed to correctly place specimens such as these into the correct clade. 21. Parazoanthus sp. 2 Figures 18B, 19 Specimens examined (n=4). RMNH Coel 40544, Snellius–II Expedition Station 4.061, east of Melolo, northeast Sumba (09°54'12"S, 120°43'30"E), depth = 50 m, collected on September 15, 1984 by rectangular dredge; RMNH Coel. 40570, station 9, reef slope of southwest Pulau Nain, Bunaken, North Sulawesi (01°46'N, 124°45'E), collected on May 8, 1998 by B.W. Hoeksema; RMNH Coel 40572, Ternate Expedition Station TER.27, Tanjung Ratemu (south of river), west Hal- mahera Sea, North Moluccas (00°54'45"N, 127°29'10"E), depth = 20 m, collected on November 8, 2009 by B.W. Hoeksema; RMNH Coel 40757, Indonesia 2012 Expedition, Station LEM.34, west Pulau Sarena Kecil Lembeh, North Sulawesi (01°27'26"N, 125°13'31"E), depth = 22 m, collected on February 17, 2012 by B.W. Hoeksema. Photographic records (n=3). West Pulau Kudingareng Keke, Spermonde Archi- pelago, South Sulawesi (05°06'20"S, 119°17'03"E), June 4, 1997; southeast Likuan, Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 41 Bunaken, North Sulawesi (01°36'N, 124°47'E), May 10, 1998; Main coast, West Bali (08°06'50"S, 114°30'40"E), May 22, 1998. y Description. Azooxanthellate, epibiotic on encrusting sponges, with 3 to 6 polyps arising in groups from a common coenenchyme, or occasionally arising in rows from stolons (Figure 18B). Polyps (oral disk, tentacles, scapus) pale yellow/cream in color. 36 to 40 tentacles, longer in length than oral disk diameter. Specimens’ preserved pol- yps (n=2 specimens, 10 polyps per specimen) averaged 5.8 mm in height (range 4 to 8 mm) and 3.3 mm in width (range 2.5 to 5 mm). g Distribution. Regions recorded in this study (Figure 19). West Bali (4), north- east Sumba (6), Spermonde Archipelago (9), west Halmahera Sea (16), Lembah Strait (17), Bunaken (18). Distribution. Regions recorded in this study (Figure 19). Northeast Sumba (6), Komodo Island (8). 22. Parazoanthus sp. 3 Figures 18C, D, 19 22. Parazoanthus sp. 3 Figures 18C, D, 19 Specimens examined (n=2). RMNH Coel 40525, Snellius–II Expedition station 4.100, east of Komodo Island (08°28.6'S, 119°37.3'E), depth 91 m, collected on Sep- tember 19, 1984 by rectangular dredge; RMNH Coel 40545, Snellius–II Expedition station 4.051, east of Melolo, northeast Sumba (09°53.5'S, 120°42.7'E), depth 75-90 m, collected on September 13, 1984 by rectangular dredge. Photographic records. NA Specimens examined (n=2). RMNH Coel 40525, Snellius–II Expedition station 4.100, east of Komodo Island (08°28.6'S, 119°37.3'E), depth 91 m, collected on Sep- tember 19, 1984 by rectangular dredge; RMNH Coel 40545, Snellius–II Expedition station 4.051, east of Melolo, northeast Sumba (09°53.5'S, 120°42.7'E), depth 75-90 m, collected on September 13, 1984 by rectangular dredge. g p Description. This putative azooxanthellate species is similar in size to Parazoanthus sp. 2 above, with polyps of average 6.1 mm in height (range 2.5 to 10 mm; n=2 colo- nies) and average width of 3.2 mm (range 2 to 4 mm). Some small dark incrustations visible on lower half (=aboral) of polyps’ scapus. Approximately 20 capitulary ridges, indicating tentacle counts of approximately 40. Polyps range from cream (RMNH Coel 40525) to tan (RMNH Coel 50545) in color when preserved. Polyps arise from a well-developed stoloniferous coenenchyme in rows, with most found along the upper and outer edges of flat, paddle-shaped sponges (Figures 18C, D). No polyps found on the lower ‘foot’ or ‘stalk’ of sponges. Distribution. Regions recorded in this study (Figure 19). Northeast Sumba (6), Komodo Island (8). James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 42 Figure 18. Images of Parazoanthus species from specimens and photographic records in this study. A Parazoanthus sp. 1 on cave ceiling at station WAK.24, Ndaa Atoll northwest outer slope, REA Waka- tobi National Park, Southeast Sulawesi, Tukang Besi Islands, Wakatobi, May 12, 2003 B Parazoanthus sp. 2 at Southeast Likuan, Bunaken, North Sulawesi, May 10, 1998; and C and D Parazoanthus sp. 3 specimen RMNH Coel 40545, Snellius–II Expedition station 4.051, east of Melolo, northeast Sumba, depth = 75 to 90 m, collected on September 13, 1984 by rectangular dredge. Scales in C and D 1 cm. Figure 18. Images of Parazoanthus species from specimens and photographic records in this study. A Parazoanthus sp. 22. Parazoanthus sp. 3 Figures 18C, D, 19 1 on cave ceiling at station WAK.24, Ndaa Atoll northwest outer slope, REA Waka- tobi National Park, Southeast Sulawesi, Tukang Besi Islands, Wakatobi, May 12, 2003 B Parazoanthus sp. 2 at Southeast Likuan, Bunaken, North Sulawesi, May 10, 1998; and C and D Parazoanthus sp. 3 specimen RMNH Coel 40545, Snellius–II Expedition station 4.051, east of Melolo, northeast Sumba, depth = 75 to 90 m, collected on September 13, 1984 by rectangular dredge. Scales in C and D 1 cm. Past records. NA. Remarks. Similar in size to Parazoanthus sp. 2 above, we have included these two specimens as a separate putative species in this study. This is partly due to speci- mens being in association with a different sponge species (compare Figures 18B, C), as host specificity may differ between species (e.g. Crocker and Reiswig 1981; Swain and Wulff 2007). As well, Parazoanthus sp. 3 specimens are from deeper depths (75 to 91 m as opposed to 20 to 50 m) than Parazoanthus sp. 2. Family Epizoanthidae Delage & Hérouard, 1901 Genus Epizoanthus Gray, 1867 23. Epizoanthus illoricatus Tischbierek, 1930 Figures 20A, 21 Family Epizoanthidae Delage & Hérouard, 1901 Genus Epizoanthus Gray, 1867 23. Epizoanthus illoricatus Tischbierek, 1930 Figures 20A, 21 Specimens examined (n=4). RMNH Coel 40533, Snellius–II Expedition Station 4.222, south of Pulau Tarupa Kecil, Taka Bone Rate (06°31'30"S, 121°08'00"E), Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 43 Figure 19. Distribution of Parazoanthus species from specimens and photographic records from this study. Parazoanthus sp. 1 specimens in red, Parazoanthus sp. 2 in green, and Parazoanthus sp. 3 in blue. Region numbers correspond to locations given in species’ information. Boxes indicate presence of speci- mens (with or without photographic records), while circles indicate only photographic records. Overlap- ping symbols indicate the same region. Figure 19. Distribution of Parazoanthus species from specimens and photographic records from this study. Parazoanthus sp. 1 specimens in red, Parazoanthus sp. 2 in green, and Parazoanthus sp. 3 in blue. Region numbers correspond to locations given in species’ information. Boxes indicate presence of speci- mens (with or without photographic records), while circles indicate only photographic records. Overlap- ping symbols indicate the same region. depth 58 m, collected on October 15, 1984 by rectangular dredge; RMNH Coel 40546, Snellius–II Expedition Station 4.051, east of Melolo, northeast Sumba (09°53'30"S, 120°42'42"E), depth = 75 to 90 m, collected on September 13, 1984 by rectangular dredge; RMNH Coel 40571, Ternate Expedition Station TER.27, Tan- jung Ratemu, south of river, west Halmahera Sea (00°54'44"N, 127°29'10"E), depth = 20 m, collected on November 08, 2007 by B.W. Hoeksema; RMNH Coel 40758, station LEM.32, north Sarena Kecil, Lembeh Strait, North Sulawesi (01°27'26"N, 125°13'38"E), depth = 30 m, collected on February 16, 2012 by B.W. Hoeksema. Photographic records (n=6). West Menjangan, West Bali (08°05'33"S, 114°29'47"E) May 22, 1998 (3 different specimens); Maluku Expedition station MAL.21, north coast Ambon Bay, Tanjung Hatupero, east of Lilibooi, Ambon (03°44'S, 128°02'E), November 20, 1996; southeast Likuan, Bunaken, North Sulawe- si (01°36'N, 124°47'E), May 10, 1998; station BER.04, south Pulau Derawan, East Kalimantan (02°17'03"N, 118°14'49”E), October 18, 2003. Description. Originally described from the Philippines. Azooxanthellate. Polyps of this species often grow at the outer bends of the zig-zag shaped tubes, and combined James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 44 with polyps’ smaller size and thin coenenchyme (Figure 20A), colonies appear to be much less crowded than as seen in E. aff. illoricatus (Figure 20B). Family Epizoanthidae Delage & Hérouard, 1901 Genus Epizoanthus Gray, 1867 f Polyps of specimens in the RMNH collection are generally less than 1 mm in di- ameter, and never more than 2 mm, and of approximately equal height. Coenenchyme generally light gray in color, oral disk and tentacles semi-translucent brown. Tentacles in images 20–22 in number, much thinner than as seen in Epizoanthus aff. illoricatus below, with orange or white colored proximal tips, longer in length than oral disk diameter. The two deeper specimens (RMNH Coel 40533 and 40546) have highly developed thin coenenchymes covering the entire worm tubes’ surface, and are both dark black in color. On the other hand, the shallower specimens had some unitary pol- yps, and colonial polyps were often in clusters of two or three with poorly developed coenenchyme. The morphological characters and dimensions observed in the specimens in this study agree well with the original description by Tischbierek (1930). Distribution. Regions recorded in this study (Figure 21). West Bali (4), north- east Sumba (6), Take Bone Rate (11), Moluccas (14), west Halmahera Sea (16), Lem- beh Strait (17), Bunaken (18), Berau Islands (19). Past records. Originally described from Manila, and subsequently reported from Taiwan (Reimer et al. 2013a), New Caledonia (Sinniger 2006), Palau (Reimer et al. 2014a), and Osprey Reef, Australia in the Coral Sea (Lindsay et al. 2012), indicating a western Indo-Pacific distribution. Found from specimens in this study as shallow as 20 m, and as deep as 90 m, similar to depths reported at Osprey Reef (=82.5 m) (Lindsay et al. 2012). Remarks. Until this report, any Epizoanthus spp. on a zig-zag shaped eunicid worm was recorded as E. illoricatus. However, from the preliminary analyses here, it appears that there may be two or more species within this group. Thus, previous re- cords must be treated with caution. 1997; Fauna Malesiana Maluku Expedition station MAL.10, south coast of Am- bon Bay, east of Eri, Ambon (03°45'S, 128°08'E), November 12, 1996; Maluku Expedition station MAL.12, north coast near Morela, Ambon (03°33'S, 128°12'E), November 13–14, 1996; Maluku Expedition station MAL.19, Tanjung Batu Dua, east of Hatu, north coast Ambon Bay, Ambon (03°43'S, 128°03'E), November 19, 1996; Fauna Malesiana Marine Sulawesi Expedition station SUL.16, bay east of Tan- jung Labuhankompeni, Pulau Lembeh, Lembeh Strait, North Sulawesi (01°26'N, 1997; Fauna Malesiana Maluku Expedition station MAL.10, south coast of Am- bon Bay, east of Eri, Ambon (03°45'S, 128°08'E), November 12, 1996; Maluku Expedition station MAL.12, north coast near Morela, Ambon (03°33'S, 128°12'E), November 13–14, 1996; Maluku Expedition station MAL.19, Tanjung Batu Dua, east of Hatu, north coast Ambon Bay, Ambon (03°43'S, 128°03'E), November 19, 1996; Fauna Malesiana Marine Sulawesi Expedition station SUL.16, bay east of Tan- jung Labuhankompeni, Pulau Lembeh, Lembeh Strait, North Sulawesi (01°26'N, 24. Epizoanthus aff. illoricatus Tischbierek, 1930 Figures 20B, 21 Specimens (n=2). RMNH Coel 40536, Snellius–II Station 4.058, east of Melolo, northeast Sumba (09°51'S, 120°45'E), depth = 180 m, collected on September 14, 1984 by rectangular dredge; RMNH Coel 40547, Snellius–II Station 4.051, east of Melolo, northeast Sumba (09°53'30"S, 120°42'42"E), depth = 75 to 90 m, collected on September 13, 1984 by rectangular dredge. Photographic records (n=12). Desa Ped, north Nusa Penida, east Bali (08°40'28"S, 115°30'50"E), May 28, 1998; 4 specimens from Tulamben, east Bali (08°16'26"S, 115°35'28"E), July 9–10, 1997; Nusa Penida, east Bali, (08°40'23"S, 115°29'13"E), May 27, 1998; Kapoposang, Spermonde Archipelago, South Sulawesi (04°41'40"S, 118°54'55"E), June 24, 1997, August 8, 1997; west Pulau Samalona, Spermonde Archipelago, South Sulawesi (05°07'30"S, 119°20'15"E), September 16, Photographic records (n=12). Desa Ped, north Nusa Penida, east Bali (08°40'28"S, 115°30'50"E), May 28, 1998; 4 specimens from Tulamben, east Bali (08°16'26"S, 115°35'28"E), July 9–10, 1997; Nusa Penida, east Bali, (08°40'23"S, 115°29'13"E), May 27, 1998; Kapoposang, Spermonde Archipelago, South Sulawesi (04°41'40"S, 118°54'55"E), June 24, 1997, August 8, 1997; west Pulau Samalona, Spermonde Archipelago, South Sulawesi (05°07'30"S, 119°20'15"E), September 16, Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 45 Figure 20. Images of Epizoanthus species from specimens and photographic records in this study. A Epizoanthus illoricatus at west Pulau Menjangan, West Bali, May 22, 1998; and B Epizoanthus aff. illoricatus at Tulamben, east coast of Bali, July 10, 1997. Figure 20. Images of Epizoanthus species from specimens and photographic records in this study. A Epizoanthus illoricatus at west Pulau Menjangan, West Bali, May 22, 1998; and B Epizoanthus aff. illoricatus at Tulamben, east coast of Bali, July 10, 1997. Figure 20. Images of Epizoanthus species from specimens and photographic records in this study. A Epizoanthus illoricatus at west Pulau Menjangan, West Bali, May 22, 1998; and B Epizoanthus aff. illoricatus at Tulamben, east coast of Bali, July 10, 1997. 1997; Fauna Malesiana Maluku Expedition station MAL.10, south coast of Am- bon Bay, east of Eri, Ambon (03°45'S, 128°08'E), November 12, 1996; Maluku Expedition station MAL.12, north coast near Morela, Ambon (03°33'S, 128°12'E), November 13–14, 1996; Maluku Expedition station MAL.19, Tanjung Batu Dua, east of Hatu, north coast Ambon Bay, Ambon (03°43'S, 128°03'E), November 19, 1996; Fauna Malesiana Marine Sulawesi Expedition station SUL.16, bay east of Tan- jung Labuhankompeni, Pulau Lembeh, Lembeh Strait, North Sulawesi (01°26'N, James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 46 Figure 21. 24. Epizoanthus aff. illoricatus Tischbierek, 1930 Figures 20B, 21 Distribution of Epizoanthus species from specimens and photographic records from this study. Epizoanthus illoricatus specimens in red, and Epizoanthus aff. illoricatus in green. Region numbers correspond to locations given in species’ information. Boxes indicate presence of specimens (with or with- out photographic records), while circles indicate only photographic records. Figure 21. Distribution of Epizoanthus species from specimens and photographic records from this study. Epizoanthus illoricatus specimens in red, and Epizoanthus aff. illoricatus in green. Region numbers correspond to locations given in species’ information. Boxes indicate presence of specimens (with or with- out photographic records), while circles indicate only photographic records. 125°11'E), October 23, 1994; west Pulau Siladen, Bunaken, North Sulawesi (01°38'N, 124°46'E), May 2, 1998. Description. Azooxanthellate. As E. illoricatus above, obligate epibiont on eunicid worms. Polyps of this putative species are at least twice as big in diameter as E. illori- catus (average 2.1 mm, compared with a maximum of 2 mm for E. illoricatus), and many times bigger in terms of volume. Additionally, both specimens have brown coe- nenchyme and scapus, different from the light gray coenenchyme and brownish oral disk reported for E. illoricatus (Figure 20B). In situ images show colonies with cream, brown, red-brown, orange-brown or tan coenenchyme and scapus, often with white tentacles that are comparatively shorter and thicker than in E. illoricatus. The coenen- chyme of this putative species is much more developed than E. illoricatus, with polyps arising from not only bends of the zig-zag shaped eunicid tube, but also from other locations. The result is a colony that has a higher density of polyps than E. illoricatus. In E. illoricatus, often the zig-zag shape of the eunicid tube is visible between polyps, but this is rarely the case in E. aff. illoricatus (Figure 20B). Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 47 Distribution. Regions recorded in this study (Figure 21): East Bali (5), northeast Sumba (6), Spermonde Archipelago (9), Moluccas (14), Lembeh Strait (17), Bunaken (18). Past records. NA. Distribution. Regions recorded in this study (Figure 21): East Bali (5), northeast Sumba (6), Spermonde Archipelago (9), Moluccas (14), Lembeh Strait (17), Bunaken (18). P d NA Past records. NA. Past records. NA. Remarks. Although the two specimens here were found at deeper depths (75 to 190 m), numerous photographic records show that this species and E. illoricatus have an overlapping depth range. 24. Epizoanthus aff. illoricatus Tischbierek, 1930 Figures 20B, 21 Examination of DNA sequences combined with detailed morphological analyses should help clear up the relationship between this putative species and E. illoricatus, although preliminary molecular analyses show differences be- tween the two groups (H. Kise and J.D. Reimer, unpubl. data). It is likely records and museum specimens identified as E. illoricatus from the central Indo-Pacific include both types mentioned in this study. Discussion Examination of the Naturalis zoantharian collection resulted in 24 species being iden- tified, 12 from suborder Brachycnemina and 12 from Macrocnemina. While by no means an extensive collection, with most specimens from Indonesia, these results in- dicate that the Central Indo-Pacific waters are at least as diverse in numbers of spe- cies, genera, and families as surrounding regions of Australia, Singapore, and Japan. In Australia, an examination of the brachycnemic shallow water zoantharians of the Great Barrier Reef indicated the presence of eight species (Burnett et al. 1997), while in Okinawa, 12 brachycnemic species have been previously reported (Reimer 2010), and in Taiwan 10 species (Reimer et al. 2013a). These previous reports did not include macrocnemic species, but from the brachycnemic results alone, Indonesia does appear to have zoantharian species diversity at least as high as Okinawa, one of the most well examined regions. Finally, as many macrocnemic species live in deeper areas or in caves and other less-examined ecosystems (Sinniger et al. 2013), we expect the number of zoantharian species in the shallow waters of Indonesia to be higher than the initial estimate in this study, and further investigations should confirm this idea.h i The discussion of total numbers of shallow water zoantharian species is clearly still in the preliminary stages given the lack of focused sampling throughout most regions of the world, as well as the continuous discovery of new species and genera (Reimer et al. 2007a, Sinniger et al. 2010, Fujii and Reimer 2011, 2013). Still, the initial species numbers from this study should provide a basis upon which future zoantharian studies can build on. Furthermore, it should not be forgotten that the previous reports listed above from other Indo-Pacific regions were all conducted by zoantharian specialists collecting specimens in the field, while the Indonesian specimens in the Naturalis col- lection constitute only part of a broad sampling effort of benthic invertebrates without the participation of any zoantharian specialists. Thus, our prediction that the total number of shallow water zoantharian species in Indonesia will be considerably higher than reported in this study is almost certainly accurate, particularly given the recent James D. Reimer et al. / ZooKeys 444: 1–57 (2014) 48 discovery of widespread yet cryptic zoantharian species from coral reef environments (Fujii and Reimer 2011, 2013) not yet reported from Indonesia. Discussion Further supporting the possibility of Indonesia harboring a diverse zoantharian fau- na is the fact that the specimens examined in the Naturalis collection are primarily from eastern Indonesia (e.g. Sulawesi and Banda Sea, Fig. 1), with few or no specimens from other regions such as the islands of Java and New Guinea, and only one locality in the Philippines and Papua New Guinea. Future collection efforts must be focused on these unexamined regions if we are to obtain a clear understanding of zoantharian diversity in the CIP. Additionally, the deep sea (>200 m depth) has been recently speculated to har- bor much undiscovered zoantharian diversity (Sinniger et al. 2013) and yet in this study only three of the zoantharian species were found in waters >50 m in depth. Exploring the deeper waters in the Indonesian region will undoubtedly result in further discoveries. g Of the 24 total species listed in this study, at least nine (and perhaps up to 12 if Paly- thoa spp. are included) are likely undescribed species. Some, such as Terrazoanthus sp. 2, have been known for years in the global aquarium trade, yet still no museum specimens exist, and thus we cannot formally describe them within this manuscript. Without for- mal descriptions and a clear understanding of species, future conservation work cannot proceed effectively, and immediate taxonomic efforts should focus on the obtaining of specimens and a formal description of this species. Similarly, many photographic records exist for Neozoanthus sp., yet no specimens are in the Naturalis collection.h Three other species that are almost certainly undescribed species are Parazoanthus sp. 1, Parazoanthus sp. 2 and Parazoanthus sp. 3. Until now, only two sponge-associ- ated Parazoanthus species has been formally described from the Pacific, and none from sub-tropical or tropical waters. Five other species, Hydrozoanthus sp. 1, Hydrozoanthus sp. 2, Parazoanthidae sp. 1, Parazoanthidae sp. 2, and Terrazoanthus sp. 1, are also very likely to be undescribed species, but with only photographic records, or one or two specimens existing for these species, additional specimens and molecular data are needed to properly describe them. Conclusions In conclusion, this study provides a starting point for zoantharian research in the Coral Triangle. We were able to discern 24 different morphological species based on speci- men examination combined with photographic records. However, based on recent previous research, phylogenetic analyses of specimens would likely result in somewhat different results due to both high levels of intraspecific morphological variation in some species (Burnett et al. 1997, Reimer et al. 2004) and also morphological con- vergence between other phylogenetically distinct species (Sinniger and Haussermann 2009). These previous studies demonstrate how difficult it often is to properly identify zoantharian species without molecular data. Furthermore, this study demonstrates that the central Indo-Pacific likely harbors very high levels of zoantharian diversity, as the numbers of putative species from this Shallow–water zoantharians (Cnidaria, Hexacorallia) from the Central Indo–Pacific 49 study (24) include a large number of undescribed species, and total numbers are as high or higher than previously reported for any other region. Finally, it is hoped that this study can serve as a template for the study of other understudied coral reef benthos in the Coral Triangle. In this study, past photographic records proved to be invaluable in aiding species identification, and understanding species distributions. Thus, while museum collections should remain the key tool in taxonomic and biogeographic research (Rainbow 2009, Hoeksema et al. 2011, Rocha et al. 2014), archived in situ images can provide additional information. Acknowledgements This study was made possible by a Temminck Fellowship Grant from Naturalis to the first author from May to June 2012. Additional support was provided by the Rising Star Program, and by the International Research Hub Project for Climate Change and Coral Reef/Island Dynamics, both at the University of the Ryukyus. At Naturalis, Mr. Koos van Egmond kindly helped with examination of specimens, and Ms. Elly Beglinger provided valuable data on the Amsterdam collection. The en- tire “Zeeteam” of NCB Naturalis is thanked for their guidance, support, and coffee throughout the term of this study. The last author received financial support for field research from the Netherlands Foundation for the Advancement of Tropical research (WOTRO grants W01–60, W77–96, WK84–354, WT87–299), the Schure Bei- jerinck Popping fund (KNAW), the Alida Buitendijkfonds (Naturalis), and the Jan Joost ter Pelkwijkfonds (Naturalis), The Nature Conservancy (Indonesia) and WWF (Malaysia). Various institutes and organizations acted as host, such as field stations of PPO–LIPI at Bitung, Halmahera, Pulau Pari, and Ambon (PPO–LIPI). Logistic support was also provided by Universitas Hasanuddin (Makassar), Universitas Bung Hatta (Padang), Universiti Malaysia Sabah (Kota Kinabalu), and Universiti Brunei Darussalam, The Nature Conservancy (Bali, Komodo and Wakatobi), the Chris- tensen Research Institute (Madang), and several dive resorts, such as Bali Hai Div- ing Adventures, Bali Blue Dive, and Derawan Dive Resort. All specimen sampling was conducted with all appropriate authorizations, and details are available in the original publications (Table 1). Comments from two anonymous reviewers greatly improved this manuscript. 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https://openalex.org/W4386561890	https://www.researchsquare.com/article/rs-3321010/latest.pdf	English	null	A measure for multiple bends and shear damage of RC rigid-frame bridge tall piers: application to seismic fragility analysis	Research Square (Research Square)	2,023	cc-by	15,979	A measure for multiple bends and shear damage of RC rigid-frame bridge tall piers: application to seismic fragility analysis Zhu MEI Wenzhou University Yang Liu (  liuyang6886@163.com ) Wenzhou University https://orcid.org/0000-0002-6610-0309 Bin WU Wuhan University of Technology Oreste S. BURSI University of Trento: Universita degli Studi di Trento Da-gang LU Harbin Institute of Technology Fabrizio PAOLACCI Roma Tre University: Universita degli Studi Roma Tre A measure for multiple bends and shear damage of RC rigid-frame bridge tall piers: application to seismic fragility analysis Zhu MEI Wenzhou University Yang Liu (  liuyang6886@163.com ) Wenzhou University https://orcid.org/0000-0002-6610-0309 Bin WU Wuhan University of Technology Oreste S. BURSI University of Trento: Universita degli Studi di Trento Da-gang LU Harbin Institute of Technology Fabrizio PAOLACCI Roma Tre University: Universita degli Studi Roma Tre Research Article Keywords: damage measure, tall pier, damage pattern, hybrid test, component fragility Posted Date: September 8th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3321010/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License A measure for multiple bends and shear damage of RC rigid-frame bridge tall piers: application to seismic fragility analysis Zhu MEI Wenzhou University Yang Liu (  liuyang6886@163.com ) Wenzhou University https://orcid.org/0000-0002-6610-0309 Bin WU Wuhan University of Technology Oreste S. BURSI University of Trento: Universita degli Studi di Trento Da-gang LU Harbin Institute of Technology Fabrizio PAOLACCI Roma Tre University: Universita degli Studi Roma Tre Research Article Keywords: damage measure, tall pier, damage pattern, hybrid test, component fragility Posted Date: September 8th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3321010/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License * liuyang6886@163.com Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li A measure for multiple bends and shear damage of RC rigid-frame bridge tall 1 piers: application to seismic fragility analysis 2 Zhu MEI1,2, Yang LIU1,3,* Bin WU4, Oreste S. BURSI5, Da-gang LU6, Fabrizio PAOLACCI7 3 1. College of Civil Engineering and Architecture, Wenzhou University, Wenzhou 325035, China 4 2. Key Laboratory of Engineering and Technology for Soft Soil Foundation and Tideland Reclamation of Zhejiang Province, 5 Wenzhou 325035, China 6 3. Zhejiang Engineering Research Center of Disaster Prevention and Mitigation for Coastal Soft Soil Foundation, WenZhou 7 325035, China. 8 4. School of Civil Engineering and Architecture, Wuhan University of Technology, Wuhan 430070, China 9 5. Department of Civil, Environment and Mechanical Engineering, University of Trento, Trento 38123, Italy 10 6. School of Civil Engineering, Harbin Institute of Technology, Harbin 150090, China 11 7. Department of Engineering, Roma Tre University, Roma, Italy 12 Abstract: The reinforced-concrete (RC) rigid-frame bridges with tall hollow piers were widely 13 constructed in Southwestern China with many seismic activities. For such bridges, the seismic 14 damages would be underestimated because of the tall piers’ multiple bends caused by their 15 high-mode shapes that can be activated under earthquakes. Moreover, the seismic damage 16 assessment can be incorrect if we ignore the tall piers’ shear damages caused by their vastly 17 weakened sectional areas and multiple bends. With this respect, this paper presents a fragility 18 analysis for such bridges. A novel seismic damage measure (piecewise drift ratio, PDR) 19 involving shear effects and multiple bends is proposed and experimentally validated. Damage 20 states congruent to the PDR are provided by statistically acquiring from the 40 existing tests of 21 hollow piers. A finite element (FE) model of an RC rigid-frame bridge with two tall piers was 22 established and adequately calibrated based on the model-updating hybrid tests. To provide 23 more comprehensive assessments of the damage measures and the seismic fragility, the 24 ground motions were selected and divided into four categories by identifying their first two 25 frequencies and their amplitude ratio. Both the 5% damped spectral acceleration of the 26 fundamental period (Sa(T1, 5%)) and the peak ground acceleration (PGA) are adopted as the 27 demand parameter to develop the seismic demand models (PSDMs). Research Article Results show the 28 effectiveness of the proposed PDR, provide the more adverse types of ground motions, and 29 reveal the high exceedance probability of the complete damage state of tall piers. 30 K d d t ll i d tt h b id t t t f ilit 31 Keywords: damage measure, tall pier, damage pattern, hybrid test, component fragility 31 A measure for multiple bends and shear damage of RC rigid-frame bridge tall 1 piers: application to seismic fragility analysis 2 Zhu MEI1,2, Yang LIU1,3,* Bin WU4, Oreste S. BURSI5, Da-gang LU6, Fabrizio PAOLACCI7 3 1. College of Civil Engineering and Architecture, Wenzhou University, Wenzhou 325035, China 4 2. Key Laboratory of Engineering and Technology for Soft Soil Foundation and Tideland Reclamation of Zhejiang Province, 5 Wenzhou 325035, China 6 3. Zhejiang Engineering Research Center of Disaster Prevention and Mitigation for Coastal Soft Soil Foundation, WenZhou 7 325035, China. 8 4. School of Civil Engineering and Architecture, Wuhan University of Technology, Wuhan 430070, China 9 5. Department of Civil, Environment and Mechanical Engineering, University of Trento, Trento 38123, Italy 10 6. School of Civil Engineering, Harbin Institute of Technology, Harbin 150090, China 11 7. Department of Engineering, Roma Tre University, Roma, Italy 12 Abstract: The reinforced-concrete (RC) rigid-frame bridges with tall hollow piers were widely 13 constructed in Southwestern China with many seismic activities. For such bridges, the seismic 14 damages would be underestimated because of the tall piers’ multiple bends caused by their 15 high-mode shapes that can be activated under earthquakes. Moreover, the seismic damage 16 assessment can be incorrect if we ignore the tall piers’ shear damages caused by their vastly 17 weakened sectional areas and multiple bends. With this respect, this paper presents a fragility 18 analysis for such bridges. A novel seismic damage measure (piecewise drift ratio, PDR) 19 involving shear effects and multiple bends is proposed and experimentally validated. Damage 20 states congruent to the PDR are provided by statistically acquiring from the 40 existing tests of 21 hollow piers. A finite element (FE) model of an RC rigid-frame bridge with two tall piers was 22 established and adequately calibrated based on the model-updating hybrid tests. To provide 23 more comprehensive assessments of the damage measures and the seismic fragility, the 24 ground motions were selected and divided into four categories by identifying their first two 25 frequencies and their amplitude ratio. Both the 5% damped spectral acceleration of the 26 fundamental period (Sa(T1, 5%)) and the peak ground acceleration (PGA) are adopted as the 27 demand parameter to develop the seismic demand models (PSDMs). Results show the 28 effectiveness of the proposed PDR, provide the more adverse types of ground motions, and 29 reveal the high exceedance probability of the complete damage state of tall piers. 30 Keywords: damage measure, tall pier, damage pattern, hybrid test, component fragility 31 1 1. Introduction 1 1. Introduction 1 1.1 background and motivation 2 With the gradual development of mountainous areas accounting for 69.1% of China’s territory, 3 reinforced concrete (RC) rigid-frame bridges with tall piers have rapidly increased in China over 4 the past decade—the number of such bridges accounts for more than 40% of the total bridge 5 number. Among them, most of the tall piers were over 60m or even 100m; the rectangular 6 hollow cross-section is particularly common, as it can decrease the distribution pier mass to 7 mitigate its adverse effects on the seismic response without too much reducing the flexural 8 capacity. At the same time, most of these western mountainous regions face severe seismic 9 activities. For instance, several large earthquakes have attacked this region, like the Tonghai 10 earthquake (M7.8, Yunnan Province, 1970), Lancang-Gengma two great earthquakes (M7.6 11 and M7.2, Yunnan Province, 1988), Wenchuan earthquake (M8.0, Sichuan Province, 2008) and 12 Ya’an earthquake (M7.0, Sichuan Province, 2013). However, the tall-pier bridges in this region, 13 the essential lifeline transport infrastructures, were built within the last 10 years and have not 14 been tested by major earthquakes. Therefore, assessing the safety of such bridges under 15 significant disastrous events appears particularly important. Then, measuring the seismic 16 damage of their tall piers, which are the supporting members of the whole bridge, is generally 17 supposed to be super important in the performance assessment of the whole bridge system. 18 Among others, developing a reasonable damage measure that can accurately characterize the 19 structural damage [1] [2] is primary. 20 Damage pattern (bending, shear, or bending-shear damage) is a crucial characteristic of 21 [3] With the gradual development of mountainous areas accounting for 69.1% of China’s territory, 3 reinforced concrete (RC) rigid-frame bridges with tall piers have rapidly increased in China over 4 the past decade—the number of such bridges accounts for more than 40% of the total bridge 5 number. Among them, most of the tall piers were over 60m or even 100m; the rectangular 6 hollow cross-section is particularly common, as it can decrease the distribution pier mass to 7 mitigate its adverse effects on the seismic response without too much reducing the flexural 8 capacity. At the same time, most of these western mountainous regions face severe seismic 9 activities. 1. Introduction 1 For instance, several large earthquakes have attacked this region, like the Tonghai 10 earthquake (M7.8, Yunnan Province, 1970), Lancang-Gengma two great earthquakes (M7.6 11 and M7.2, Yunnan Province, 1988), Wenchuan earthquake (M8.0, Sichuan Province, 2008) and 12 Ya’an earthquake (M7.0, Sichuan Province, 2013). However, the tall-pier bridges in this region, 13 the essential lifeline transport infrastructures, were built within the last 10 years and have not 14 been tested by major earthquakes. Therefore, assessing the safety of such bridges under 15 significant disastrous events appears particularly important. Then, measuring the seismic 16 damage of their tall piers, which are the supporting members of the whole bridge, is generally 17 supposed to be super important in the performance assessment of the whole bridge system. 18 Among others, developing a reasonable damage measure that can accurately characterize the 19 structural damage [1] [2] is primary. 20 Damage pattern (bending, shear, or bending-shear damage) is a crucial characteristic of 21 Damage pattern (bending, shear, or bending-shear damage) is a crucial characteristic of 21 structural damage and must be involved[3] in a damage measure. Though giving us the 22 Damage pattern (bending, shear, or bending-shear damage) is a crucial characteristic of 21 structural damage and must be involved[3] in a damage measure. Though giving us the 22 2 impression of being slender and flexible, some RC tall-hollow piers’ shear-dominant brittle 1 failures were highlighted by experimental results, even if their aspect ratios are larger than 2[7- 2 8] or reached even 5[9, 11]. Therefore, the shear contribution must be correctly involved in a 3 damage measure for RC tall-hollow piers. In addition, the analysis and experimental results 4 show that the seismic performance of tall-pier bridges is significantly affected by higher-order 5 modes [14, 15]; tall piers under earthquakes can form some other plastic hinges along the pier 6 height except for the hinges at the bottom and top of the piers [16, 17]. Hence, the seismic 7 damage measure must have the ability to consider the effects of this specific damage 8 pattern—multiple bends along the pier height when tall piers’ seismic damages are evaluated. 9 For most ordinary RC bridge piers where bending failure is anticipated under 10 earthquakes, the quantities related to section curvature at the pier base are naturally selected 11 as the damage measure, for it can theoretically reflect the piers’ seismic damage. 1. Introduction 1 Accordingly, 12 the corresponding thresholds of section-curvature-related quantities for dividing damage 13 states are studied and provided [4-6]. However, the section-curvature-related damage measure 14 could not embrace the shear damage. Even so, the authors believe that the section curvature 15 could still be used for RC tall-hollow piers as long as the proportion of bending damage to the 16 total damage remains constant during the linear-nonlinear process under different 17 earthquakes. Then, the total damage can be evaluated through the section curvature and this 18 proportionality coefficient. In this respect, it needs reevaluation on whether the section 19 curvature is a suitable seismic damage measure for RC tall-hollow piers. 20 More generally, the pier-top displacement derivative quantities, such as the drift ratio, 21 are taken as the damage measure [5, 6, 12], as they could respond synchronically with the section 22 More generally, the pier-top displacement derivative quantities, such as the drift ratio, 21 are taken as the damage measure [5, 6, 12], as they could respond synchronically with the section 22 More generally, the pier-top displacement derivative quantities, such as the drift ratio, 21 are taken as the damage measure [5, 6, 12], as they could respond synchronically with the section 22 3 curvature and are more friendly to apply. Unlike the section curvature, the drift ratio includes 1 the shear effects and fit for RC piers facing bending and shear-dominant failure. Consequently, 2 in much research, we almost reached a consensus on the limit state values corresponding to 3 the drift ratio of RC solid piers [12, 13]. However, the pier-top drift ratio will ignore the multi- 4 bends effects, which may result in a contradiction that only a small pier-top displacement 5 appears despite severe damage. This will further lead to an underestimation of seismic 6 damage to tall piers, providing a false safety assessment that the piers are safe when they, in 7 fact, have already suffered serious damage. Nevertheless, the authors believe the pier-top drift 8 ratio could be used for tall-hollow piers if the increment of damage measure (DM) increases 9 in direct proportion to the increment of intense measure (IM). Therefore, the pier-top drift 10 ratio must also be further estimated for RC tall-hollow piers. 11 curvature and are more friendly to apply. 1. Introduction 1 Unlike the section curvature, the drift ratio includes 1 the shear effects and fit for RC piers facing bending and shear-dominant failure. Consequently, 2 in much research, we almost reached a consensus on the limit state values corresponding to 3 the drift ratio of RC solid piers [12, 13]. However, the pier-top drift ratio will ignore the multi- 4 bends effects, which may result in a contradiction that only a small pier-top displacement 5 appears despite severe damage. This will further lead to an underestimation of seismic 6 damage to tall piers, providing a false safety assessment that the piers are safe when they, in 7 fact, have already suffered serious damage. Nevertheless, the authors believe the pier-top drift 8 ratio could be used for tall-hollow piers if the increment of damage measure (DM) increases 9 in direct proportion to the increment of intense measure (IM). Therefore, the pier-top drift 10 ratio must also be further estimated for RC tall-hollow piers. 11 Except for the experiments carried out by other researchers, the damage patterns 12 associated with multiple bends and shear were observed obviously from our hybrid tests on a 13 rigid-frame tall-pier bridge. So, we reevaluated the two damage measures mentioned above. 14 Our evaluation results presented in the following parts show that the section curvature and 15 the pier-top drift ratio are unsuitable for those RC tall-hollow piers of rigid-frame bridges. 16 Therefore, a reasonable seismic damage measure that could include the two specific damage 17 patterns is necessary for such RC piers’ damage analysis and fragility assessment. 18 Except for the experiments carried out by other researchers, the damage patterns 12 associated with multiple bends and shear were observed obviously from our hybrid tests on a 13 rigid-frame tall-pier bridge. So, we reevaluated the two damage measures mentioned above. 14 Our evaluation results presented in the following parts show that the section curvature and 15 the pier-top drift ratio are unsuitable for those RC tall-hollow piers of rigid-frame bridges. 16 Therefore, a reasonable seismic damage measure that could include the two specific damage 17 patterns is necessary for such RC piers’ damage analysis and fragility assessment. 18 Developing seismic fragility curves is now an effective way to assess the seismic 19 performance of bridges [18-20]; they provide the probability of exceeding a damage threshold 20 over a range of potential earthquake ground motion intensities [21]. 1. Introduction 1 Significant bias in fragilities 21 can mislead the performance assessments and post-disaster rehabilitation strategies. The 22 Developing seismic fragility curves is now an effective way to assess the seismic 19 performance of bridges [18-20]; they provide the probability of exceeding a damage threshold 20 over a range of potential earthquake ground motion intensities [21]. Significant bias in fragilities 21 can mislead the performance assessments and post-disaster rehabilitation strategies. The 22 4 accuracy of fragility analysis explicitly depends on the damage limit states closely related to 1 the damage measure. The damage limit states are the limit states marked by the upper 2 thresholds when dividing the structural seismic damage into several states, i.e., damage states, 3 according to the damage measure. Therefore, the damage limit states must be provided along 4 with the new proposed damage measure to give assessments on the piers’ seismic safety. 5 1 2 Objective and methodology 6 In view of the multiple bends and shear damage pattern, a novel damage measure named the 7 piecewise drift ratio (PDR) is defined for RC tall-hollow piers. The limitation threshold values 8 for four seismic damage states are extrapolated through the statistical analysis based on test 9 data of 40 RC pier specimens. The damage measure (PDR) associated with the threshold values 10 is then used to assess the seismic damage of the tall-hollow pier belonging to a typical RC rigid- 11 frame bridge and validated by the bridge’s hybrid test results. Then, the standard fragility 12 analysis method under the PBEE framework proposed by PEER is performed to assess the 13 seismic damage of the bridge under various earthquakes so as further to present the 14 effectiveness and availability of the PDR. In the fragility analysis, the uncertainties from the 15 ground motions and the structure are calculated in terms of ‘record-to-record’ and ‘capacity 16 dispersion’, respectively. The FE model is built and validated by the hybrid tests for the analysis 17 of the damage measure and fragility curves. 18 In view of the multiple bends and shear damage pattern, a novel damage measure named the 7 piecewise drift ratio (PDR) is defined for RC tall-hollow piers. The limitation threshold values 8 for four seismic damage states are extrapolated through the statistical analysis based on test 9 data of 40 RC pier specimens. 1. Introduction 1 The damage measure (PDR) associated with the threshold values 10 is then used to assess the seismic damage of the tall-hollow pier belonging to a typical RC rigid- 11 frame bridge and validated by the bridge’s hybrid test results. Then, the standard fragility 12 analysis method under the PBEE framework proposed by PEER is performed to assess the 13 seismic damage of the bridge under various earthquakes so as further to present the 14 effectiveness and availability of the PDR. In the fragility analysis, the uncertainties from the 15 ground motions and the structure are calculated in terms of ‘record-to-record’ and ‘capacity 16 dispersion’, respectively. The FE model is built and validated by the hybrid tests for the analysis 17 of the damage measure and fragility curves. 18 The excited higher-mode shapes and the hollow section can be blamed for the multiple 19 bends and shear damage pattern of RC tall piers. Therefore, by identifying their first two 20 frequencies and the amplitude ratio between them, the ground motions were finely selected 21 and divided into four categories so that the bridge’s first- and second-mode shapes could be 22 The excited higher-mode shapes and the hollow section can be blamed for the multiple 19 bends and shear damage pattern of RC tall piers. Therefore, by identifying their first two 20 frequencies and the amplitude ratio between them, the ground motions were finely selected 21 and divided into four categories so that the bridge’s first- and second-mode shapes could be 22 5 fully activated. Consequently, the effectiveness of the proposed PDR can be evaluated more 1 comprehensively by deriving the PDR-based fragility curves of the tall-pier bridge under 2 different kinds of earthquakes. Though the fragility analysis of the bridge in this paper is a case 3 study, the relationship between the ground motions’ spectral characteristics and the degree 4 of the specific multi-bends-shear damage can be quantitatively discussed. 5 The superiority of hybrid testing for large structures, such as bridges, has been displayed by 7 many studies[22]-[24]. Among them, the authors’ previous study[16] shows that the model- 8 updating hybrid test can provide credible results, especially in capturing higher-mode shapes 9 of tall piers. 1. Introduction 1 To analyze the effectiveness of the existing and proposed damage measures and 10 then conduct the fragility analysis, an RC continuous bridge with tall-hollow piers constructed 11 in southwestern China is taken to be a case study prototype bridge. Its FE model was built and 12 calibrated based on the results of the model-updating hybrid tests. 13 calibrated based on the results of the model-updating hybrid tests. 13 2.1 Prototype bridge and brief introduction of the hybrid tests 14 The prototype bridge has three spans of 90m+170m+90m, shown as Part A in Figure 1. The 15 two piers are 126.06m in height and fixed with the beam. The hollow cross-sections of the two 16 tall piers are adopted with the width changing from 6.7m to 10.7m along the height of the pier, 17 2.1 Prototype bridge and brief introduction of the hybrid tests 14 2.1 Prototype bridge and brief introduction of the hybrid tests 14 2.1 Prototype bridge and brief introduction of the hybrid tests 14 The prototype bridge has three spans of 90m+170m+90m, shown as Part A in Figure 1. The 15 two piers are 126.06m in height and fixed with the beam. The hollow cross-sections of the two 16 tall piers are adopted with the width changing from 6.7m to 10.7m along the height of the pier, 17 while the length is a constant of 12m and the thickness is 0.8m. For more details on the bridge, 18 please see reference[16]. Several model-updating hybrid tests have been conducted, where the 19 physical substructure was the bottom part of the left pier with a 31.2m height (Figure 1 – Part 20 A), and its 1:12 scaled specimen (Figure 1 – Part B) was tested in the Structural and Seismic 21 Testing Center, Harbin Institute Technology. We build two OpenSees finite element models: i) 22 The prototype bridge has three spans of 90m+170m+90m, shown as Part A in Figure 1. The 15 two piers are 126.06m in height and fixed with the beam. The hollow cross-sections of the two 16 tall piers are adopted with the width changing from 6.7m to 10.7m along the height of the pier, 17 while the length is a constant of 12m and the thickness is 0.8m. For more details on the bridge, 18 please see reference[16]. 1. Introduction 1 Several model-updating hybrid tests have been conducted, where the 19 physical substructure was the bottom part of the left pier with a 31.2m height (Figure 1 – Part 20 A), and its 1:12 scaled specimen (Figure 1 – Part B) was tested in the Structural and Seismic 21 Testing Center, Harbin Institute Technology. We build two OpenSees finite element models: i) 22 6 one is for the numerical substructure containing the beam and the other parts of the pier, 1 illustrated as Part C in Figure 1; ii) the other is for the physical substructure, which is used to 2 identify the concrete model parameters during the tests. The results manifest that the 3 accuracy of the numerical substructure can be much improved by updating the concrete 4 parameters with the identified ones. The tests found that the failure of rigid-frame tall-pier 5 bridges characterized by the piers’ brittle shear-dominant failure. For readers interested in the 6 principle and details of the hybrid tests, please refer to the literature [16]. 7 principle and details of the hybrid tests, please refer to the literature [16]. 7 1 8 Figure 1 Overview of the hybrid tests carried on the prototype Bridge 9 Figure 1 Overview of the hybrid tests carried on the prototype Bridge 2.2 Model calibration with hybrid test data 10 7 An OpenSees model of the prototype bridge is established for the following analysis of damage 11 measure and seismic fragility. In order to ensure its accuracy, the model is improved from the 12 numerical substructure (NS) of the bridge’s hybrid tests, where the NS’s accuracy has been 13 validated through parameter identification. Compared to the NS, i) the part of the physical 14 substructure is complemented, as shown in Figure 2, ii) the element number of the beam and 15 7 An OpenSees model of the prototype bridge is established for the following analysis of damage 11 measure and seismic fragility. In order to ensure its accuracy, the model is improved from the 12 numerical substructure (NS) of the bridge’s hybrid tests, where the NS’s accuracy has been 13 validated through parameter identification. 1. Introduction 1 Compared to the NS, i) the part of the physical 14 substructure is complemented, as shown in Figure 2, ii) the element number of the beam and 15 piers is increased to describe the high-order features more precisely, where each pier was 1 modelled by 25 force-based beam-column elements, and the girder beam by 8 such elements. 2 2 3 Figure 2 Validated FE model of the bridge based on hybrid tests 4 3 Figure 2 Validated FE model of the bridge based on hybrid tests 4 Figure 2 Validated FE model of the bridge based on hybrid tests Except for the above two points, the other factors of modelling are all the same as those in the 5 NS. Such as, the cone-shaped cross-sections of the piers and the deck were modelled using 6 piecewise uniform subsections; a fully constrained fixed end was used to simulate the pier 7 base and the boundaries between the deck and piers; sliding bearings were used to simulate 8 the boundaries between the superstructure end and two piers of the approach bridge; the 9 Concrete01 model (Kent–Scott–Park model) and the Steel02 model (Giuffre–Menegotto–Pinto 10 model), as well as the parameter values in these two material model. It is worth noting that 11 the values of the parameters in the Concrete01 model, fc (compressive strength), fu (crushing 12 strength) and their corresponding strain (ε0 and εu), are those identified from the physical 13 substructure and updated to the numerical substructure in the hybrid tests. In this way, we 14 can ensure the accuracy of the bridge model for seismic fragility analysis. 15 Except for the above two points, the other factors of modelling are all the same as those in the 5 NS. Such as, the cone-shaped cross-sections of the piers and the deck were modelled using 6 piecewise uniform subsections; a fully constrained fixed end was used to simulate the pier 7 base and the boundaries between the deck and piers; sliding bearings were used to simulate 8 the boundaries between the superstructure end and two piers of the approach bridge; the 9 Concrete01 model (Kent–Scott–Park model) and the Steel02 model (Giuffre–Menegotto–Pinto 10 model), as well as the parameter values in these two material model. 1. Introduction 1 It is worth noting that 11 the values of the parameters in the Concrete01 model, fc (compressive strength), fu (crushing 12 strength) and their corresponding strain (ε0 and εu), are those identified from the physical 13 substructure and updated to the numerical substructure in the hybrid tests. In this way, we 14 can ensure the accuracy of the bridge model for seismic fragility analysis. 15 To comprehensively analyze the damage measure and develop the fragility curves, the tall 17 To comprehensively analyze the damage measure and develop the fragility curves, the tall 17 8 piers high-mode shapes must be activated in varying degrees, as they are significantly related 1 to the piers’ damage pattern. Therefore, we propose to select earthquake records by 2 identifying their frequencies around the pier’s first and second frequency and divide them into 3 two main categories: i) the ground motion whose dominant frequency is close to the bridge’s 4 first frequency; ii) the ground motion whose dominant frequency is close to the bridge’s 5 second frequency. As illustrated in Figure 3(a), the main frequencies of the first-type 6 earthquake records range from ‘f1- ‘ to ‘f1+ ’. Here f1 represents the bridge’s fundamental 7 frequency, in this case, 0.45Hz, and  equals 0.05Hz. Similarly, the main frequency of the 8 second-type ground motions can be determined as presented in Figure 3(b), where f2 is 1.21Hz. 1. Introduction 1 9 10 (a) Type-1 The dominant frequency close to the f1 (b) Type-2 The dominant frequency close to the f2 11 Figure 3 Categories of the earthquake records for seismic fragility analysis of a tall-pier bridge 12 10 10 (a) Type-1 The dominant frequency close to the f1 (b) Type-2 The dominant frequency close to the f2 11 Figure 3 Categories of the earthquake records for seismic fragility analysis of a tall-pier bridge 12 Then, two minor groups were refined for each main category according to the ratio of the 13 Then, two minor groups were refined for each main category according to the ratio of th first two frequencies, i) for the first main category, 14 𝐴2 𝐴1 ⁄ < 0.5 Type 1-1, 𝐴2 𝐴1 ⁄ ≥0.5 Type 1-2 15 ii) for the second main category, 16 𝐴1 𝐴2 ⁄ < 0.5 Type 2-1, 𝐴1 𝐴2 ⁄ ≥0.5 Type 2-2 17 𝐴1 𝐴2 ⁄ < 0.5 Type 2-1, 𝐴1 𝐴2 ⁄ ≥0.5 Type 2-2 17 where, A1 is the amplitude of the ground motion’s first frequency, f1’; A2 is the amplitude of 18 , 1 p g q y, f1 ; 2 p their second frequency, f2’; f1’ and f2’ are the frequency around f1 and f2 with a difference of 19 their second frequency, f2’; f1’ and f2’ are the frequency around f1 and f2 with a difference of 19 9 ‘±’’, respectively; Finally, ten earthquake records of each minor type, 40 in total, are selected 1 and presented in Table 1Table 1. One earthquake record for each type is taken out, and the 2 results of their frequencies obtained by the Fast Fourier Transform are shown in Figure 4. 3 and presented in Table 1Table 1. One earthquake record for each type is taken out, and the 2 results of their frequencies obtained by the Fast Fourier Transform are shown in Figure 4. 3 Table 1 Characteristics of ground motions 4 No. Earth. 𝐴2 𝐴1 ⁄ (%) MW Distance (km) No Earth. 1. Introduction 1 Tests show that hollow piers 11 with an aspect ratio larger than 2[7]-[8] or even 4[9], [10] still suffer shear-dominant failure. Besides, 12 many diagonal cracks were still observed for a hollow RC pier with an aspect ratio even 13 reaching 8. This is because the hollow cross-section area is considerably reduced, significantly 14 decreasing shear strength, and hence, non-negligible shear damage appears. Moreover, the 15 maximum shear stress on a hollow cross-section greatly increases due to the different shear 16 stress distributions, while the maximum bending stress remains almost unchanged. So, the 17 shear stress can reach the allowable stress first. This further increases the shear effects. 18 ii) The high-order responses of the tall-pier bridges can be activated, leading to more plastic 19 hinges formed along the pier height. The multiple bends cut a tall pier into several short piers, 20 further decreasing the aspect ratio of tall piers. In our hybrid tests, the aspect ratio of the fixed- 21 end tall pier was supposed to be 5.25, computing through the half height of the pier divided 22 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers 3 There are two kinds of commonly used seismic damage measures for RC piers: section 4 curvature and pier-top displacement. As mentioned above, they need reevaluation for RC tall- 5 hollow piers exhibiting high-mode shapes and shear-dominant failure under earthquakes. 6 Some RC tall-hollow piers face shear-dominant brittle failure, though they give us the 8 impression of being slender and flexible. This phenomenon can be attributed to two main 9 factors based on our previous study[10]: 10 Some RC tall-hollow piers face shear-dominant brittle failure, though they give us the 8 impression of being slender and flexible. This phenomenon can be attributed to two main 9 factors based on our previous study[10]: 10 Some RC tall-hollow piers face shear-dominant brittle failure, though they give us the 8 impression of being slender and flexible. This phenomenon can be attributed to two main 9 factors based on our previous study[10]: 10 i) The hollow cross-section is the preferable choice for tall piers. Tests show that hollow piers 11 with an aspect ratio larger than 2[7]-[8] or even 4[9], [10] still suffer shear-dominant failure. Besides, 12 many diagonal cracks were still observed for a hollow RC pier with an aspect ratio even 13 reaching 8. This is because the hollow cross-section area is considerably reduced, significantly 14 decreasing shear strength, and hence, non-negligible shear damage appears. Moreover, the 15 maximum shear stress on a hollow cross-section greatly increases due to the different shear 16 stress distributions, while the maximum bending stress remains almost unchanged. So, the 17 shear stress can reach the allowable stress first. This further increases the shear effects. 18 i) The hollow cross-section is the preferable choice for tall piers. Tests show that hollow piers 11 with an aspect ratio larger than 2[7]-[8] or even 4[9], [10] still suffer shear-dominant failure. Besides, 12 many diagonal cracks were still observed for a hollow RC pier with an aspect ratio even 13 reaching 8. This is because the hollow cross-section area is considerably reduced, significantly 14 decreasing shear strength, and hence, non-negligible shear damage appears. Moreover, the 15 maximum shear stress on a hollow cross-section greatly increases due to the different shear 16 stress distributions, while the maximum bending stress remains almost unchanged. So, the 17 shear stress can reach the allowable stress first. This further increases the shear effects. 1. Introduction 1 𝐴1 𝐴2 ⁄ (%) MW Distance (km) Type1-1 𝐴2 𝐴1 < 0.5 1 RSN38 38 6.63 199.84 Type2-1 𝐴1 𝐴2 < 0.5 21 RSN8 26 6.4 44.52 2 RSN39 30 6.63 207.14 22 RSN14 25 7.36 81.3 3 RSN53 23 6.61 111.88 23 RSN24 8 5 7.28 4 RSN76 31 6.61 109.01 24 RSN108 31 4.79 12.07 5 RSN90 34 6.61 124.38 25 RSN110 28 4.37 13.37 6 RSN532 43 6.06 77.98 26 RSN121 7 6.5 49.13 7 RSN1114 38 6.9 3.31 27 RSN130 16 5.91 10.99 8 RSN1356 27 7.62 97.5 28 RSN165 36 6.53 7.29 9 RSN1365 37 7.62 119.31 29 RSN392 39 5.38 12.05 10 RSN3044 21 6.2 119.18 30 RSN608 11 5.99 26.3 Type1-2 𝐴2 𝐴1 ≥0.5 11 RSN7 54 6.6 91.15 Type2-2 𝐴1 𝐴2 ≥0.5 31 RSN6 50 6.59 6.09 12 RSN9 88 6.5 56.88 32 RSN10 83 5.6 24.58 13 RSN12 63 7.36 114.62 33 RSN15 56 7.36 38.42 14 RSN175 65 6.53 17.94 34 RSN163 85 6.53 23.17 15 RSN181 93 6.53 0 35 RSN182 56 6.53 0.56 16 RSN183 91 6.53 3.86 36 RSN293 59 6.9 59.63 17 RSN186 97 6.53 35.64 37 RSN1101 70 6.9 11.34 18 RSN187 66 6.53 12.69 38 RSN1244 74 7.62 9.94 19 RSN575 87 7.3 56.87 39 RSN1491 88 7.62 7.64 20 RSN584 72 7.3 58 40 RSN1496 57 7.62 10.48 5 (a) RSN1356 (Type 1-1) (b) RSN181 (Type 1-2) (c) RSN608 (Type 2-1) (d) RSN1101 (Type 2-2) 6 (a) RSN1356 (Type 1-1) (b) RSN181 (Type 1-2) (c) RSN608 (Type 2-1) (d) RSN1101 (Type 2-2) 6 10 Figure 4 Fast Fourier transform of the earthquake record of each minor type 1 11 4. Seismic damage measure for RC tall-hollow piers 2 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers 3 There are two kinds of commonly used seismic damage measures for RC piers: section 4 curvature and pier-top displacement. As mentioned above, they need reevaluation for RC tall- 5 hollow piers exhibiting high-mode shapes and shear-dominant failure under earthquakes. 6 4.1.1 Section curvature 7 Some RC tall-hollow piers face shear-dominant brittle failure, though they give us the 8 impression of being slender and flexible. This phenomenon can be attributed to two main 9 factors based on our previous study[10]: 10 i) The hollow cross-section is the preferable choice for tall piers. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers However, if the effect of multiple bends was involved, the aspect 1 ratio of the tall pier in our tests is actually 2.1. 2 If we combine the two factors, the aspect ratio of the tall pier is only 1.53, which may 3 lead to shear-dominant failure. The tall pier actually appears to have a shear-dominant brittle 4 failure, marked by a main wide diagonal crack and the fracture of a few stirrups. Regarding the 5 combination computing approach of the aspect ratio of hollow RC tall piers, please refer to our 6 previous work[10]. 7 If we combine the two factors, the aspect ratio of the tall pier is only 1.53, which may 3 lead to shear-dominant failure. The tall pier actually appears to have a shear-dominant brittle 4 failure, marked by a main wide diagonal crack and the fracture of a few stirrups. Regarding the 5 combination computing approach of the aspect ratio of hollow RC tall piers, please refer to our 6 previous work[10]. 7 Therefore, a seismic damage measure for such shear-dominant RC tall piers must 8 appropriately involve the shear effects and accurately describe the shear damage pattern. For 9 those RC tall piers where bending failure is anticipated, like medium-height RC piers, the 10 section curvature can still be selected, for it can naturally describe the damage pattern[25]. For 11 the RC tall piers facing shear-dominant damage, the authors believe that the section curvature 12 could be used as a damage measure if the seismic damage deduced by bending is linear with 13 the total damage caused by both the bending and shear effects. To this end, the time-history 14 analysis is carried out with the FE model of the physical substructure whose parameters are 15 the identified ones. Selecting only the physical substructure as a study case can exclude the 16 influence of multiple bends. Two records of ground motions, the RSN6 and RSN24, are chosen 17 randomly, and their PGAs are scaled from 0.07g to 2.0g. We use the total displacement at the 18 top to describe the whole damage of the pier and the displacement associated with shear to 19 represent the shear damage. For each PGA, the maximum total displacement and the shear 20 displacement are presented in Figure 5. It shows that the proportion of bending displacement 21 to the total displacement differs from one PGA to another. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers 18 ii) The high-order responses of the tall-pier bridges can be activated, leading to more plastic 19 hinges formed along the pier height. The multiple bends cut a tall pier into several short piers, 20 further decreasing the aspect ratio of tall piers. In our hybrid tests, the aspect ratio of the fixed- 21 end tall pier was supposed to be 5.25, computing through the half height of the pier divided 22 11 by the cross-section height. However, if the effect of multiple bends was involved, the aspect 1 ratio of the tall pier in our tests is actually 2.1. 2 If we combine the two factors, the aspect ratio of the tall pier is only 1.53, which may 3 lead to shear-dominant failure. The tall pier actually appears to have a shear-dominant brittle 4 failure, marked by a main wide diagonal crack and the fracture of a few stirrups. Regarding the 5 combination computing approach of the aspect ratio of hollow RC tall piers, please refer to our 6 previous work[10]. 7 Therefore, a seismic damage measure for such shear-dominant RC tall piers must 8 appropriately involve the shear effects and accurately describe the shear damage pattern. For 9 those RC tall piers where bending failure is anticipated, like medium-height RC piers, the 10 section curvature can still be selected, for it can naturally describe the damage pattern[25]. For 11 the RC tall piers facing shear-dominant damage, the authors believe that the section curvature 12 could be used as a damage measure if the seismic damage deduced by bending is linear with 13 the total damage caused by both the bending and shear effects. To this end, the time-history 14 analysis is carried out with the FE model of the physical substructure whose parameters are 15 the identified ones. Selecting only the physical substructure as a study case can exclude the 16 influence of multiple bends. Two records of ground motions, the RSN6 and RSN24, are chosen 17 randomly, and their PGAs are scaled from 0.07g to 2.0g. We use the total displacement at the 18 top to describe the whole damage of the pier and the displacement associated with shear to 19 represent the shear damage. For each PGA, the maximum total displacement and the shear 20 displacement are presented in Figure 5. It shows that the proportion of bending displacement 21 by the cross-section height. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers Finding a constant coefficient to 22 12 gain the total structural damage is hard. This indicates that the section curvature is not a 1 suitable measure to assess the damage of shear-dominant RC piers. It is worth noting that 2 directly using the section curvature as the damage measure would dramatically underestimate 3 the seismic damage of such piers. 4 5 Figure 5 The proportion of bending displacement to total displacement 6 5 Figure 5 The proportion of bending displacement to total displacement 6 Figure 5 The proportion of bending displacement to total displacement 6 Figure 5 The proportion of bending displacement to total displacement 4.1.2 Displacement at the pier top 7 The pier-top drift ratio, a displacement-related application-friendly quantity, is another widely 8 used seismic damage measure, for it can respond in phase with the pier-base section curvature. 9 In addition, it can also take the shear effects into account when applied to shear-dominant RC 10 piers. However, some investigations found that several plastic hinges may be formed along the 11 height of tall piers due to the influence of high-order modes[17], [26]. A similar phenomenon was 12 observed in our hybrid tests, where three main vibration shapes appeared, as shown in Figure 13 6 by the solid lines. For comparison, the first-order vibration shapes with the same 14 displacement at the pier top are drawn by dashed lines. The locations of the plastic hinges, 15 around 35m-, 55m-, 80m- and 105m-height, are determined based on the section curvature. 16 The pier-top drift ratio, a displacement-related application-friendly quantity, is another widely 8 used seismic damage measure, for it can respond in phase with the pier-base section curvature. 9 In addition, it can also take the shear effects into account when applied to shear-dominant RC 10 piers. However, some investigations found that several plastic hinges may be formed along the 11 height of tall piers due to the influence of high-order modes[17], [26]. A similar phenomenon was 12 observed in our hybrid tests, where three main vibration shapes appeared, as shown in Figure 13 6 by the solid lines. For comparison, the first-order vibration shapes with the same 14 displacement at the pier top are drawn by dashed lines. The locations of the plastic hinges, 15 around 35m-, 55m-, 80m- and 105m-height, are determined based on the section curvature. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers 16 13 13 1 Figure 6 Vibration shapes of the bridge pier and distribution of plastic hinges 2 Figure 6 Vibration shapes of the bridge pier and distribution of plastic hinges Take the vibration shape at 1.85s as an example, shown in Figure 7, we use Dtop and Hfull 3 to represent the displacement at the pier top and the full height of the pier; then, the drift 4 ratio at the pier top, Rd, can be computed as follows, 5 𝑅d = 𝐷top/𝐻full (1) 𝑅d = 𝐷top/𝐻full 6 𝑅d = 𝐷top/𝐻full (1) 6 (1) It is seen that, by the same drift ratio Rd, the tall bridge pier could be in different damage states 7 with or w/o multiple bends, which can lead the pier-top displacement to be an in 8 with or w/o multiple bends, which can lead the pier-top displacement to be an invalid damage 8 measure for tall piers. 9 10 Figure 7 Two possible damage states with the same drift ratio 11 Figure 7 Two possible damage states with the same drift ratio Figure 7 Two possible damage states with the same drift ratio 14 Even so, the authors believe the pier-top drift ratio could be used for tall piers as long as 12 the increment of damage measure (DM) increases in direct proportion to the increment of 13 intense measure (IM), as shown in Figure 8 and Formula (2). In other words, though the 14 displacement responses at the pier top, according to the existing thresholds, are inconsistent 15 with the actual damage, the drift ratio could be used as long as it can keep pace with the 16 14 damage states and represent it. 1 𝛥𝐷𝑀𝑖 𝛥𝐼𝑀𝑖= 𝐷𝑀𝑖−𝐷𝑀𝑖−1 𝐼𝑀𝑖−𝐼𝑀𝑖−1 = 𝑘𝑖> 0 (2) 𝛥𝐷𝑀𝑖 𝛥𝐼𝑀𝑖= 𝐷𝑀𝑖−𝐷𝑀𝑖−1 𝐼𝑀𝑖−𝐼𝑀𝑖−1 = 𝑘𝑖> 0 (2) 2 (2) where DMi indicates the damage measure of the ith damage state, and IMi defines the ith level 3 where DMi indicates the damage measure of the ith damage state, and IMi defines the ith level 3 of the seismic intensity. Among all the cases where ki>0, ki=ki-1 is the ideal scenario, implying 4 that the relationship between DM and IM is linear. 5 that the relationship between DM and IM is linear. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers 5 6 Figure 8 Reasonable relationship between damage measure and intense measure 7 Figure 8 Reasonable relationship between damage measure and intense measure To this end, the time history analysis based on the validated model of the RC tall piers is 8 conducted. Twenty Type2-2 earthquake records presented in Table 1 are adopted to activate 9 the second-order responses better. The PGA of each earthquake record is adjusted from 0.07g 10 to 2g, the drift ratios at the pier top, , are calculated. The  versus PGA relationship curves 11 are obtained and illustrated in Figure 9. For clarity, the drift ratios on Y-axis were normalized, 12 where the value corresponding to the case of PGA=2g was set to be 1. The -PGA curves we 13 obtained can be broadly divided into three types: i) most curves have a segment with negative 14 ki, as highlighted in the red segment lines in Figure 9; ii) several curves have the ki > 0 at all the 15 ith damage states, such as those by RSN6, RSN24, RSN182 and RSN1180; iii) few -PGA curves, 16 only by RSN22, are approximately linear. 17 To this end, the time history analysis based on the validated model of the RC tall piers is 8 conducted. Twenty Type2-2 earthquake records presented in Table 1 are adopted to activate 9 the second-order responses better. The PGA of each earthquake record is adjusted from 0.07g 10 to 2g, the drift ratios at the pier top, , are calculated. The  versus PGA relationship curves 11 are obtained and illustrated in Figure 9. For clarity, the drift ratios on Y-axis were normalized, 12 where the value corresponding to the case of PGA=2g was set to be 1. The -PGA curves we 13 obtained can be broadly divided into three types: i) most curves have a segment with negative 14 ki, as highlighted in the red segment lines in Figure 9; ii) several curves have the ki > 0 at all the 15 ith damage states, such as those by RSN6, RSN24, RSN182 and RSN1180; iii) few -PGA curves, 16 only by RSN22, are approximately linear. 17 15 The results indicate that the pier-top drift ratio cannot accurately characterize the 18 increasing damage of tall piers under earthquakes. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers Thereby, a novel damage 15 14 16 16 measure, named the piecewise drift ratio (PDR), is proposed as follows, 1 measure, named the piecewise drift ratio (PDR), is proposed as follows, 1 measure, named the piecewise drift ratio (PDR), is proposed as follows, 1 𝑅d,𝑖= 𝑖/𝐻𝑖 𝑅d,𝑖= 𝑖/𝐻𝑖 (5) 2 𝑅d p = max( 𝑅d 𝑖) (6) 3 (5) d,𝑖 𝑖/ 𝑖 ( ) 𝑅d p = max( 𝑅d,𝑖) (6) 3 𝑅d p = max( 𝑅d,𝑖) (6) where Rd,i is the drift ratio for each pier piece i. For all i, the maximum value of Rd,i , max(Rd,i), 4 is the proposed piecewise drift ratio of a tall pier, 𝑅d p. For the calculation of Rd,i, i is the relative 5 displacement between the two adjacent plastic hinges, and Hi is the height of each pier 6 segment. This way, the multiple-bends factor can be involved by converting the tall pier into 7 several short-to-medium piers. Meanwhile, the shear effects can be also taken into account by 8 continuously using the drift ratio as a damage measure. 9 (b) Locations of plastic hinges 10 The piecewise drift ratio (PDR) is defined based on the locations of plastic hinges. The 11 feasibility of the PDR can be much improved if the positions of the plastic hinges remain 12 unchanged along the pier height under various ground motions. We all know that the 13 superimposed number of structural mode shapes influences the plastic-hinge positions. For 14 regular RC piers, the mechanism associated with higher modes is possible but not likely to 15 develop during known realistic ground motions [27], [28]. So, the first-order vibration shape 16 strongly contributes to the locations of plastic hinges, where the location at the pier base for 17 the free-end piers, at both the pier base and pier top for the fixed-end piers. 18 The piecewise drift ratio (PDR) is defined based on the locations of plastic hinges. The 11 feasibility of the PDR can be much improved if the positions of the plastic hinges remain 12 unchanged along the pier height under various ground motions. We all know that the 13 superimposed number of structural mode shapes influences the plastic-hinge positions. For 14 regular RC piers, the mechanism associated with higher modes is possible but not likely to 15 develop during known realistic ground motions [27], [28]. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers It is not an effective damage measure for 19 15 The results indicate that the pier-top drift ratio cannot accurately characterize the 18 increasing damage of tall piers under earthquakes. It is not an effective damage measure for 19 15 tall piers with multiple bends. When the negative segment appears, the estimated seismic 1 damage based on the pier-top drift ratio will be incorrect. 2 damage based on the pier-top drift ratio will be incorrect. 2 damage based on the pier-top drift ratio will be incorrect. 2 damage based on the pier-top drift ratio will be incorrect. 2 3 Figure 9 Increments of drift ratio versus PGA 4 Figure 9 Increments of drift ratio versus PGA 4 Figure 9 Increments of drift ratio versus PGA 4.2 Damage measure incorporating the multiple-bends factor and the shear effects 5 4.2.1 Damage measure defined as piecewise drift ratio 6 A reasonable damage measure for tall piers should involve the multiple-inflexions factor and 8 the shear effects. As mentioned above, we found through hybrid tests that more than one 9 plastic hinge will form along the pier height of a tall pier. With this respect, we propose 10 partitioning the tall pier into several segments by the plastic hinges, as shown in Figure 10. 11 A reasonable damage measure for tall piers should involve the multiple-inflexions factor and 8 the shear effects. As mentioned above, we found through hybrid tests that more than one 9 plastic hinge will form along the pier height of a tall pier. With this respect, we propose 10 partitioning the tall pier into several segments by the plastic hinges, as shown in Figure 10. 11 12 Figure 10 Schematic diagram of the piecewise drift ratio (𝑅d p) 13 12 Figure 10 Schematic diagram of the piecewise drift ratio (𝑅d p) 13 Figure 10 Schematic diagram of the piecewise drift ratio (𝑅d p) 13 Then, each piece of the tall pier could be considered a low- or medium-height pier. Th Then, each piece of the tall pier could be considered a low- or medium-height pier. The 14 drift ratio that involves the shear effects can continue to be used. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers So, the first-order vibration shape 16 strongly contributes to the locations of plastic hinges, where the location at the pier base for 17 the free-end piers, at both the pier base and pier top for the fixed-end piers. 18 For tall-pier bridges, their first frequencies become smaller due to the flexible tall piers, 19 and their second frequencies get closer to the fundamental frequency of the regular bridges. 20 Consequently, ground motions are expected to activate their second-mode shape [28]. The 21 third-mode shape then becomes the one that is hard to be activated. Hence, the tall piers’ 22 For tall-pier bridges, their first frequencies become smaller due to the flexible tall piers, 19 and their second frequencies get closer to the fundamental frequency of the regular bridges. 20 Consequently, ground motions are expected to activate their second-mode shape [28]. The 21 third-mode shape then becomes the one that is hard to be activated. Hence, the tall piers’ 22 17 plastic-hinge positions are influenced only by the superposition of the first two mode shapes. 1 With this respective, 40 times analyses were conducted on the validated model using the 40 2 ground motions listed in Table 1. Results are drawn in Figure 11. It shows that there is almost 3 no change in the plastic-hinge positions. The possible positions of the tall pier are 0m-, 35m-, 4 55m-, 80m-, 106m- and 126.06m-height along the pier. Accordingly, the Hi used for computing 5 the piecewise drift ratio is 35m, 20m, 25m, 26m and 26.06m when i equals 1, 2, 3, 4 and 5. So, 6 we do not need to calculate the Hi for every ground motion at every intense level. 7 we do not need to calculate the Hi for every ground motion at every intense le 7 8 Figure 11 Distribution of section curvature along pier height 9 8 Figure 11 Distribution of section curvature along pier height 9 Figure 11 Distribution of section curvature along pier height Figure 11 Distribution of section curvature along pier height 9 4.2.2 Statistic damage limit states for the PDR 10 4.2.2 Statistic damage limit states for the PDR 10 As mentioned above, plastic hinges along the pier height can divide the tall pier into several 11 pieces of medium-height piers. The piecewise drift ratio is the maximum value among the drift 12 ratio of each piece-pier. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers For each piece-pier, the damage measure is precisely the drift ratio of 13 a medium-height pier. However, the most widely used threshold values of the damage states 14 (i.e. the damage limit state) are derived from the statistics of the medium-height piers with 15 solid cross-sections. This study provides the statistic capacity thresholds of hollow piers for 16 reference through 40 existing tests of hollow piers. The pier-top drift ratio of each pier 17 specimen at each damage state is obtained by analyzing the test results from the references. 18 As mentioned above, plastic hinges along the pier height can divide the tall pier into several 11 pieces of medium-height piers. The piecewise drift ratio is the maximum value among the drift 12 ratio of each piece-pier. For each piece-pier, the damage measure is precisely the drift ratio of 13 a medium-height pier. However, the most widely used threshold values of the damage states 14 (i.e. the damage limit state) are derived from the statistics of the medium-height piers with 15 solid cross-sections. This study provides the statistic capacity thresholds of hollow piers for 16 reference through 40 existing tests of hollow piers. The pier-top drift ratio of each pier 17 specimen at each damage state is obtained by analyzing the test results from the references. 18 18 The main damage characteristics of each damage state are presented in Error! Reference 1 source not found.. The statistical results on the drift ratio are illustrated in Table 2. 2 source not found.. The statistical results on the drift ratio are illustrated in Table 2. 2 source not found.. The statistical results on the drift ratio are illustrated in Table 2. 3 Table 2 Statistical values of the thresholds for each damage state 4 19 3 Table 2 Statistical values of the thresholds for each damage state 4 Table 3 Damage characteristics and conditions for determination of  5 Damage state Damage characteristics Slight A few bending cracks distributed along the pier height Moderate The Yield of longitudinal bars, increased number of bending cracks, appeared and expanded inclined cracks Severe The specimen strength decreased 20% of the peak strength, the width of inclined cracks reached 2mm, the cover concrete spalled Complete The specimen strength decreased 40% of the peak strength, the cover concrete completely spalled It manifests that the seismic statistic capacities follow a lognormal distribution. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers The mean 6 values are adopted in this paper to be the thresholds of each damage state and lists in Table 7 4, where the logarithmic standard deviation for each damage state is also listed. The standard 8 deviations are relatively small and meet the regular: the dispersion of the seismic capacities 9 decreases with the increase of damage level. This is because the slight damage state is more 10 accessible through different mechanical paths, which leads to different capacity values. For 11 detailed information on the specimens from the references, please see the Appendix. 12 Table 4 Thresholds for damage states 13 Damage state Mean Value (/H) Thresholds (/H) Standard Deviation of ln(/H) Slight 0.002 ＜0.002 0.412 Table 2 Statistical values of the thresholds for each damage state Table 3 Damage characteristics and conditions for determination of  5 Damage state Damage characteristics Slight A few bending cracks distributed along the pier height Moderate The Yield of longitudinal bars, increased number of bending cracks, appeared and expanded inclined cracks Severe The specimen strength decreased 20% of the peak strength, the width of inclined cracks reached 2mm, the cover concrete spalled Complete The specimen strength decreased 40% of the peak strength, the cover concrete completely spalled It manifests that the seismic statistic capacities follow a lognormal distribution. The mean 6 values are adopted in this paper to be the thresholds of each damage state and lists in Table 7 4, where the logarithmic standard deviation for each damage state is also listed. The standard 8 deviations are relatively small and meet the regular: the dispersion of the seismic capacities 9 decreases with the increase of damage level. This is because the slight damage state is more 10 accessible through different mechanical paths, which leads to different capacity values. For 11 detailed information on the specimens from the references, please see the Appendix. 12 Table 4 Thresholds for damage states 13 Damage state Mean Value (/H) Thresholds (/H) Standard Deviation of ln(/H) Slight 0.002 ＜0.002 0.412 19 Moderate 0.009 ≥0.002, ＜0.009 0.231 Severe 0.016 ≥0.009, ＜0.0016 0.128 Complete 0.023 ≥0.023 0.111 4.2.3 Comparison of the damage measures 1 To further compare the three seismic damage measures, the piecewise drift ratio (PDR), 2 the drift ratio (DR) and the section curvature (SC), we carried out the time history analysis on 3 the bridge model under eight earthquake records. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers The records are randomly selected from 4 Table 1, with two for each type. They are RSN39, RSN76, RSN187, RSN575, RSN165, RSN392, 5 RSN10 and RSN6, among which the RSN6 is the one for model-updating hybrid tests. 6 Using the PDR and the limit states provided in this paper, the damage assessment results 7 of the RC tall pier under the RSN6 were proved to be accurate by the test results, as they are 8 consistent with the observed damage of the physical substructure. Then, based on the PDR, 9 the seismic damage under the other earthquakes with different PGAs is assessed and 10 illustrated in Figure 12. Take the PDR assessment results as the criterion: the same-, over- or 11 under-estimations of the damage based on the DR or SC are also presented in Figure 12. The 12 ‘same-estimation’ represents that the estimated damage state based on the DR or SC is the 13 same as that based on the PDR, which is illustrated in Figure 12 by solid grey; the ‘over- 14 estimation’, illustrated by solid green, represents that the estimated result is more severe; the 15 ‘under-estimation’, illustrated by solid red, is slighter. The rectangle with a triangle describes 16 that, though the estimated damage state is correct, the estimated damage value at the current 17 PGA is smaller than the value at the previous PGA. 18 4.2.3 Comparison of the damage measures 1 To further compare the three seismic damage measures, the piecewise drift ratio (PDR), 2 the drift ratio (DR) and the section curvature (SC), we carried out the time history analysis on 3 the bridge model under eight earthquake records. The records are randomly selected from 4 Table 1, with two for each type. They are RSN39, RSN76, RSN187, RSN575, RSN165, RSN392, 5 RSN10 and RSN6, among which the RSN6 is the one for model-updating hybrid tests. 6 To further compare the three seismic damage measures, the piecewise drift ratio (PDR), 2 the drift ratio (DR) and the section curvature (SC), we carried out the time history analysis on 3 the bridge model under eight earthquake records. The records are randomly selected from 4 Table 1, with two for each type. They are RSN39, RSN76, RSN187, RSN575, RSN165, RSN392, 5 RSN10 and RSN6, among which the RSN6 is the one for model-updating hybrid tests. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers 6 To further compare the three seismic damage measures, the piecewise drift ratio (PDR), 2 the drift ratio (DR) and the section curvature (SC), we carried out the time history analysis on 3 the bridge model under eight earthquake records. The records are randomly selected from 4 Table 1, with two for each type. They are RSN39, RSN76, RSN187, RSN575, RSN165, RSN392, 5 RSN10 and RSN6, among which the RSN6 is the one for model-updating hybrid tests. 6 Using the PDR and the limit states provided in this paper, the damage assessment results 7 of the RC tall pier under the RSN6 were proved to be accurate by the test results, as they are 8 consistent with the observed damage of the physical substructure. Then, based on the PDR, 9 the seismic damage under the other earthquakes with different PGAs is assessed and 10 illustrated in Figure 12. Take the PDR assessment results as the criterion: the same-, over- or 11 under-estimations of the damage based on the DR or SC are also presented in Figure 12. The 12 ‘same-estimation’ represents that the estimated damage state based on the DR or SC is the 13 same as that based on the PDR, which is illustrated in Figure 12 by solid grey; the ‘over- 14 estimation’, illustrated by solid green, represents that the estimated result is more severe; the 15 ‘under-estimation’, illustrated by solid red, is slighter. The rectangle with a triangle describes 16 that, though the estimated damage state is correct, the estimated damage value at the current 17 PGA is smaller than the value at the previous PGA. 18 Using the PDR and the limit states provided in this paper, the damage assessment results 7 of the RC tall pier under the RSN6 were proved to be accurate by the test results, as they are 8 consistent with the observed damage of the physical substructure. Then, based on the PDR, 9 the seismic damage under the other earthquakes with different PGAs is assessed and 10 illustrated in Figure 12. Take the PDR assessment results as the criterion: the same-, over- or 11 under-estimations of the damage based on the DR or SC are also presented in Figure 12. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers assess if the demand based on the PDR is well 21 𝑃(𝐷≥𝐶\|𝐼𝑀) = 𝛷[ ln ( 𝑚𝐷)−ln (𝑚𝐶) √𝛽𝐷\|𝐼𝑀 2 +𝛽𝐶 2 ] (7) 3 𝑃(𝐷≥𝐶\|𝐼𝑀) = 𝛷[ ln ( 𝑚𝐷)−ln (𝑚𝐶) √𝛽𝐷\|𝐼𝑀 2 +𝛽𝐶 2 ] (7) 3 (7) where (▪) is the cumulative distribution function of the standard normal distribution; mD and 4 where (▪) is the cumulative distribution function of the standard normal distribution; mD and 4 D\|IM are the median and the dispersion of the seismic demand, respectively, and can be 5 where (▪) is the cumulative distribution function of the standard normal distribution; mD and 4 D\|IM are the median and the dispersion of the seismic demand, respectively, and can be 5 estimated based on DM data generated by an incremental dynamic analysis (IDA); mC and C 6 are the median and dispersion of the seismic capacity, and can be obtained by statistical 7 calculation using the extrapolated experimental damage limit state data. 8 D\|IM are the median and the dispersion of the seismic demand, respectively, a estimated based on DM data generated by an incremental dynamic analysis (IDA); mC and C 6 are the median and dispersion of the seismic capacity, and can be obtained by statistical 7 calculation using the extrapolated experimental damage limit state data. 8 The Sa(T1,5%) and PGA are the widely employed IMs when developing the probabilistic 9 seismic demand models (PSDSs) due to their efficiency and sufficiency that have been proved. 10 Some studies show that taking Sa as IM may lead to a more efficient PSDM for long-period 11 structures, as the correlation between the seismic responses and the Sa is stronger. However, 12 taking the PGA as IM can provide the damages more directly related to seismic intensity so 13 that the seismic responses and damages can be compared under a unified seismic intensity. 14 Therefore, two PSDMs are developed in this research, respectively, based on Sa and PGA. 15 22 Then, the responses of the bridge were computed through IDA analysis of the 40 ground 16 motions. The PSDS is established by linear fitting of the seismic responses. The parameters of 17 PSDM are listed in Table 6. The ln(a) and b are the regression parameters, and the D\|IM is the 18 logarithmic standard deviation of the seismic demand conditioned on IM. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers The 12 ‘same-estimation’ represents that the estimated damage state based on the DR or SC is the 13 same as that based on the PDR, which is illustrated in Figure 12 by solid grey; the ‘over- 14 estimation’, illustrated by solid green, represents that the estimated result is more severe; the 15 ‘under-estimation’, illustrated by solid red, is slighter. The rectangle with a triangle describes 16 that, though the estimated damage state is correct, the estimated damage value at the current 17 PGA is smaller than the value at the previous PGA. 18 20 20 Figure 12 Statistical values of the thresholds for each damage state Figure 12 Statistical values of the thresholds for each damage state Figure 12 Statistical values of the thresholds for each damage state It is seen from Figure 12 that the results are worrying when the DR was adopted for 3 assessing the seismic damage of RC tall-hollow piers, as the seismic damage will be 4 underestimated. If the SC was adopted for such piers where the shear effects are non- 5 negligible, the results would be incorrect and unstable; for some earthquakes, it will give over- 6 estimated results, while for some others, under-estimated results, or even over- and under- 7 estimation appear in one earthquake. The SC used in this paper is the curvature ductility, a 8 commonly used damage measure, as follows, 9 𝜇𝜑= ∅ ∅𝑦 10 𝜇𝜑= ∅ ∅𝑦 10 where ∅ is the section curvature, ∅𝑦 is the curvature for the first yielding of longitudinal steel 11 where ∅ is the section curvature, ∅𝑦 is the curvature for the first yielding of longitudinal steel 11 bars. The damage states were from reference [25] and presented in Table 5 for convenience. 12 bars. The damage states were from reference [25] and presented in Table 5 for convenience. 12 Table 5 Damage states for curvature ductility (SC)[25] 13 Damage state Slight Moderate Severe Complete Curvature ductility () ≥1, ＜1.70 ≥1.70, ＜3.67 ≥3.67, ＜14.19 ≥14.19 5. Seismic fragility analysis 14 5.1 Fragility analysis based on the piecewise drift ratio 15 Table 5 Damage states for curvature ductility (SC)[25] 13 Damage state Slight Moderate Severe Complete Curvature ductility () ≥1, ＜1.70 ≥1.70, ＜3.67 ≥3.67, ＜14.19 ≥14.19 and capacity (C) are often assumed following the lognormal distribution[29]. Thus, the fragility 1 and capacity (C) are often assumed following the lognormal distribution[29]. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers Thus, the fragility 1 and capacity (C) are often assumed following the lognormal distribution[29]. Thus, the fragility 1 can be calculated using the following function 2 can be calculated using the following function 2 𝑃(𝐷≥𝐶\|𝐼𝑀) = 𝛷[ ln ( 𝑚𝐷)−ln (𝑚𝐶) √𝛽𝐷\|𝐼𝑀 2 +𝛽𝐶 2 ] (7) 3 where (▪) is the cumulative distribution function of the standard normal distribution; mD and 4 D\|IM are the median and the dispersion of the seismic demand, respectively, and can be 5 estimated based on DM data generated by an incremental dynamic analysis (IDA); mC and C 6 are the median and dispersion of the seismic capacity, and can be obtained by statistical 7 calculation using the extrapolated experimental damage limit state data. 8 The Sa(T1,5%) and PGA are the widely employed IMs when developing the probabilistic 9 seismic demand models (PSDSs) due to their efficiency and sufficiency that have been proved. 10 Some studies show that taking Sa as IM may lead to a more efficient PSDM for long-period 11 structures, as the correlation between the seismic responses and the Sa is stronger. However, 12 taking the PGA as IM can provide the damages more directly related to seismic intensity so 13 that the seismic responses and damages can be compared under a unified seismic intensity. 14 Therefore, two PSDMs are developed in this research, respectively, based on Sa and PGA. 15 Then, the responses of the bridge were computed through IDA analysis of the 40 ground 16 motions. The PSDS is established by linear fitting of the seismic responses. The parameters of 17 PSDM are listed in Table 6. The ln(a) and b are the regression parameters, and the D\|IM is the 18 logarithmic standard deviation of the seismic demand conditioned on IM. The R2 and the 19 difference between the two logarithmic standard deviations are tiny, as gathered in Table 6. 20 We can now carry a reverse analysis: i.e. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers The R2 and the 19 difference between the two logarithmic standard deviations are tiny, as gathered in Table 6. 20 We can now carry a reverse analysis: i.e. assess if the demand based on the PDR is well 21 correlated with both the IM, i.e. Sa(T1) and PGA; in other words, understand if the PDR is 22 22 22 reasonable to be the seismic damage measure for tall-hollow piers, as it can reflect more 1 information of ground motions acting on the piers. 2 Since the difference relevant to R2 between PGA and Sa is small, the PGA is chosen to be 3 the IM. Then following Eq. (7), the seismic fragility curves of the RC bridge under four types of 4 ground motions can then be finally identified and are presented in Figure 13. 5 Since the difference relevant to R2 between PGA and Sa is small, the PGA is chosen to be 3 the IM. Then following Eq. (7), the seismic fragility curves of the RC bridge under four types of 4 ground motions can then be finally identified and are presented in Figure 13. 5 Since the difference relevant to R2 between PGA and Sa is small, the PGA is chosen to be 3 the IM. Then following Eq. (7), the seismic fragility curves of the RC bridge under four types of 4 ground motions can then be finally identified and are presented in Figure 13. 5 ground motions can then be finally identified and are presented in Figure 13. different ground motions. Moreover, the damage occurrence probability of the 10 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers 5 Table 6 Parameters of PSDMs IM ln(a) b D\|IM R2 PGA -3.30 0.94 0.45 0.81 Sa(T1,5%) -3.22 0.79 0.43 0.85 (a) Slight damage (b) Moderate damage (c) Severe damage (d) Complete damage Figure 13 Fragility curves of a tall pier for different type of ground motion Table 6 Parameters of PSDMs 6 IM ln(a) b D\|IM R2 PGA -3.30 0.94 0.45 0.81 Sa(T1,5%) -3.22 0.79 0.43 0.85 (a) Slight damage ( ) S d (a) Slight damage (b) Moderate damage (b) Moderate damage (d) Complete damage (d) Complete damage (d) Complete damage Figure 13 Fragility curves of a tall pier for different type of ground motio Figure 13 Fragility curves of a tall pier for different type of ground motion The obtained fragility curves fit the widely accepted experience: the exceedance 8 probability across structural damage limit state decreases with the increased intensity level of 9 different ground motions. Moreover, the damage occurrence probability of the tall pier has 10 The obtained fragility curves fit the widely accepted experience: the exceedance 8 probability across structural damage limit state decreases with the increased intensity level of 9 different ground motions. Moreover, the damage occurrence probability of the tall pier has 10 23 apparent differences among the four types of ground motions, indicating the significant effects 1 of the ground motions’ frequency factor on the damage level of tall piers. Several specific 2 phenomena can be found by comparing the fragility curves of four types of ground motions: 3 i) ‘Type 1-1’ versus ‘Type 2-1’: We can draw that the first-mode shape is the control factor to 4 the damage exceedance probability of tall-hollow piers when the combination of first- and 5 second-mode shapes is weak. ii) ‘Type 2-1’ versus ‘Type 2-2’: We can draw that the 6 contribution of the first-mode shape is necessary to generate more considerable damage 7 when the second-mode shape is dominant. iii) Comparing all the fragility curves, we can draw 8 that the second-mode shape can cause the largest damage exceedance probability with the 9 same IM. In brief, for high-intensity ground motions whose PGA is larger than 0.3g in this study 10 case, the tall pier’s seismic damage reaches the maximum when the second-mode shape can 11 be significantly activated and coupled with the fundamental shape. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers As mentioned above, the 12 tall-pier bridge’s first frequencies become smaller, and their second frequencies get closer to 13 the fundamental frequencies of the regular bridges. Therefore, the ground motions that can 14 activate their second-mode shape became common. 15 apparent differences among the four types of ground motions, indicating the significant effects 1 of the ground motions’ frequency factor on the damage level of tall piers. Several specific 2 phenomena can be found by comparing the fragility curves of four types of ground motions: 3 i) ‘Type 1-1’ versus ‘Type 2-1’: We can draw that the first-mode shape is the control factor to 4 the damage exceedance probability of tall-hollow piers when the combination of first- and 5 second-mode shapes is weak. ii) ‘Type 2-1’ versus ‘Type 2-2’: We can draw that the 6 contribution of the first-mode shape is necessary to generate more considerable damage 7 when the second-mode shape is dominant. iii) Comparing all the fragility curves, we can draw 8 that the second-mode shape can cause the largest damage exceedance probability with the 9 same IM. In brief, for high-intensity ground motions whose PGA is larger than 0.3g in this study 10 case, the tall pier’s seismic damage reaches the maximum when the second-mode shape can 11 be significantly activated and coupled with the fundamental shape. As mentioned above, the 12 tall-pier bridge’s first frequencies become smaller, and their second frequencies get closer to 13 the fundamental frequencies of the regular bridges. Therefore, the ground motions that can 14 activate their second-mode shape became common. 15 As the ‘Type 2-2’ ground motions are the most unfavorable for this rigid-frame bridge 16 with tall-hollow piers, the seismic fragility curves based on both the Sa and PGA as IM are 17 summed up, respectively, and presented in Figure 14. The exceedance probability for the 18 severe damage state is 84%, 90% and 96%, with the PGA values equal to 0.7g, 0.8g and 1.0g, 19 respectively. The exceedance probability for the complete damage state is 61%, 71% and 85%, 20 respectively. If Sa is taken as IM, the exceedance probability is a little bigger. The results present 21 a high probability that the bridge will be unsafe under ‘Type 2-2’ ground motions. 4.1 Evaluation of the commonly used damage measures for RC tall-hollow piers 22 As the ‘Type 2-2’ ground motions are the most unfavorable for this rigid-frame bridge 16 with tall-hollow piers, the seismic fragility curves based on both the Sa and PGA as IM are 17 summed up, respectively, and presented in Figure 14. The exceedance probability for the 18 severe damage state is 84%, 90% and 96%, with the PGA values equal to 0.7g, 0.8g and 1.0g, 19 respectively. The exceedance probability for the complete damage state is 61%, 71% and 85%, 20 respectively. If Sa is taken as IM, the exceedance probability is a little bigger. The results present 21 a high probability that the bridge will be unsafe under ‘Type 2-2’ ground motions. 22 24 1 2 (a) PGA as IM (b) Sa as IM 3 Figure 14 Fragility curves of the tall pier under ‘type 2-2’ ground motions 4 Figure 14 Fragility curves of the tall pier under ‘type 2-2’ ground motions 5.2 Comparison to the fragility curves based on the common damage measures 5 5.2 Comparison to the fragility curves based on the common damage measures 5 The new proposed PDR has been proven to be reasonable and adequate for tall-hollow piers 6 of rigid-frame bridges, as it can consider the multiple-bends and shear damage appropriately. 7 The seismic damage and safety assessment of such piers based on the section-curvature- or 8 displacement-related damage measures would be unstable, repeating between the 9 underestimated results and overestimated ones. 10 p f g y g The new proposed PDR has been proven to be reasonable and adequate for tall-hollow piers 6 of rigid-frame bridges, as it can consider the multiple-bends and shear damage appropriately. 7 The seismic damage and safety assessment of such piers based on the section-curvature- or 8 displacement-related damage measures would be unstable, repeating between the 9 underestimated results and overestimated ones. 10 The new proposed PDR has been proven to be reasonable and adequate for tall-hollow piers 6 of rigid-frame bridges, as it can consider the multiple-bends and shear damage appropriately. 7 The seismic damage and safety assessment of such piers based on the section-curvature- or 8 displacement-related damage measures would be unstable, repeating between the 9 underestimated results and overestimated ones. 10 The number of experimental specimens and human factors affected the seismic capacity 11 model. However, this type of uncertainty has no concern with the effectiveness of the PDR, 12 though it would impact the quantity of the fragility curves. The fragility analysis based on the 13 PDR can be further used to select of the most unfavorable ground motions used for tall-hollow 14 piers’ earthquake fortification and seismic design. It is necessary to improve the quality of 15 fragility curves further based on the PDR; in particular, one can improve the FE model of the 16 tall-hollow piers to better consider the shear effects, discuss the sensibility of the PDR on 17 ground motion parameters, select the ‘Type2-2’ ground motions more carefully. 18 6. Conclusions 19 The number of experimental specimens and human factors affected the seismic capacity 11 model. However, this type of uncertainty has no concern with the effectiveness of the PDR, 12 though it would impact the quantity of the fragility curves. The fragility analysis based on the 13 PDR can be further used to select of the most unfavorable ground motions used for tall-hollow 14 piers’ earthquake fortification and seismic design. 5.2 Comparison to the fragility curves based on the common damage measures 5 It is necessary to improve the quality of 15 fragility curves further based on the PDR; in particular, one can improve the FE model of the 16 tall-hollow piers to better consider the shear effects, discuss the sensibility of the PDR on 17 ground motion parameters, select the ‘Type2-2’ ground motions more carefully. 18 6 Conclusions 19 25 reevaluated. A novel damage measure, namely the piecewise drift ratio (PDR), with the 1 corresponding damage states, is proposed to fit their significant multiple-bends and shear 2 damage pattern. The PDR is then applied to determine a tall-hollow pier’s seismic fragility 3 curves, through which the relationship between the ground motions’ spectral characteristics 4 and the pier’s damage degree is observed. The results show that the ‘type2-2’ earthquakes 5 are necessary for assessing such piers’ seismic damage and are recommended for earthquake- 6 fortification-related work. Specific conclusions are as follows, 7 1. The proposed PDR can involve the apparent multiple-bends and shear damage to provide 8 accurate assessment results. The fragility curves based on the new damage measure are 9 reasonable and fit the widely accepted experience. 10 1. The proposed PDR can involve the apparent multiple-bends and shear damage to provide 8 accurate assessment results. The fragility curves based on the new damage measure are 9 reasonable and fit the widely accepted experience. 10 2 The first-mode shape is the control factor to the damage exceedance probability of tall- 11 hollow piers when the combination of first- and second-mode shape is weak; its contribution 12 is necessary to generate more considerable damage when the second-mode shape is 13 dominant. The second-mode shape can cause the largest damage exceedance probability with 14 the same IM. 15 2 The first-mode shape is the control factor to the damage exceedance probability of tall- 11 hollow piers when the combination of first- and second-mode shape is weak; its contribution 12 is necessary to generate more considerable damage when the second-mode shape is 13 dominant. The second-mode shape can cause the largest damage exceedance probability with 14 the same IM. 15 3. There is a high probability that the tall-pier bridge would be unsafe under 'Type2-2' ground 16 motions for the quite large exceedance probability of severe and complete damage. The 17 exceedance probability for the severe damage state is 84%, 90% and 96%, with the PGA values 18 equal to 0.7g, 0.8g and 1.0g, respectively. 5.2 Comparison to the fragility curves based on the common damage measures 5 The exceedance probability for the complete 19 damage state is 61%, 71% and 85%, respectively. 20 3. There is a high probability that the tall-pier bridge would be unsafe under 'Type2-2' ground 16 motions for the quite large exceedance probability of severe and complete damage. The 17 exceedance probability for the severe damage state is 84%, 90% and 96%, with the PGA values 18 equal to 0.7g, 0.8g and 1.0g, respectively. The exceedance probability for the complete 19 damage state is 61%, 71% and 85%, respectively. 20 3. There is a high probability that the tall-pier bridge would be unsafe under 'Type2-2' ground 16 motions for the quite large exceedance probability of severe and complete damage. The 17 exceedance probability for the severe damage state is 84%, 90% and 96%, with the PGA values 18 equal to 0.7g, 0.8g and 1.0g, respectively. The exceedance probability for the complete 19 damage state is 61%, 71% and 85%, respectively. 20 The authors declare that there is no conflict of interest regarding the publication of this paper. 22 The authors declare that there is no conflict of interest regarding the publication of this paper. 22 26 Acknowledgements 1 The authors gratefully acknowledge the financial supports from the National Natural Science 2 Foundation of China (Grant No. 51908384) and Zhejiang Provincial Natural Science Foundation 3 of China (Grant No. LTGG23E080004). This paper is also financed by the National Key Research 4 and Development Program of China (Grant No. 2021YFB2600500), Wenzhou Basic Social 5 Development Science and Technology Project (Grant No. S20220006), and the National 6 Natural Science Foundation of China (Grant No. 52078398). The fourth author acknowledges 7 the Italian Ministry of Education, Universities and Research (MUR) in the framework of the 8 project DICAM-EXC (Departments of Excellence 2023-2027, grant L232/2016). 9 project DICAM-EXC (Departments of Excellence 2023-2027, grant L232/2016). 9 REFERENCES [1] Cosenza E & Manfredi G. Damage indices and damage measures, Progress in Structural 11 erringeering and Materials, 2000, 2(1): 50-59. 12 [2] Pan Y, Ventura C E & Tannert T. Damage index fragility of low-rise light-frame wood 13 buildings under long duration subduction earthquakes. Structural Safety, 2020, 84(2020): 14 101940. 15 [3] Park Y J & Ang A H S. Mechanistic seismic damage model for reinforced concrete. 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If the specimen top is the free end, the height given in the last column is twice 6 REFERENCES Reference Specimen Tag Dimension of cross-section (mm) Wall thickness T (mm) Height of between two adjacent plastic hinges H (mm) 1 [30] D1 500×500 120 7900 2 [30] D2 500×500 120 5900 3 [31] A1 500×500 120 3900 4 [31] A2 500×500 120 5900 5 [31] A3 500×500 120 7900 6 [31] B1 500×500 120 5900 7 [31] B2 500×500 120 5900 8 [31] C1 500×500 120 5900 9 [31] C2 500×500 120 5900 10 [31] D1 500×500 120 3900 11 [31] D2 500×500 120 5900 12 [31] D3 500×500 120 7900 13 [31] E1 500×500 120 5900 14 [31] E2 500×500 120 5900 15 [31] F1 500×500 120 5900 16 [31] F2 500×500 120 5900 17 [31] G1 500×500 120 3900 18 [31] G2 500×500 120 5900 19 [31] G3 500×500 120 7900 20 [31] H1 500×500 120 5900 21 [31] H2 500×500 120 5900 22 [31] I1 500×500 120 5900 23 [31] I2 500×500 120 5900 24 [32] S1 500×360 120 5760 25 [32] S2 500×360 120 5760 26 [32] S3 500×360 120 5760 27 [32] S4 500×360 120 5760 28 [32] S5 500×360 120 5760 29 [33] S1 500×360 120 2880 30 [33] S2 500×360 120 2880 31 [33] S8 500×360 120 7200 32 [33] S9 500×360 120 7200 33 [33] S10 500×360 120 7200 34 [33] S11 500×360 120 7200 35 [33] S12 500×360 120 7200 36 [11] A1 400×250 80 4000 37 [11] A2 400×250 80 4000 38 [11] A3 400×250 80 4000 39 [11] A4 400×250 80 4000 40 [34] A1 400×250 80 3600 Note: If the specimen top is the fixed end, the height given in the last column of Table A1 is the full height of 5 the specimen. If the specimen top is the free end, the height given in the last column is twice the height of the 6 f ll h i ht f th i 7 APPENDIX 30
W2900605294.txt	https://www.shs-conferences.org/articles/shsconf/pdf/2018/15/shsconf_icolgas2018_07002.pdf	en		Critisism of The Juridical Positivism Paradigm on The Meaning of Pornography In The Judge Mindset	SHS web of conferences	2,018	cc-by	2,846	SHS Web of Conferences 54, 07002 (2018) ICoL GaS 2018 https://doi.org/10.1051/shsconf/20185407002 Critisism of The Juridical Positivism Paradigm on The Meaning of Pornography In The Judge Mindset Erni Wulandari1 and Rini Fidiyani2* 1 Law Student of Doctoral Program, Sebelas Maret University, Surakarta, Indonesia of Law and Society, Faculty of Law, Semarang State University, Semarang, Indonesia 2 Department Abstract. Pornographical in the way of lex spesialis was regulated in The Law No. 44/2008 and lex generalis loaded on The Criminal Code. The Judge interpreting pornography refer to textual definition of pornography according to the Law No. 44/2008 according the data founded that dominantly on juridical positivist paradigm. Juridical positivist paradigm is not the only one paradigm that used by the judge, moreover related about pornographical, need the change of appropriate paradigm concerning the judge mindset in interpreting pornography recorded to the judge considerations. The aim of this writing is to criticize the judge mindset and social sensitivity in interpreting and handling pornography. This study used qualitative and socio legal research to reveal the judicial considerations textual-contextually. With exposing the textual-contextual meaning of judge’s considerations, it can be traced to the legal paradigm used by judges and need to use appropriate legal paradigm related to the use of social theories that support it. The judge needs to have a non-doctrinal legal science perspective on the correct legal paradigm reform in giving judges consideration to pornographic cases. Judges are more likely shackled to the institutional structure and establishment of the juridical positivism paradigm. 1 Introduction and Literature Review The issue of pornography has long been as old as humans inhabit this earth in various forms and ways so that others listen and enjoy and even circulate widely. The etymology of pornography from the word porne means bitch and graphos or graphien, which means pictures or writing and pornography refer to pictures or photos that show the prohibited body parts of women [1]. Often pornography problems are embedded in the elements of eroticism in accordance with the encyclopedia of Britain, pornography is anything that has material material in the form of films, newspapers, writings, etc., which causes or arises. Today's information technology for the forms and ways of pornography is increasingly varied and adept at modifying aims to attract many viewers to be aroused so that it becomes an acute addictive phenomenon. Included in a large scope is called cbyerporno. The target extends across age, and the main national borders sell well and spread throughout the area. The pornography arrangement is classically contained in the Criminal Code, especially regarding the moral offenses of articles 282, 283, 532 and 533. But the development of information technology loaded with globalization with a variety of social media, regulating pornography needs to be made specific regulations in the form of laws on pornography. Law No. 44 year 2008 seeks to accommodate the content of pornography in a variety of movements and time and space, which of course applies uniformly formally according to the principle of unification adopted by the Indonesian state. Pornography law enforcement using Law No. 44 year 2008 was accompanied by an increase in legal knowledge by judges as the last guardian law enforcement officers in making judges 'decisions reflected in the judges' consideration. Judges' consideration is a description of the logic and juristicche juristiche ethics of the judge on the response to legal cases, especially pornography. The judge's paradigm is still dominated by legal positivism which is easier and more measurable using legal interpretation methods and which surfaces in grammatical interpretation of wording and sentences. Whereas the whole method of interpretation needs to be empowered including referring to the cultural and humanitarian approaches that are critical of the legal positivism paradigm. * Corresponding author: fidiyani.rini@gmail.com © 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/). SHS Web of Conferences 54, 07002 (2018) ICoL GaS 2018 https://doi.org/10.1051/shsconf/20185407002 1.1 Definition of Pornography Understanding pornography involves the opening of moral norms and cultural values of the local community. The parameter of the definition of pornography is formulated uniformly and definitively as in Law No. 44 year 2008, pornography is a picture, sketch, illustration, photo, writing, sound, sound, moving picture, animation, cartoon, conversation, gesture, or other message form through various forms of communication media and/or performances in public, which contain obscenity or sexual exploitation that violate moral norms in the community. From the contents of the definition or understanding above forms and pornographic media metamorphosed with the development of information technology in this globalization period. Whereas in substance refers to content concerning sexual exploitation which has the weaknesses of subjectivity in the words: "contains:" moral norms "and" society " [2]. Scoping space can someday shift, change and even disappear according to the demands of community cultural development. Oral culture and written culture in the scope of culture will speak active movements can fade and strengthen according to the style and manner conveyed to the community. Including the word "loading" the ability to accommodate during the period of critical questioning, then the phrase moral norms and speech communities also questioned the understanding and ability of cross-country roaming [2]. Questioning the decency norms on cross-term cruising refers to the word meaning regarding the noble values that are believed and adhered to by local people that are easy to occur between communities and between different regions in defining the meaning and content of moral norms inherent in the local community. Moral norms are one manifestation of a cultural framework within the sphere of cultural systems and social systems initiated by Koentjaraningrat [3]. Moral norms that are easy to experience a shift that used to be said to be immoral or pornographic acts in the future are neither assusila or vice versa [4]. Therefore, the definition or definition of pornography is extended to the subject, location, situation when the content of the decency norm is used by the local community within the scope of the meaning of pornography. This happens because Indonesian society is a plural society [5], where they have different morality, customs, norms and religions and even different types of law. To be understood the meaning of pornography needs to be seen in the text and context. Concrete examples of Sukuh temple relief works in the Karanganyar Regency of Central Java and Borobudur temple have carved human body carvings and carvings between the opposite sex which are displayed in real terms, as an observation of the stages of human life that still prioritize the lust of biological pleasure. The discovery of lingga and yoni artifacts at the shrine that is interpreted is not merely concrete but a symbol of balance and calm from the encounter of Lord Shiwa and Dewi Parvati. Furthermore, the literary works of the book Kamasutra in India take the form of narratives and pictures of the art of making love, in the homeland of the novel Ronggeng Dukuh Panarukan about the life of Ronggeng dancers in the 1960s the condition of being a ronggeng dancer according to the local tradition tradition of giving up her virginity to a man who is not a legitimate husband. The meaning of pornography is relative, because in the scope of meaning includes an understanding of the cultural values adopted by the community and local area. Likewise, the meaning of cross-community pornography and across regions cannot be equated in a region or nation. The meaning of pornography contains the text and context, not just fixed on the definition or understanding of pornography. A position that views the meaning of pornography cannot be uniformed in every law case, among others, cultural artist, feminist and critical legal observer. The feminism movement is oriented towards the liberation of women's position in order to eliminate and find the formula for equal rights of women and men in all fields according to the potential they possess [6]. Related to the meaning of pornography for the feminist movement is the result of superior male ideology and politics dominated and designed by male logic. Like the sexual politics of both women and men, Plato prioritizes the content of the head rather than the groin section but actually there is a relationship between being ruled and mastering having an intimate erotic encounter in a secret room in the sense of physical and non-physical geographical. Plato tried to sort out erotic encounters in the body of men and women not from mimicry in a beautiful body but authentic ideas in the form of beauty that resides in the body as well as in other bodies. Here Plato already implies that the body experiences fragmentation into spaces that we can enter in various ways. And the right way for the Plato regime is to enter the space of beauty - not the beauty that arises from intimacy, but from the thoughts or ideas of the beautiful. A beautiful body is just a step. If the idea of beauty is what makes the body beautiful, there is nothing more erotic than loving an idea that is eternal and beyond all forms, namely the beauty itself, therefore Plato agrees to promiscuity behavior [7]. 1.2 Criticism of the Law Positivism Paradigm Positivism published this issue from Comte comes from a positive word to be positivism which means knowledge should not exceed the facts [6]. Therefore adherents of legal postivism see the rule of law as only valid because the law gets its positive form from an authorized agency [8]. The law of positivism paradigm views the types and 2 SHS Web of Conferences 54, 07002 (2018) ICoL GaS 2018 https://doi.org/10.1051/shsconf/20185407002 forms of law which are the most superior in the form of state law (state-law) in writing derived from the human ratio. This type of natural law is not a major study especially concerning the legal purpose of justice which is considered to have existed by itself. Whereas aspects of social, cultural, political, economic and ideological sensitivity for the operation of state law are not the main and urgent matters because they can damage the working system of state law. Because the social sensitivity of the legal apparatus is easily dull even though the state law is dealing with fellow human beings, humanizing human beings in law needs to be considered in the enforcement of pornography law. 2 Objective of Study The purpose of this study is to map and analyze the mindset of judges through the consideration of judges who strongly adhere to the paradigm of positivism and criticize it in handling legal cases of pornography. 3 Research Method Research methods used are qualitative. This approach refers to normative and empirical law to obtain a comprehensive description of the object under study. Primary data sources in the form of humans in actions, events and documents and related archives and others. Data sources that have been obtained are then processed using interactive and non interactive models then analyzed using interactive analysis models. 4 Discussion Judges are the determinants of justice for legal cases handled because the public court is the last bastion of justice for litigants who use litigation. All legal remedies are taken for parties who have litigated in winning legal cases, including pornography cases. Pornography contains an injury to the norms of public decency and experiences a shift in meaning and reality at all times, therefore legal apparatuses, especially judges, are required to have excessive mastery in carrying out legal interpretation. This legal interpretation experiences a shift due to changing situations and demands of the times. The interpretation that dominates the judge in the mindset or paradigm of positivism in the form of grammatical words and sentences is described according to the origin of the word and language. The mastery of grammatical interpretation is no longer sufficient in pornographic cases because the judges consider the content to be biased in meaning and bias of the perpetrator. Judges need to master hermeneutics to uncover first; text, second; text maker, and reader or interpreter of the text. Text interpreters are tasked with exploring what is explicit and implicit behind the hidden meaning of a series of words in the form of text sentences. Hermeneutical work reveals thoughts through words as a medium of delivery. Decisions of the Constitutional Court of the Republic of Indonesia 10-17-23 / PUU-VII / 2009 have made decisions in a judicial review, among others, the Constitutional Court recognizes the exceptions of pornography according to expert testimony submitted by the government by Tjipta Lesmana (hukumonline.com, colored dissent, MK states Act Constitutional Pornography, accessed August 12, 2018): a. Art needs b. Literary needs c. Custom / custom requirements d. The need for knowledge e. Sports needs For artists and cultural observers understand the meaning of pornography, as stated in the table below: Meaning Giver Painting Artist Sculpture Artist Meaning of Pornography The meaning of pornography is reinforced and not biased meaning because, the painting of naturalist flow that accentuates the beauty of the human body and natural forms are popular and are enjoyed by local and foreign consumers Agree the existence of Law Number 44 of 2008 provided that the legal apparatus including judges have extensive legal knowledge, especially interpreting art because the art of serving art is not a 3 Description The results of naturalist painting are limited by the rules of decency and the rules of decency precisely the aesthetic value disappears and is not sold in the market Interpretation of judges or the mindset of judges need to be expanded because related sculpture works accentuate the body of mansia as a sign of gratitude as a perfect being. SHS Web of Conferences 54, 07002 (2018) ICoL GaS 2018 Fiction Artist Drama Artist https://doi.org/10.1051/shsconf/20185407002 pornographic product. Sculpture requires high creativity so that a legal culture and high legal authority are needed. Fiction artists really appreciate the multicultural society that can be promoted into fiction according to the culture that lives in their respective regions. Example of Noevl Ronggeng Dukuh Penarukan by Ahmad Tohari. Pornography only takes care of human sex which has been taken over by the State so that it kills the creativity of artists and cultural observers in expressing their artistic ideas. Legal certainty that is pursued by the legal apparatus is not the main thing precisely in justice and expediency because the expression of artists written on fictional art is related to the uniqueness of local culture. Like the intercourse scene is packed and framed the beauty of art through songs, dances and other symbols Naked bathing scenes on the river in the art of drama are natural because morality rules allow. The part judge of the justice apparatus determines justice, so it is necessary to look at Lawrence Friedman's legal system theory, structure, substance, and culture [9, 10]. the three elements above are first attached to the judge, the two judiciary institutions as a forum for the operation of state law designed with a system of certain judicial mechanisms and thirdly attached to the soul of the community affected by lawsuits. The perspective of the judge in carrying out his legal knowledge in the judiciary cannot be separated from the quality aspect of the judge himself in the form of a mindset or legal paradigm which the judge believes and adheres to when dealing with pornography lawsuits. Based on the cases of pornography criminal acts investigated, namely: No. 751/Pid.B/2017/PN. JKT. BRT, No. 210/Pid.B/2018/PN Mlg, No, 512/Pid.B /2017/PN Mjk (Mojokerto, East Java), and No. 123 PK / Pid.Sus / 2015 (Judicial Review), clearly the judge did not give pornography meaning to Law No. 44 of 2008 only on grammatical interpretation only. Judges do not dare to think and argue differently from what is contained in the law. This is the form of the positivism paradigm in the judiciary which is still held firmly by the judge. There needs to be a change of mind in their mindset so that the judge's decision - not only in a pornography case - is able to give meaning to every case he handles. 5 Conclusion The judge is dominated by the mastery of the legal positivism paradigm as a result of Indonesian legal higher education in giving the consideration to pornographic cases in accordance with Law No. 44 year 2008. Pornography is not a simple matter because it generally involves many parties such as the distribution of pornographic vcd and judges are easier to use legal positvism paradigm more likely to reject the criticism paradigm on the critical approach of legal hermeneutics. Reference 1. 2. 3 4. 5. 6. 7. 8. 9. 10. Y.S. Hamid, Pornografi merusak masa depan bangsa, Juni 2018, access in www.dwp.or.id J. Situmpul, JHP, 45, 3 (2015) Koentjaraningrat, Pengantar Antropologi I (Rineka Cipta, Jakarta, 2001) E. Wulandari, JDH, 15, 1 (2015) Nasikun, Sistim Sosial Indonesia (RajaGrafindo, Jakarta, 1995) E. Wahyuni, JAM, 1, 1 (2012) F.B. Hardiman, SPT, 9, 1 (2009) T. Huijbers, Filsafat Hukum dalam Lintas Sejarah (Pustaka Filsafat Kanisius, Yogyakarta, 1982) L.M. Friedman, The Legal System: A Social Sciences Perspective (Russel Sage Foundation, New York: 1986) L.M. Friedman, American Law, An Introduction, 2nd Edition (Tatanusa, Jakarta: 1998) 4
https://openalex.org/W2029287038	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0102017&type=printable	English	null	Up-Regulation of MicroRNA-145 Associates with Lymph Node Metastasis in Colorectal Cancer	PloS one	2,014	cc-by	7,791	Wei Yuan1., Chenguang Sui1., Qian Liu2., Wanyan Tang1, Huaying An1, Jie Ma1* 1 State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China, 2 Department of Abdominal Surgical Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China 1 State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences, Peking Un 2 Department of Abdominal Surgical Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences, Peking Union M Abstract Metastasis is the main cause of mortality in patients with solid tumours. Identifying the exact molecules associated with CRC metastasis may be crucial to understand the process, which might also be translated to the diagnosis and treatment of CRC. In this study, we investigate the association of microRNA expression patterns with the lymph node metastasis of colorectal cancer. Among these candidate miRNAs, the expression of miRNA-145 was significantly related to lymph node metastasis of CRC. Both in vitro and in vivo study demonstrated that up-regulation of miR-145 could improve the ability of migration and invasion of colorectal cancer cell, while no effect on proliferation was observed. The mechanism of this promotion is associated with the stabilization of Hsp-27, a protein which plays an important role in the promotion of metastasis. These results may be crucial to understanding CRC metastasis and may be translated to the diagnosis and treatment of CRC. Citation: Yuan W, Sui C, Liu Q, Tang W, An H, et al. (2014) Up-Regulation of MicroRNA-145 Associates with Lymph Node Metastasis in Colorectal Cancer. PLoS ONE 9(7): e102017. doi:10.1371/journal.pone.0102017 Editor: Rolf Mu¨ller, Philipps University, Germany Received April 10, 2013; Accepted June 14, 2014; Published July 14, 2014 Copyright: 2014 Yuan 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 study was supported by the National Basic Research Program of China (Grant no. 2011CB911004), The Beijing Training Project for The Leading Talents (Z131107000513001), Beijing Nova Program (Z131107000413066) and Beijing Natural Science Foundation of China (Grant no. 7122150). 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. . These authors contributed equally to this work. Introduction carcinogenesis could prevent liver metastasis in CRC [11]. However, metastasis resulted from a complex cascade of biological processes and the exact molecular mechanisms underlying CRC metastasis are far from being fully understood. Colorectal cancer (CRC) ranks the third most common tumor and the fourth leading cause of cancer mortality worldwide [1]. Although many achievements have been made in the treatment of CRC in the past decades, the overall survival rate of patients with CRC has marginally changed. Poor prognosis and survival rate are mainly due to metastasis, thus more than one-third of patients with CRC will ultimately develop metastatic diseases [2]. Therefore, identifying the exact molecules associated with CRC metastasis may be crucial to understand the process, which might also be translated to the diagnosis and treatment of CRC. g y In this study, the miRNA expression profiles in primary CRC lesion with or without lymph node metastasis were analyzed by using a miRNA microarray and quantitative reverse-transcription polymerase chain (qRT-PCR). In this regard, miR-145 was selected as it displayed dramatic up-regulation in CRC with lymph node metastasis in comparison to that without lymph node metastasis. Both in vitro and in vivo study demonstrated that up- regulation of miR-145 could improve the ability of migration and invasion of colorectal cancer cell. In addition, iTRAQ (isobaric tag for relative and absolute quantification) labeling and 2DLC-ESI- MS/MS (liquid chromatography tandem MS) were employed to identify cellular proteins which were directly or indirectly regulated by miR-145. These results suggested that miR-145 might play an important role in the metastasis of CRC by stabilization of Hsp-27. MicroRNAs (miRNAs) are 21- to 25-nucleotide single-stranded, non-coding RNA molecules that exert their functions by binding to the 39-untranslated regions of their corresponding mRNA targets [3]. It has been estimated that one-third of the total human genes may be regulated by miRNAs, indicating that miRNAs have pivotal roles in physiological and pathological processes [4–5]. A large number of findings show that miRNAs are implicated in human cancers. The inappropriate expression of miRNAs can lead to the aberrant expression of gene products that may contribute to acquisition of the hallmarks of cancer. These observations suggested the function of miRNAs as tumor suppressors or oncogenes [6–8]. July 2014 \| Volume 9 \| Issue 7 \| e102017 5. Analysis of differentially expressed proteins The above findings indicated that miR-145 acts as a prometa- static miRNA in CRC. To aim to gain a better understanding of proteins affected either directly or indirectly by miR-145 overexpression, HCT-8-miR-145 and HCT-8-NC cells were lysed, and subjected to iTRAQ labeling, 2DLC-ESI-MS/MS analysis. A total of 1117 distinct proteins were identified and quantified, which were subsequently filtered with manually selected filter exclusion parameters. We took a 1.3 fold change cut-off for the iTRAQ ratio to classify proteins as up or down regulation. This cut-off was applied because several previous iTRAQ studies conducted in our laboratory demonstrated that the technical variation was consistently below 30%, the criterion of cutoff was also accepted by previous research [12]. The proteins were considered up or down-regulated only when their expression ratios (HCT-8-miR-145 cells vs. HCT-8-NC cells) were .1.3 (161.3) or ,0.77 (161.3) AND showed statistically significance. To further confirm the miR-145 expression profile, qRT-PCR analysis was performed in additional 202 CRC samples (99 CRC patients with lymph node metastasis and 103 CRC patients with no lymph node metastasis) (see Table 1 and Table S1). As shown in Fig. 1C, miR-145 was underexpressed in colorectal cancer specimens with or without metastasis compared to adjacent normal tissues respectively, which is consistent with previous reports by others [14]. However, the expression of miR-145 was significantly up-regulated in CRC with lymph node metastasis than that without lymph node metastasis. This change suggested that miR-145 may play an important role in CRC lymph node metastasis. 3. Overexpression of miR-145 has no effect on proliferation of HCT-8 cells Thus 13 proteins were screened out as differentially expressed proteins, including 7 significantly up-regulated proteins and 6 remarkably down-regulated proteins (Table S2, S3). Among 13 differentially expressed proteins, the up-regulation of heat shock protein 27 (Hsp-27) was validated using western blotting (Fig. 4A). We therefore chose Hsp-27 for further investigation. To determine whether the expression of miR-145 affect the biological function of CRC cells, the miR-145 overexpressed model was created in HCT-8 cells using a lentivirus system, which was referred to as HCT-8-miR-145 cells in this paper. The miR- 145 levels of HCT-8-miR-145 cells and mock control cells (HCT- 8-NC) were determined using qRT-PCR (Fig. 2A). A significant up-regulation of miR-145 in HCT-8-miR-145 cells was observed compared to that in HCT-8-NC cells. To observe the effect of miR-145 on the HCT-8 cells, cell growth rate or cell cycle was evaluated by CCK-8 or FACS assay, respectively. As shown in Fig. 2B and 2C, the proliferation rate or cell cycle of HCT-8-miR- 145 cells did not change as compared with HCT-8-NC. This result suggested that overexpression of miR-145 did not significantly influence the proliferation or cell cycle of HCT-8 cells. To further validate the association between Hsp-27 protein and miR-145 expression in CRC, we analyzed their expression profile in primary human tissue samples (including 41 CRC with LNM, 43 CRC without LNM, 47 adjacent non-tumor tissue) using western blotting or qRT-PCR respectively. Among the 131 human tissue samples, the expression of Hsp-27 was significantly up- regulated in CRC with lymph node metastasis compared to that without lymph node metastasis. This trend is consistent with miR- 145 expression profile. There was a strong, positive correlation (Spearman) between Hsp-27 protein and miR-145 expression (r = 0.402; P,0.0001) (Fig.4B, Fig. S2 and S3 in File S1). These results indicated that miR-145 is involved, at least partially, in up- regulating of Hsp-27 protein expression. 2. Verification of miRNA expression by real-time PCR analysis To validate the miRNA microarray data, we performed quantitative real-time PCR (qRT-PCR) to analyze the expression level of 11 miRNAs which were the most significantly dysregulated miRNAs or which were not reported its association with metastasis, including miR-99b,-125b,-100,-99a,-152,-199b-5p,- 145,-376c,-29b,-95 and -1274a. We examined the expression of miRNAs in the same tissues used for microarray analysis. After normalization with the endogenous control U6, qRT-PCR data confirmed that the expression of 8 miRNAs showed to be consistent with microarray result. Among them, the expression of miRNA-145 displayed the most significant difference between cancer tissues of the two groups (Fig. 1B). MiR-145 Promotes Metastasis of Colorectal Cancer MiR-145 Promotes Metastasis of Colorectal Cancer similar result was also observed in a cell invasion assay (Fig. 3B). We also determined the ability of miR-145 to promote migration in other CRC cell lines (SW480 and SW620). As showed in Fig. S1 in File S1, overexpression of miR-145 significantly increased the migratory ability of sw620 cells, whereas no apparent change in sw480 cells (data not shown). Collectively, these results provide strong evidence that up-regulation of miR-145 could promote cell migration and invasion in vitro. Because the expression of miR-145 in HCT-8 cells exhibited the lowest expression compared with that in other CRC cells, the rest of the work was focused on this CRC cell line to illustrate typical validation. determined using Agilent miRNA microarray. Among the unique 851 human miRNA probe, 32 miRNAs were identified differen- tially expressed in CRC tissues between lymph node metastasis positive and negative group (P,0.05). Twenty one of them were observed considerably increased expression in CRC with lymph node metastasis. On the other hand, 11 of them were under- expressed significantly in lymph node metastasis positive CRC tissues. Unsupervised clustering analysis with these 32 significantly dysregulated miRNAs (21 miRNAs with significant overexpression and 11 miRNAs with significant down-regulation) was able to distinguish the CRCs with or without lymph node metastasis (Fig. 1A). To further confirm this notion, an orthotropic transplantation nude mouse model was established to study whether overexpres- sion of miR-145 could promote tumor metastasis in vivo. We found no significant difference of the weight or volume of the primary tumors in the liver transplanted by both HCT-8-miR-145 cells and HCT-8-NC cells. We also found that 100% of mice in the HCT-8- miR-145 group had the mesenteric lymph node metastasis and the mean number of metastatic nodules reached 149615. However, there was none of mice in the HCT-8-NC control group displaying mesenteric lymph node metastasis (Fig. 3C, 3D). Taken together, these results confirmed that high level of miR-145 could promote CRC migration and invasion in vitro and in vivo. 2. Verification of miRNA expression by real-time PCR analysis July 2014 \| Volume 9 \| Issue 7 \| e102017 1. Different miRNA expression profiles of CRC with or without lymph node metastasis Recently, convincing evidence showed that a series of miRNAs play crucial roles in CRC metastasis. For example, Asangani et al. identified mir-21 as metastasis promoter in CRC [9]. Liu et al reported that miR-499-5p enhanced cellular invasion and tumor metastasis in CRC by targeting FOXO4 and PDCD4 [10]. Okamoto K, demonstrated that up-regulation of miR-493 during To investigate the association of microRNA expression patterns with the lymph node metastasis of colorectal cancer, eight primary colorectal cancer tissues derived from stage II–III colorectal cancer patients with (n = 4) or without (n = 4) lymph node metastasis were collected and the miRNA expression profiles of them were PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e102017 1 4. miR-145 promoted CRC migration and invasion in vitro and in vivo We subsequently analyzed whether miR-145 contributed to change the migratory mobility of CRC cells. Compared with the mock group, cell migration was significantly increased in HCT-8- miR-145 cells in a transwell cell migration assay (Fig. 3A). A July 2014 \| Volume 9 \| Issue 7 \| e102017 PLOS ONE \| www.plosone.org 2 MiR-145 Promotes Metastasis of Colorectal Cancer 6. Enhancement of Hsp-27 stability by miR-145 in CRC cells not mRNA of Hsp-27 was modulated by miR-145, suggesting this regulation is post- transcriptional. To further explore the protein l i f H 27 ff d b iR 14 d d Figure 1. MiRNA expression profiles in CRC with or without lymph node metastasis. A. The certified result of microarray analysis. Hierarchical clustering of 32 significantly dysregulated miRNAs expression profiles in human primary colorectal cancer tissues derived from colorectal cancer patients with (LNM-P, n = 4) or without (LNM-N, n = 4) lymph node metastasis. B.Validation of selected miRNAs predicted to be dysregulated in CRC with or without lymph node metastasis using qRT-PCR in the same tissues used for microarray analysis. Data shown in B is representative of three independent experiments, and presented as fold expression normalized to U6 6 SD (standard deviation).C. QRT-PCR analysis of the relative expression of miR-145 in additional 202 (LNM-N = 99; LNM-P = 103) cases of human CRC tissues, including tumor sample (T) and matching non-tumor tissue sample (NT) from the same patient. Each sample was analyzed in triplicate and normalized to U6. * P,0.05, ** P,0.01. doi:10.1371/journal.pone.0102017.g001 Figure 1. MiRNA expression profiles in CRC with or without lymph node metastasis. A. The certified result of microarray analysis. Hierarchical clustering of 32 significantly dysregulated miRNAs expression profiles in human primary colorectal cancer tissues derived from colorectal cancer patients with (LNM-P, n = 4) or without (LNM-N, n = 4) lymph node metastasis. B.Validation of selected miRNAs predicted to be dysregulated in CRC with or without lymph node metastasis using qRT-PCR in the same tissues used for microarray analysis. Data shown in B is representative of three independent experiments, and presented as fold expression normalized to U6 6 SD (standard deviation).C. QRT-PCR analysis of the relative expression of miR-145 in additional 202 (LNM-N = 99; LNM-P = 103) cases of human CRC tissues, including tumor sample (T) and matching non-tumor tissue sample (NT) from the same patient. Each sample was analyzed in triplicate and normalized to U6. 4. miR-145 promoted CRC migration and invasion in vitro and in vivo * P,0.05, ** P,0.01. doi:10.1371/journal.pone.0102017.g001 Discussion Metastasis is the key hallmark of malignance. Although there are many reports of the expression of miR-145 in cancers, the report of the function of miR-145 in the metastasis development of CRC is rarely few. In current study, we investigated the expression of miR-145 in the primary cancer tissue of CRC patients with or without lymph node metastasis. The miR-145 was identified to be underexpressed in CRC specimens with or without lymph node metastasis compared with adjacent normal tissues respectively, which is consistent with previous reports by others [14–16]. However, the expression of miR-145 displayed a dramatic up- regulation in CRC with lymph node metastasis, which was out of our expectation. Because to our knowledge, the expression trend of tumor associated genes usually move toward one direction along the development of tumor. To confirm the result of our preliminary observation, the precise expression of miR-145 in the primary CRC tissue of 103 patients with lymph node metastasis and 99 patients without lymph node metastasis was determined using qRT-PCR. Each sample was analyzed in triplicate and normalized against an endogenous control U6. Strict calibration standards and large quantity of tumor specimens used in this study ensured the credibility of our results. We found no significant difference in comparison of miR-145 expression level in adjacent normal tissues between CRC with or without lymph node metastasis. However, a clear association between miR-145 expression and lymphatic metastasis was observed. In these CRC patient samples, miR-145 showed significant higher expression in cancer with lymph node metastasis than those without lymph node metastasis, which further confirmed our array result. Furthermore, as we analyzed data of several other miRNAs, we also observed similar expression profile of decrease, restoration, 6. Enhancement of Hsp-27 stability by miR-145 in CRC cells not mRNA of Hsp-27 was modulated by miR-145, suggesting this regulation is post- transcriptional. To further explore the protein up-regulation of Hsp-27 affected by miR-145, we detected whether miR-145 enhanced the stability of Hsp-27 protein. There was no detectable change of Hsp-27 transcriptional level after miR-145 up-regulation (data not shown). Only protein but PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e102017 3 MiR-145 Promotes Metastasis of Colorectal Cancer Table 1. The clinicopathologic characteristics of 202 cases of primary CRC patients used in this study. Clinicopathologic characteristics Case numbers % Age(Years) ,60 100 49.5 .60 102 50.5 Gender Male 117 57.9 Female 85 42.1 Differentiation Well 22 10.9 Moderate 161 79.9 Poor 19 9.4 Tumor size ,5 105 52.0 .5 97 48.0 TNM Stage I 35 17.3 II 66 32.7 III 83 41.1 IV 18 8.9 Lymph node metastasis Negative 99 49.0 Positive 103 51.0 doi:10.1371/journal.pone.0102017.t001 Table 1. The clinicopathologic characteristics of 202 cases of primary CRC patients used in this study. doi:10.1371/journal.pone.0102017.t001 HCT-8-miR-145 cells and HCT-8-NC cells were treated with either the protein synthesis inhibitor, cycloheximide (CHX), or proteasome inhibitor, MG-132, respectively. As illustrated in Fig.4C and 4D, the enhanced expression of Hsp-27 in miR-145 overexpressed cells was abolished when incubated with MG132. Such phenomenon was not found when incubated with CHX. These results indicated that miR-145 could not influence the protein synthesis of Hsp-27, but could reduce the rate of Hsp-27 degradation, which enhanced its stability. July 2014 \| Volume 9 \| Issue 7 \| e102017 7. Down-regulation of Hsp-27 attenuated the oncogenic effect of miR-145 For example, Sachdeva et al found that the down-regulation of miR-145 was more prominent in CRC than in breast cancer [17]. All of these observations indicated that some miRNAs can be multitasking by interacting with different target genes in various cells and tissues [18]. but not further down-regulation, along with the development of CRC (unpublished data). These observations of other CRC related miRNAs confirmed the accuracy of our results, which suggested that one miRNA maybe display different expression profile in different stages of cancer. Besides stage difference, the expression of miR-145 seems to be dependent on the type of tissue. For example, Sachdeva et al found that the down-regulation of miR-145 was more prominent in CRC than in breast cancer [17]. All of these observations indicated that some miRNAs can be multitasking by interacting with different target genes in various cells and tissues [18]. This study provides the first evidence that miR-145 functions primarily as a prometastatic miRNA in advanced CRC. It is in general that single specific miRNA can regulate multiple target genes, which suggests that single miRNA could carry out a variety of functions by targeting different genes in various cellular contexts [22]. At the early stage of CRC, miR-145 is down-regulated, indicating its target related to cell proliferation. At the advanced stage, high expression of miR-145 might relate to targets against metastasis. MiR-17-5p, a well-investigated miRNA, could target pro- and anti-proliferative genes and act as both an oncogene and a tumor suppressor in different cancers [13,23–24]. MiR-143, another cancer associated miRNA, its expression is always consistent with miR-145 (we did not exam its co-expression with miR-145 in our study), showed multifunction in cancer metastasis [25]. Taken together, these findings support the hypothesis that miR-145 may play a complex function in the development of CRC. To investigate the relationship of up-regulation of miR-145 with metastasis of colorectal cancer, miR-145 gain-of-function studies were performed in human CRC cells using lentivirus system. Our results showed that up-regulation of miR-145 could promote CRC migration and invasion in vitro and in vivo, while no effect on cell growth was observed. These data were inconsistent with some other reports which showed anti-oncogenic role of miR-145 in the metastasis. However, data derived from these observations were either from tissues other than colon [17,19–20]. Arndt et al reported an oncogenic role of miR-145 in CRC, which is in good agreement with our results [21]. 7. Down-regulation of Hsp-27 attenuated the oncogenic effect of miR-145 To verify whether the up-regulation of Hsp-27 contributes to the miR-145-induced motility in CRC cell, a series of assays were carried out in vitro. Hsp-27 siRNA or control siRNA was initially transfected into HCT-8-miR-145 cells. The reduction of protein expression of Hsp-27 was confirmed via western blotting (Fig. 5A). Cell migration was then observed using wound healing assay (Fig. 5B). These results showed that the mobility of HCT-8-miR- 145 cells transfected with Hsp-27 siRNA was remarkably slower than that of cells transfected with control siRNA. A similar result was also obtained in a cell invasion assay. Transwell-Matrigel penetration assay experiments were performed with HCT-8-miR- 145 cells transfected with Hsp-27 siRNA or control siRNA. In comparison to the control, knock down of Hsp-27 inhibited the invasion ability of HCT-8-miR-145 cells (Fig. 5C). The other three different siRNA oligos of Hsp-27 were able to recapitulate similar phenotypes (Fig. S4 in File S1). These results manifested that Hsp- 27 was an important downstream mediator during the prometas- tasis related to miR-145. PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e102017 4 MiR-145 Promotes Metastasis of Colorectal Cancer Figure 2. The effect of miR-145 overexpression on the HCT-8 cells. (A) qRT-PCR analysis of miR-145 expression in HCT-8 cells transfected with the lenti-miR-145 expression vector or the miRNA negative control vector using a lentivirus system. (B) Proliferation rates of HCT-8-miR-145 or HCT-8- NC cells detected by CCK-8 assay. (C) Cell cycle analysis of HCT-8-miR-145 or HCT-8-NC cells by Flow cytometry. Data represents average +SD of three independent experiments. doi:10.1371/journal.pone.0102017.g002 Figure 2. The effect of miR-145 overexpression on the HCT-8 cells. (A) qRT-PCR analysis of miR-145 expression in HCT-8 cells transfected with the lenti-miR-145 expression vector or the miRNA negative control vector using a lentivirus system. (B) Proliferation rates of HCT-8-miR-145 or HCT-8- NC cells detected by CCK-8 assay. (C) Cell cycle analysis of HCT-8-miR-145 or HCT-8-NC cells by Flow cytometry. Data represents average +SD of three independent experiments. doi:10.1371/journal.pone.0102017.g002 confirmed that the function of miR-145 related to cancer development is tissue-type specific. but not further down-regulation, along with the development of CRC (unpublished data). These observations of other CRC related miRNAs confirmed the accuracy of our results, which suggested that one miRNA maybe display different expression profile in different stages of cancer. Besides stage difference, the expression of miR-145 seems to be dependent on the type of tissue. July 2014 \| Volume 9 \| Issue 7 \| e102017 Microarray analysis Eight primary colorectal cancer tissues derived from stage II–III colorectal cancer patients with (n = 4) or without (n = 4) lymph node metastasis were collected and the expression profiles of miRNA were determined using Agilent miRNA microarray. Briefly, total RNA was extracted from tumor samples using the miRVana miRNA Isolation Kit (Ambion Inc., TX, USA). The quality and quantity of RNA samples were assessed by a 2100 Bioanalyzer using the RNA 6000 Pico LabChip kit (Agilent Technologies, Santa Clara, CA). The microarray contains probes for 851 human miRNAs from the Sanger database v.12.0. The microarray experiments were performed at ShanghaiBio Corpo- ration using Agilent miRNA labeling reagent and Hybridization Kits, Agilent human miRNA array (V2) and Agilent microarray scanner. All original microarray data is deposited in the NCBI GEO database [GSE48074]. Knockdown of Hsp-27 gene expression by siRNA was found to reverse miR-145-mediated induction of CRC cell migration in our study. The role of miR-145 in the maintenance of high level of Hsp-27 was not through direct gene targeting but stabilization of Hsp-27. Although the role and clinical outcome of Hsp-27 in primary tumors has been well studied and documented [33], its function in metastasis invasion is still unclear. Further investiga- tions to identify the mechanism of Hsp-27 involved in CRC metastasis will further enrich our understanding on the up- regulation of miR-145 in CRC. MiR-145 Promotes Metastasis of Colorectal Cancer MiR-145 Promotes Metastasis of Colorectal Cancer Figure 4. The expression profiles of Hsp-27 were detected in CRC cells or CRC tissues. (A) The expression levels of Hsp-27 were examined in HCT-8-miR-145 or HCT-8-NC cells by western blotting analysis. (B) The expression of Hsp-27 protein was detected in CRC and adjacent normal tissues by western blot assay. The relative Hsp27/actin ratios of individual bands are shown as the mean + SD of values derived from all patient samples (Non-tumor tissue, n = 47; LNM-P tumor tissue, n = 41; LNM-N tumor tissue, n = 43). (C–D) MiR-145 Enhanced Hsp-27 Stability in CRC Cells.HCT-8-miR-145 or HCT-8-NC cells were incubated with the protein synthesis inhibitor cycloheximide (CHX, 0.5 mg/mL) (C) or proteasome inhibitor MG-132 (5 mM) (D) for 24 hours. The level of total Hsp-27 was detected by western blotting analysis. The relative Hsp27/actin ratios of individual bands are shown as the mean 6 SD of values normalized to beta-actin. doi:10.1371/journal.pone.0102017.g004 Medical Sciences, Beijing, China. The samples were used with the written informed consents from patients and with the approval of the Chinese Academy of Medical Sciences Cancer Hospital. None of the patients received chemotherapy or radiotherapy prior to surgical resection. The whole samples reflect the natural distribu- tion of clinicopathological characteristics of CRC patients. Resected specimens were histologically examined by hematoxylin and eosin staining. Primary tumor tissues and corresponding adjacent non-tumor tissues were immediately collected after surgical removal and snap-frozen in liquid nitrogen for further use. We divided this patient cohort into two groups. Those with confirmed LNM were termed as lymph node positive (LNP) group and those without detectable LNM were termed the lymph node negative (LNN) group. All cases were reviewed and confirmed by two experienced pathologists. The clinical characteristics of these specimens are shown in Table 1. Figure 4. The expression profiles of Hsp-27 were detected in CRC cells or CRC tissues. (A) The expression levels of Hsp-27 were examined in HCT-8-miR-145 or HCT-8-NC cells by western blotting analysis. (B) The expression of Hsp-27 protein was detected in CRC and adjacent normal tissues by western blot assay. The relative Hsp27/actin ratios of individual bands are shown as the mean + SD of values derived from all patient samples (Non-tumor tissue, n = 47; LNM-P tumor tissue, n = 41; LNM-N tumor tissue, n = 43). Cell culture The human CRC cell line HCT-8 was purchased from Institute of Basic Medical Sciences Chinese Academy of Medical Sciences’ cell culture center (Beijing, China). The cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (Gibco, CA), 100 U/ml of penicillin and 100 mg/ml of streptomycin. Cells were incubated at 37uC and supplemented with 5% CO2 in a humidified chamber. 2DLC-ESI-MS/MS. Our analyses showed that Hsp-27 was up- regulated in miR-145 overexpressed CRC cells. Heat shock protein 27 (Hsp-27), an important member of the small Hsp family, is ubiquitously expressed in various cell types and involved in cellular responses for a variety of stresses such as heat shock, hypertonic stress, oxidative stress [26–27]. High levels of Hsp-27 have been found to be associated with the metastasis of several tumor types including CRC, prostate cancer, gastric cancer, hepatocellular cancer, head and neck squamous cell cancer [28– 30]. In particular, it has been proved that increased expression level of Hsp-27 in CRC was related to the lymph node metastasis [31–32]. Our results showed that there was a strong, positive correlation between Hsp-27 protein and miR-145 expression in primary human tissue samples, which indicated that miR-145 was involved, at least partially, in up-regulating of Hsp-27 protein expression. MiR-145 Promotes Metastasis of Colorectal Cancer (C–D) MiR-145 Enhanced Hsp-27 Stability in CRC Cells.HCT-8-miR-145 or HCT-8-NC cells were incubated with the protein synthesis inhibitor cycloheximide (CHX, 0.5 mg/mL) (C) or proteasome inhibitor MG-132 (5 mM) (D) for 24 hours. The level of total Hsp-27 was detected by western blotting analysis. The relative Hsp27/actin ratios of individual bands are shown as the mean 6 SD of values normalized to beta-actin. doi:10.1371/journal.pone.0102017.g004 RNA extraction and real-time quantitative reverse transcription–polymerase chain reaction The miRNA was purified from cultured cells or tissues using the Qiagen miRNeasy Mini Kit. Reverse-transcription reactions were performed using MiScript Reverse Transcription Kit (Qiagen, Germany.). MiScript SYBR Green PCR Kit in combination with miRNA-specific primers (mir-29b-1* cat.no. MS00009289, mir-95 cat.no. MS00010906, mir-100 cat.no. MS00003388, mir-125b cat.no. MS00006629, mir-152 cat.no. MS00003591, mir-376c cat.no. MS00004046, mir-199b cat.no. MS00003731, mir-145 cat.no. MS00003528, mir-99a cat.no. MS00003374, mir-1274a cat.no. MS00014420, mir-99b cat.no. MS00032165, Qiagen, Germany.) were used to detect mature miRNAs on LightCycler 480 (Roche, Basel, Switzerland). The relative expression of miRNA compared with U6 (cat. no. MS00033740, Qiagen, Germany.) was calculated using the -DCt method. All qRT-PCR reactions were performed in triplicate. In summary, the current study demonstrated that up-regulation of miR-145 contributed to lymph node metastasis of CRC. The mechanism of this contribution associated with the stabilization of Hsp-27, a protein which plays an important role in the promotion of metastasis. Future direction of evaluation of miR-145 should focus on the mechanism study which might lead to its application in metastasis diagnosis and treatment of CRC. 7. Down-regulation of Hsp-27 attenuated the oncogenic effect of miR-145 These discoveries further In order to disclose possible effector genes participating in this function, we identified cellular proteins which were directly or indirectly regulated by miR-145 using iTRAQ labeling and July 2014 \| Volume 9 \| Issue 7 \| e102017 PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e102017 PLOS ONE \| www.plosone.org 5 MiR-145 Promotes Metastasis of Colorectal Cancer ted invasion and metastasis of CRC cells in vitro and in vivo. (A) Migration and invasion es were representatives of at least three independent experiments. Average number of migr dent experiments 6 SD is shown by column figure. P,0.01. (C) Photo images of mesente njected into the liver with HCT-8-miR-145 or HCT-8-NC cells followed by surgical suture. Anima n. (D) Incidence of mesenteric lymph node metastasis in mice (table) and mean number of vis P,0.01. 02017.g003 145 promoted invasion and metastasis of CRC cells in vitro and in vivo. (A) Migration and invasion (B) assay of HCT-8-m ls. The images were representatives of at least three independent experiments. Average number of migration cell number pe ree independent experiments 6 SD is shown by column figure. P,0.01. (C) Photo images of mesenteric lymph node met which was injected into the liver with HCT-8-miR-145 or HCT-8-NC cells followed by surgical suture. Animals were killed 2 week ll inoculation. (D) Incidence of mesenteric lymph node metastasis in mice (table) and mean number of visible metastatic nod umn figure). P,0.01. rnal.pone.0102017.g003 ww.plosone.org 6 July 2014 \| Volume 9 \| Issue 7 \| e1 Figure 3. MiR-145 promoted invasion and metastasis of CRC cells in vitro and in vivo. (A) Migration and invasion (B) assay of HCT-8-miR-145 or HCT-8-NC cells. The images were representatives of at least three independent experiments. Average number of migration cell number per field from at least three independent experiments 6 SD is shown by column figure. P,0.01. (C) Photo images of mesenteric lymph node metastasis from nude mice which was injected into the liver with HCT-8-miR-145 or HCT-8-NC cells followed by surgical suture. Animals were killed 2 weeks post intra-hepatic cell inoculation. (D) Incidence of mesenteric lymph node metastasis in mice (table) and mean number of visible metastatic nodules in mesentery (column figure). P,0.01. doi:10.1371/journal.pone.0102017.g003 July 2014 \| Volume 9 \| Issue 7 \| e102017 PLOS ONE \| www.plosone.org 6 MiR-145 Promotes Metastasis of Colorectal Cancer Clinical samples The tissue samples analyzed in this study were obtained from 202 patients (117 males and 85 females) undergoing surgical resection for CRC at Cancer Hospital, Chinese Academy of July 2014 \| Volume 9 \| Issue 7 \| e102017 PLOS ONE \| www.plosone.org 7 MiR-145 Promotes Metastasis of Colorectal Cancer Generation of lentivirus to achieve gain of miR-145 PCR assays. Infected populations exhibiting between 70–90% fl ll d f l i i Figure 5. Knockdown of Hsp-27 by siRNA attenuated the prometastatic effect of miR-145. (A) HCT-8-mir-145 (mir-145) or HCT-8-NC (NC) cells were transfected with Hsp-27 siRNA (mir-145+si-hsp27) or a negative control siRNA (mir-145+mock). The expression of Hsp-27 protein was detected by western blot assay. (B) Wound-healing assay to evaluate the effect of Hsp-27 siRNA in HCT-8-miR-145 cells. (C) Knockdown of Hsp-27 by siRNA in HCT-8-miR-145 cells significantly inhibited cell invasion. The images were representatives of at least three independent experiments. Average number of invasion cell number per field from at least three independent experiments 6 SD is shown by column figure. P,0.01. doi:10.1371/journal.pone.0102017.g005 Figure 5. Knockdown of Hsp-27 by siRNA attenuated the prometastatic effect of miR-145. (A) HCT-8-mir-145 (mir-145) or HCT-8-NC (NC) cells were transfected with Hsp-27 siRNA (mir-145+si-hsp27) or a negative control siRNA (mir-145+mock). The expression of Hsp-27 protein was detected by western blot assay. (B) Wound-healing assay to evaluate the effect of Hsp-27 siRNA in HCT-8-miR-145 cells. (C) Knockdown of Hsp-27 by siRNA in HCT-8-miR-145 cells significantly inhibited cell invasion. The images were representatives of at least three independent experiments. Average number of invasion cell number per field from at least three independent experiments 6 SD is shown by column figure. P,0.01. doi:10.1371/journal.pone.0102017.g005 Figure 5. Knockdown of Hsp-27 by siRNA attenuated the prometastatic effect of miR-145. (A) HCT-8-mir-145 (mir-145) or HCT-8-NC (NC) cells were transfected with Hsp-27 siRNA (mir-145+si-hsp27) or a negative control siRNA (mir-145+mock). The expression of Hsp-27 protein was detected by western blot assay. (B) Wound-healing assay to evaluate the effect of Hsp-27 siRNA in HCT-8-miR-145 cells. (C) Knockdown of Hsp-27 by siRNA in HCT-8-miR-145 cells significantly inhibited cell invasion. The images were representatives of at least three independent experiments. Average number of invasion cell number per field from at least three independent experiments 6 SD is shown by column figure. ** P,0.01. doi:10.1371/journal.pone.0102017.g005 PCR assays. Infected populations exhibiting between 70–90% green fluorescent cells were used for later experimentation. Generation of lentivirus to achieve gain of miR-145 function Lentiviral pGCsil-GFP Vector was used to carrying human pre- miR-145 (miR-145) or nonfunctional control (NC). Lentiviral vector construction and production of high-titer lentiviral particles were made by Genechem biology company. The generated lentiviruses were used to infect HCT-8 for 24–48 h at 1–5 MOI, and the expression levels of miR-145 were determined by qRT- Supporting Information Table S1 Clinical and pathological characteristics of patients. iTRAQ labeling and LC-ESI-MS/MS analysis Statistical analysis was performed using SPSS program version 17.0. In vitro and in vivo data were evaluated by Student’s t-test. Unpaired t-test were used for analysis in Fig 1B, 1C (LNM-N(T) vs LNM-P(T)) and 4B. Paired t-test were used for analysis in Fig 1C (LNM-N(NT) vs LNM-N(T); LNM-P(NT) vs LNM- P(T)),2,3,4D,5,S1 and S4. Data are presented as mean 6 standard error of the mean. Error bars are representative of at least three independent experiments. The relationship between miRNA expression levels and various clinicopathologic characteristics were employed using the Mann–Whitney test. Correlations were determined using the Spearman correlation. All P values ,0.05 were considered significant. g y The cells were washed twice with PBS, collected with a cell scraper, and centrifuged. The pellet was vigorously washed with PBS. After centrifugation at 15,000 rpm for 30 minutes at 4uC, the clarified supernatant was transferred to fresh microtubes and the 2-D Quant Kit (GE Healthcare) was used for the accurate determination of protein concentration in samples followed by analysis in terms of SDS-PAGE. Each sample (100 mg protein) was digested with 0.2 mL of trypsin solution (50 mg/mL) at 37uC. After trypsin digestion, peptides were dried by vacuum centrifu- gation, reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for 8-plex iTRAQ (Applied Biosys- tems). Briefly, one unit of iTRAQ reagent (defined as the amount of reagent required to label 100 mg of protein) was thawed and reconstituted in 70 mL isopropanol. Peptides from treatment (or disease) and control subgroup were labeled with 114 and 121 iTRAQ tags, respectively, by incubation at room temperature for 2 h. The peptide mixtures were subsequently pooled and dried by vacuum centrifugation. The pooled mixtures of iTRAQ-labeled peptides were fractionated by SCX chromatography (Phenomen- ex),tandem mass spectrometry (MS/MS) in a LTQ Orbitrap Velos (Thermo fisher) coupled online to the HPLC. The protein experiments were performed at BGI Corporation. Proliferation assay Cells were grown in RPMI-1640 medium containing 10% fetal serum. 16103 cells were seeded in flat-bottom 96 well plates and incubated at 37uC in 5% CO2. Cell viability was measured using Cell Counting Kit-8 (Dojindo Laboratories) at day1,day3 and day5. For cell cycle analysis, cells were washed and fixed with ice- PLOS ONE \| www.plosone.org July 2014 \| Volume 9 \| Issue 7 \| e102017 8 MiR-145 Promotes Metastasis of Colorectal Cancer on the same membrane was used as a loading control. Mouse monoclonal anti-Hsp27 antibody (G31) was purchased from Cell Signaling Technology, anti-b-actin Ab (A5411) from Sigma. cold 75% (v/v) ethanol at 220uC for 2 h, then stained with PI at the concentration of 50 mg/mL in the presence of RNase A (100 mg/mL). DNA content was analyzed by Flow cytometry analysis (Beckman Coulter, USA). Cell migration/invasion assays Cell motility and invasiveness were determined by a 24 well transwell plate (8 mM pore size; Costar), as described previously.10 Briefly, for transwell migration assays, 16104 cells were placed on the top chamber lined with the noncoated membrane. For invasion assays, 36104 cells were placed on the upper chamber of each insert coated with 200 mg/ml of Matrigel (BD Biosciences, CA, USA). The siRNA specifically targeting Hsp-27 (Sense: 59-ACGGU- CAAGACCAAGGAUGdTdT-39; Anti-sense: 59-CAUCCUUG- GUCUUGACCGUdTdT-39) and control siRNAs were designed as described and were synthesized as 29-O-methyl modification by GenePharma (Shanghai, China) [13]. HCT-8-miR-145 cells were transfected with Hsp-27 siRNA or control siRNAs (200 nM) using Lipofectamine 2000 (Invitrogen) reagent according to the manu- facturer’s instructions. Lysates or cells were harvested 24 h later and subjected to western blotting or transwell-matrigel penetration assay respectively. In vivo metastasis assays For in vivo metastasis assays, 56104 HCT-8-miR-145 cells or HCT-8-NC cells were injected into the liver of nude mice followed by surgical suture (three in each group, female nu/nu). After 2 weeks, the mice were killed, their livers were dissected, and the mesenteric lymph node metastases were counted. The nude mice were purchased from Vital River (Beijing, China) and raised in a specific pathogen free (SPF) animal laboratory. All experiments involving animals were approved by Chinese Academy of Medical Sciences and Peking Union Medical College Ethical Committee and performed according to the legal requirements. Wound-healing assay The cells were grown to confluence, and a wound was made through the monolayer using a p200 pipette tip. After wounding, the culture medium was removed, and cells were washed at least twice to eliminate detached cells. Wound closure was imaged by an inverted microscope at 0, 6, 12 and 24 h after wounding. Three independent experiments were performed. Inhibition of protein synthesis and proteasome After incubation with protein synthesis inhibitor Cycloheximide (CHX, 0.5 mg/ml, Beyotime, Shanghai, China) or proteasome inhibitor MG-132 (5 mM, Beyotime, Shanghai, China) for 24 h, cells were collected and processed for western blotting analysis. File S1 Supporting Figures. Figure S1, Migration assay of sw620 or sw480 cells transfected with the lentimiR-145-expression vector or the control vector. Figure S2, Hsp-27 protein expression profile in primary human tissue samples (including 41 CRC with LNM, 43 CRC without LNM, 47 adjacent non-tumor tissue) by western blot. Figure S3, Correlation between Hsp-27 protein and miR-145 expression in primary human tissue samples (including 41 CRC with LNM, 43 CRC without LNM, 47 adjacent non-tumor tissue). Figure S4, Knockdown of Hsp-27 by other three different siRNA oligos in HCT-8-miR-145 cells significantly inhibited cell migration and invasion. (DOC) Knockdown of Hsp-27 using small interfering RNA Knockdown of Hsp-27 using small interfering RNA (siRNA) Table S2 Most significant differentially expressed pro- teins identified in iTRAQ. (DOC) Table S2 Most significant differentially expressed pro- teins identified in iTRAQ. (DOC) References (2010) MicroRNA let-7a inhibits proliferation of human prostate cancer cells in vitro and in vivo by targeting E2F2 and CCND2. PLoS One 5: e10147. 25. Zhang X, Liu S, Hu T, Liu S, He Y, et al. (2009) Up-regulated microRNA-143 transcribed by nuclear factor kappa B enhances hepatocarcinoma metastasis by repressing fibronectin expression. Hepatology 50(2):490–499. 9. Asangani IA, Rasheed SA, Nikolova DA, Leupold JH, Colburn NH, et al. (2008) MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 27: 2128–2136. 26. 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Chen X, Gong J, Zeng H, Chen N, Huang R, et al. (2010) MicroRNA145 targets BNIP3 and suppresses prostate cancer progression. Cancer Res 70: 2728–2738. 23. He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, et al. (2005) A microRNA polycistron as a potential human oncogene. Nature 435: 828–833. 24. O’Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT (2005) C-myc regulated microRNAs modulate E2F1 expression. Nature 435: 839–843. 8. Dong Q, Meng P, Wang T, Qin W, Wang F, et al. MiR-145 Promotes Metastasis of Colorectal Cancer QL. Contributed reagents/materials/analysis tools: WY CGS QL WYT HYA. Wrote the paper: WY CGS QL JM. Author Contributions Conceived and designed the experiments: WY CGS QL JM. Performed the experiments: WY CGS QL WYT HYA. Analyzed the data: WY CGS Western blot Cell proteins were extracted using RIPA buffer (1xPBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM Na3VO4 and 1 mM aprotinin and 1 mM phenylmethylsulfonyl fluoride) and determined using 10% SDS- PAGE. Western blot analysis was performed according to standard procedures as previously described [12]. The expression of b-actin July 2014 \| Volume 9 \| Issue 7 \| e102017 PLOS ONE \| www.plosone.org 9 QL. Contributed reagents/materials/analysis tools: WY CGS QL WYT HYA. Wrote the paper: WY CGS QL JM. References Cancer Res 68(15):6416–6424. 31. Pei H, Zhu H, Zeng S, Graham M, Prince ME, et al.(2007)Proteome analysis and tissue microarray for profiling protein markers associated with lymph node metastasis in colorectal cancer. J Proteome Res 6(7):2495–2501. 15. Bandre´s E, Cubedo E, Agirre X, Malumbres R, Za´rate R, et al. (2006) Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues. Mol Cancer 5: 29–39. 32. Tweedle EM, Khattak I, Ang CW, Nedjadi T, Jenkins R, et al. (2010) Low molecular weight heat shock protein Hsp-27 is a prognostic indicator in rectal cancer but not colon cancer. Gut 59(11):1501–1510. p 16. Akao Y, Nakagawa Y, Hirata I, Iio A, Itoh T, et al. (2010) Role of anti-oncomirs miR-143 and -145 in human colorectal tumors. Cancer Gene Ther 17(6): 398– 408. 33. Huang Q, Ye J, Chen W, Wang L, Lin W, et al. (2010) Heat shock protein 27 is overexpressed in tumor tissues and increased in sera of patients with gastric adenocarcinoma. Clin Chem Lab Med 48: 263–269. 17. Sachdeva M, Mo YY (2010) MicroRNA-145 suppresses cell invasion and metastasis by directly targeting mucin 1. Cancer Res 70(1):378–387. July 2014 \| Volume 9 \| Issue 7 \| e102017 PLOS ONE \| www.plosone.org 10
https://openalex.org/W2905179958	https://www.nature.com/articles/sdata2018286.pdf	English	null	On the privacy-conscientious use of mobile phone data	Scientific data	2,018	cc-by	4,840	Comment: On the privacy- conscientious use of mobile phone data Yves-Alexandre de Montjoye1,2, Sébastien Gambs3, Vincent Blondel4, Geoffrey Canright5, Nicolas de Cordes6, Sébastien Deletaille7, Kenth Engø-Monsen5, Manuel Garcia-Herranz8, Jake Kendall9, Cameron Kerry2, Gautier Krings4,7, Emmanuel Letouzé2,10, Miguel Luengo-Oroz11, Nuria Oliver10,12, Luc Rocher4, Alex Rutherford11, Zbigniew Smoreda6, Jessica Steele13,14, Erik Wetter14,15,16, Alex “Sandy” Pentland2 & Linus Bengtsson14 Yves-Alexandre de Montjoye1,2, Sébastien Gambs3, Vincent Blondel4, Geoffrey Canright5, Nicolas de Cordes6, Sébastien Deletaille7, Kenth Engø-Monsen5, Manuel Garcia-Herranz8, Jake Kendall9, Cameron Kerry2, Gautier Krings4,7, Emmanuel Letouzé2,10, Miguel Luengo-Oroz11, Nuria Oliver10,12, Luc Rocher4, Alex Rutherford11, Zbigniew Smoreda6, Jessica Steele13,14, Erik Wetter14,15,16, Alex “Sandy” Pentland2 & Linus Bengtsson14 Received: 29 January 2018 Accepted: 26 October 2018 Published: 11 December 2018 Received: 29 January 2018 Accepted: 26 October 2018 Published: 11 December 2018 The breadcrumbs we leave behind when using our mobile phones—who somebody calls, for how long, and from where—contain unprecedented insights about us and our societies. Researchers have compared the recent availability of large-scale behavioral datasets, such as the ones generated by mobile phones, to the invention of the microscope, giving rise to the new ﬁeld of computational social science. With mobile phone penetration rates reaching 90%1 and under-resourced national statistical agencies2, the data generated by our phones—traditional Call Detail Records (CDR) but also high-frequency x-Detail Record (xDR)—have the potential to become a primary data source to tackle crucial humanitarian questions in low- and middle-income countries. For instance, they have already been used to monitor population displacement after disasters3, to provide real-time trafﬁc information, and to improve our understanding of the dynamics of infectious diseases4. These data are also used by governmental and industry practitioners in high-income countries. While there is little doubt on the potential of mobile phone data for good, these data contain intimate details of our lives: rich information about our whereabouts, social life, preferences, and potentially even ﬁnances. A BCG study showed, e.g., that 60% of Americans consider location data and phone number history—both available in mobile phone data—as “private”. Historically and legally, the balance between the societal value of statistical data (in aggregate) and the protection of privacy of individuals has been achieved through data anonymization. While hundreds of different anonymization algorithms exist, most of them are variations and improvements of the seminal k-anonymity algorithm introduced in 19985. Recent studies have, however, shown that pseudonymization and standard de-identiﬁcation are not sufﬁcient to prevent users from being re-identiﬁed in mobile phone data. www.nature.com/scientificdata www.nature.com/scientificdata Received: 29 January 2018 Accepted: 26 October 2018 Published: 11 December 2018 Comment: On the privacy- conscientious use of mobile phone data This was echoed in the recent report of the [US] President’s Council of Advisors on Science and Technology on Big Data Privacy which consider de- identiﬁcation to be useful as an “added safeguard, but [emphasized that] it is not robust against near-term future re-identiﬁcation methods”. The limits of the historical de-identiﬁcation framework to adequately balance risks and beneﬁts in the use of mobile phone data are a major hindrance to their use by researchers, development practitioners, humanitarian workers, and companies. This became particularly clear at the height of the Ebola crisis, when qualiﬁed researchers (including some of us) were prevented from accessing relevant mobile phone data on time despite efforts by mobile phone operators, the GSMA, and UN agencies8, with privacy being cited as one of the main concerns. These privacy concerns are, in our opinion, due to the failures of the traditional de-identiﬁcation model and the lack of a modern and agreed upon framework for the privacy-conscientious use of mobile phone data by third-parties especially in the context of the EU General Data Protection Regulation (GDPR). Such frameworks have been developed for the anonymous use of other sensitive data such as census, household survey, and tax data9. The positive societal impact of making these data accessible and the technical means available to protect people’s identity have been considered and a trade-off, albeit far from perfect9, has been agreed on and implemented. This has allowed the data to be used in aggregate for the beneﬁt of society. Such thinking and an agreed upon set of models has been missing so far for mobile phone data10. This has left data protection authorities, mobile phone operators, and data users with little guidance on technically sound yet reasonable models for the privacy-conscientious use of mobile phone data. This has often resulted in suboptimal tradeoffs if any8. p y In this paper, we propose four models for the privacy-conscientious use of mobile phone data (Fig. 1). All of these models 1) focus on a use of mobile phone data in which only statistical, aggregate information is ultimately needed by a third-party and, while this needs to be conﬁrmed on a per-country basis, 2) are designed to fall under the legal umbrella of “anonymous use of the data”. Examples of cases in which only statistical aggregated information is ultimately needed by the third-party are discussed below. Comment: On the privacy- conscientious use of mobile phone data Four data points—approximate places and times where an individual was present—have been shown to be enough to uniquely re-identify them 95% of the time in a mobile phone dataset of 1.5 million people6. Furthermore, re-identiﬁcation estimations using unicity—a metric to evaluate the risk of 1Department of Computing, Imperial College London, London SW7 2AZ, UK. 2MIT Media Lab, 20 Ames St, Cambridge, MA 02139, USA. 3Université du Québec à Montréal, Département d’informatique, Case postale 8888, succ. Centre-ville, Montréal (Québec), H3C 3P8, Canada. 4Université catholique de Louvain, Place de l’Université 1, 1348 Louvain-la-Neuve, Belgium. 5Telenor Research, Snarøyveien 30, 1360 Fornebu, Norway. 6Orange, 44 avenue de la République, 92320 Châtillon, France. 7Riaktr, 5 Place du Champs de Mars, 1050 Brussels, Belgium. 8UNICEF, Ofﬁce of Innovation, 3 UN Plaza, New York, NY 10017, USA. 9University of Washington, Dept. of Computer Science, 708b 11th Avenue East, Seattle, WA 98102, USA. 10Data-Pop Alliance, 99 Madison Avenue, 15th Floor, New York, NY 10016, USA. 11UN Global Pulse, 370 Lexington Avenue, New York, NY 10017, USA. 12Vodafone Research, Paddington Central, London, W2 6BY, UK. 13University of Southampton, Geography and Environment, Building 44, University Road, Southampton, SO17 1BJ, UK. 14Flowminder Foundation, Roslagsgatan 17, SE-11355, Stockholm, Sweden. 15Stockholm School of Economics, Sveavägen 65, 113 83 Stockholm, Sweden. 16Asian Institute of Management, 123 Paseo de Roxas, 1229 Metro Manila, Philippines. Correspondence and requests for materials should be addressed to Y.-A.d.M. (email: demontjoye@imperial.ac.uk) SCIENTIFIC DATA \| 5:180286 \| DOI: 10.1038/sdata.2018.286 www.nature.com/sdata/ Remote access Question-and- answer Limited release Pre-computed indicators and synthetic data Applicative Medium to large number of users and/or open data Exploratory Low to medium number of users Development stage Secure access to pseudonymized data Direct access to limited or aggregated data release Data protected through Figure 1. Matrix of the four models for the privacy-conscientious use of mobile phone data. Figure 1. Matrix of the four models for the privacy-conscientious use of mobile phone data. re-identiﬁcation in large-scale datasets6—and attempts at k-anonymizing mobile phone data7 ruled out de-identiﬁcation as sufﬁcient to truly anonymize the data. This was echoed in the recent report of the [US] President’s Council of Advisors on Science and Technology on Big Data Privacy which consider de- identiﬁcation to be useful as an “added safeguard, but [emphasized that] it is not robust against near-term future re-identiﬁcation methods”. re-identiﬁcation in large-scale datasets6—and attempts at k-anonymizing mobile phone data7 ruled out de-identiﬁcation as sufﬁcient to truly anonymize the data. Comment: On the privacy- conscientious use of mobile phone data They would include, e.g., disaster management, mobility analysis, or the training of AI algorithms11 in which only aggregate information on people’s mobility is ultimately needed by agencies, and exclude cases in which individual-level identiﬁable information is needed such as targeted advertising or loans based on behavioral data. First, it is important to insist that none of these models is a silver bullet. However, we believe that each one, depending on the stage of development of the project and the release cycle of the data, provides a reasonable balance between utility and privacy. They can all be used as a basis to use mobile phone data for positive social impact in a privacy-conscientious way, with costs deemed reasonable to telco’s data philanthropy efforts. Other models however also exist e.g. contractual arrangements that do not rely on anonymization including the pooling of data from several stakeholders through a trusted intermediary. We however do not discuss these models here as their privacy and security guarantees are non-technical and stem solely from contractual relationships between institutions. While our analysis and recommendations focus on mobile phone data, some of the challenges we highlight and the models we propose are likely to be applicable to other types of data. For instance, URL data were shown to have a high unicity12 making them likely to be re-identiﬁable, and the remote access model described below is used by the Secure Access Data Center (CASD) infrastructure in France to grant third-parties access to sensitive health data. Finally, our models focus on providing ways for mobile phone data to be anonymously used. Other risks related to ethics and membership inference attacks exist13,14. While such SCIENTIFIC DATA \| 5:180286 \| DOI: 10.1038/sdata.2018.286 www.nature.com/sdata/ risks typically have to be legally addressed in data protection impact assessments (DPIA), some organizations go further, e.g., by setting up an external ethics committee to review data uses. We will now review the four models, emphasizing their applicability to different data uses, pros and cons, and implementation challenges. We will also discuss potential threats and resulting information leaks for each model. Limited release is the closest model to traditional sharing of data. A mobile phone dataset is transformed in-house and a copy of the data is given to third-parties under a legal contract. Comment: On the privacy- conscientious use of mobile phone data The transformation aims at both adding technical difﬁculties to attempts at re-identifying individuals and at limiting the amount of information that could be uncovered if the data were to be re-identiﬁed. The transformed data are, however, still fairly close to the raw data. Transformations typically consist of 1) data sampling and longitudinal resampling with new identiﬁers - either using correspondence tables, properly salted hashes or the use of key-hash function - as well as 2) limited data coarsening along the temporal axis and Voronoi translation of antennas (spatial axis)10. We recommend limited spatial and temporal coarsening as it has been shown to only marginally help prevent re-identiﬁcation while restricting the general useof the data6. Transformations affect, in general, the quality and quantity of the data available to researchers thereby limiting statistical power and potentially preventing important research questions from being explored. The main implementation challenge of the limited release model is probably the choice of the transformation. It requires an in-depth understanding of all of the current but also future uses of the data, as anonymization can usually only be performed once. Furthermore, as discussed before, these transformations alone are increasingly not sufﬁcient to make the data subject “no longer identiﬁable” and, consequently, to release the data openly. Appropriate non-disclosure and data use agreements (DUA) are therefore required. q In the limited release model, the transformed data is released directly to the users. The data controller therefore loses technical control over the data. This signiﬁcantly increases the risk of the data to be stolen, uploaded online, or to be part of a data breach. It puts a lot of weight on the data anonymization procedure. Because of this, we consider re-identiﬁcation using auxiliary location information to be the main privacy threat in the limited release model: re-identiﬁcation would allow an attacker to link the released data about one to all of the users back to their identities. We therefore recommend the limited release model for data sharing with a small to medium-sized group of moderately trusted third-parties, for initial and exploratory data analysis. An example of limited releases are Orange’s D4D challenges, in which data were transformed (sampled, limited longitudinality, slightly coarsened, etc.) before being released to selected teams of researchers under strict DUA15. Pre-computed indicators and synthetic data. Comment: On the privacy- conscientious use of mobile phone data Despite the limits of data anonymization, there are cases in which one would like to (or has to by law) release data without restrictions on users or access. In the pre- computed indicators model, indicators derived from mobile phone data are released to third-parties. These indicators can be computed at individual level (e.g., number of calls, radius of gyration)16 or aggregated across individuals (e.g., number of users per tower over time, long or short-term mobility matrices, and matrices of inter-towers communications). Because indicators are 1) much more disconnected from both the raw and potential auxiliary data, and 2) potentially aggregated across individuals, they can often be properly anonymized. However, it should be noted that recent work in the privacy literature has started to question the level of protection that is really provided by aggregation methods17. Similarly, synthetic data representations can be parameterized using mobile phone data and the parameters released openly along with the model. Synthetic data generated by the model and preserving pre-deﬁned statistical properties of the original data can equivalently be released. However, little work, so far, exists in synthetic mobile phone data representations and the development of representative and useful synthetic data in other ﬁelds has proven challenging18. On the privacy side, we see the main privacy threats for pre-computed indicators and synthetic data to be questions around the notion of “group privacy”13,19, which pertains to all release types. Deﬁnitions vary but, intuitively, the idea is that one’s individual privacy might be violated if information about a group he belongs to is revealed. Aggregated or anonymized data might indeed reveal sensitive information on groups and could lead to stigmatization or discrimination. In the case of mobile phone data, the privacy of a speciﬁc ethnic, or religious or minority group might, for example, be endangered if information about their behavior were to be revealed. We therefore recommend the pre-computed indicators model for the open release of well-established and stable-across-time metrics of interest such as mobility and behavioral indicators for applicative purposes. Examples would include the release of ﬂow maps parameterized using mobile phone data by Flowminder as part of the ﬁght against Ebola20 or the release of tourism statistics by Statistics Netherlands21. Remote access is our ﬁrst model using the privacy-through-security approach. Comment: On the privacy- conscientious use of mobile phone data This is the model that was used by Flowminder when studying people’s mobility directly after the Nepal earthquake. A small number of registered researchers analysed pseudonymized mobile phone data remotely with security measures in place. CASD is an example of the type of infrastructure needed to support these kind of analyses. It allows researchers to access the data through a virtual desktop system with dedicated authentication hardware while any data taken out of the system are manually veriﬁed. Question-and-answer Last but not least, the question-and-answer (QA) model pushes the privacy- through-security approach one step further: the data stays within the premises of the operator but third- parties now only access the data through a question-and-answer system (e.g., SafeAnswers22 or SQL queries23,24). Questions are asked in the form of a piece of code whose answers are computed using the pseudonymized data. These are validated by the system before being sent back to the user through the API. Answers can be at the level of individuals or, more often, groups of individuals. A question could be, for example, “How many people have been travelling from city A to city B between this date and this date”. The results are then aggregated and validated, and the answer, e.g., “3159”, is shared with the third- party through the API. The same security mechanisms than for the remote access are put in place: registration of users, restrictions on IP addresses, etc. On top of this, because the framework and language used to ask the questions as well as the user-facing API are standardized, more advanced and automated security and auditing mechanisms can be put in place. For instance, the system can ensure that the code runs for each user independently within a sandbox and can, manually or automatically, validate it25. If answers are aggregated over groups of individuals, the system can also ensure that the aggregation mechanism protects individuals’ privacy ensuring, e.g., that k individuals have contributed to each answer, that a certain level of coarsening or noise addition is added, or guaranteeing differential privacy26. Finally, every question asked (both algorithm and parameters) can be fully logged. y y q g p y gg In practice, the implementation details of these techniques will depend on the trust we place in users, how many users there are, and the estimated sensitivity of the data. Comment: On the privacy- conscientious use of mobile phone data Here, the data are not released but instead stay within the premises and under the control of the operator (or an authorized entity) and are analysed remotely. The data processing takes place within the operator’s premises and only aggregated data leave the secure area. In contrast to the data anonymization-based models we presented SCIENTIFIC DATA \| 5:180286 \| DOI: 10.1038/sdata.2018.286 www.nature.com/sdata/ previously, the data controller does not have to relinquish all control over the data. The controller can supervise who accesses the data (having users registering, signing a DUA, setting restrictions on IP addresses), how the data are being used (e.g., through active monitoring of the secured environment or by controlling the output), and can ensure that no individual-level or raw data leave the server (through a manual approval process or by monitoring the amount of data leaving the server). While they do not remove all possible risks, these security-based mechanisms already strongly limit the risks of the data to be re-identiﬁed en masse and misused. This, in turn, allows the data controller to transform the data less aggressively, for instance only removing phone numbers and other direct personal identiﬁers, potentially along with limited temporal and spatial coarsening. This limited transformation as well as the ability to access data in near-real time strongly increase the utility and possible uses of the data. We see the main privacy threat for the remote access model to be the risk of a targeted user to be re- identiﬁed. Because the data analysis happens within a secured and controlled environment, the mass re- identiﬁcation of users and exﬁltration of their data is very unlikely. A secondary threat would be for the server holding the data to be compromised. While not impossible, we do not consider this risk to be signiﬁcantly higher than the risk of the server currently holding the data to be compromised. From a practical perspective, we see the funding and the development of such appropriately secured infrastructure—yet ﬂexible enough to support a variety of research questions and tools—as the main practical challenge for the remote access model especially as it requires signiﬁcant human investments from the telco. We therefore recommend the remote access model to allow near real-time data to be used by highly- trusted third-parties under a DUA for conﬁrmatory or applicative analysis including the training of AI algorithms. SCIENTIFIC DATA \| 5:180286 \| DOI: 10.1038/sdata.2018.286 References In Privacy- Preserving Data Mining: Models and Algorithms Aggarwal (eds. Aggarwal, C. C. & Yu, P. S.) 415–431 (Springer US, 2008). g g g gg ( gg ) ( p 26. Dwork, C. Differential privacy. Encyclopedia of Cryptography and Security, 338–340. (Springer US, 2011). 27. Chaum, D., Crépeau, C. & Damgard, I. Multiparty Unconditionally Secure Protocols. In Proceedings of the Twentieth Annual ACM Symposium on Theory of Computing 11–19 (ACM, 1988). 8. Gascón, A. et al. Privacy-preserving distributed linear regression on high-dimensional data. Proceedings on Privacy Enhancing Technologies 2017, 345–364 (2017). References 1. International Telecommunication Union. The World in 2014: ICT Facts and Figures (2014). 1. 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Quantifying Surveillance in the Networked Age: Node-based Intrusions and Group Privacy. Preprint at, https://arxiv.org/abs/1803.09007 (2018). de-based Intrusions and Group Privacy. Preprint at, https://arxiv l k l h b l b k 20. Wesolowski, A. et al. Commentary: Containing the Ebola Outbreak – the Potential and Challenge of Mobile Network Data. P Curr 6 (2014). 21. Heerschap, N., Ortega, S., Priem, A. & Offermans, M. Innovation of tourism statistics through the use of new big data sources. In 12th Global Forum on Tourism Statistics, Prague, CZ (2014). 22. de Montjoye, Y.-A., Shmueli, E., Wang, S. S. & Pentland, A. S. openPDS: protecting the privacy of metadata through SafeAnswers. PLoS One 9, e98790 (2014). 3. Francis, P., Probst Eide, S. & Munz, R. Difﬁx: High-Utility Database Anonymization. in Privacy Technologies and Policy 141–158 (Springer International Publishing, 2017). 24. Johnson, N., Near, J. P. & Song, D. Towards Practical Differential Privacy for SQL Queries. Proceedings VLDB Endowment 11, 526–539 (2018). 25. Nabar, S. U., Kenthapadi, K., Mishra, N. & Motwani, R. A Survey of Query Auditing Techniques for Data Privacy. Acknowledgements S.G. is supported by a Discovery Grant and a Discovery Accelerator Supplement Grant from NSERC as well by the Canada Research Chair program, J.E.S. by the Bill & Melinda Gates Foundation (OPP1106936) and Belgian Federal Science Policy Ofﬁce (BELSPO), S.P. by MIT Media Lab Consortium: 2746036, Y.-A. dM., N.dC., E.L. and S.P. are supported by a grant from the Agence française de développement (AFD), L. R. by the Belgian Fund for Scientiﬁc Research (F.R.S.-FNRS), M.G.-H. by the UNICEF Venture Fund. Comment: On the privacy- conscientious use of mobile phone data We would consider reasonable a system with 1) some validation (manual or semi-automatic) of the code being used—potentially through a bank of open-source algorithms such as in the OPAL project—, 2) a strict control of the aggregation mechanisms used for each question, and 3) carefully added noise. If the data are distributed (across users or pieces of information), tools such as secure multiparty computation can be used to compute aggregated results27 or to run statistical analysis such as correlations28. We see the need for open-source software and practical privacy mechanisms to be the main challenges to the implementation of the question-and-answer model. q Since the use of the data is tightly controlled, we consider the server being compromised to be the main privacy threat. However, as for the remote access model, we do not consider this risk to be signiﬁcantly higher than the risk of any places where the data would be digitally stored (server, laptops, etc.) to be compromised. While the likelihood of an attacker being able to infer information about a speciﬁc re-identiﬁed user through the QA API is not null (these attacks served as motivation for mechanisms such as differential privacy26), we consider this risk to be moderate when combined with defense-in-depth mechanisms. In both the remote access and question-and-answer model, the data controller does not lose technical control over the data and measures can always be taken as response to a potential privacy breach. We therefore recommend the question-and-answer model for more formalized uses of mobile phone data by a medium to high numbers of third-parties in near real or real time. To conclude, mobile phone data has a great potential for good but its high dimensionality limits the applicability of traditional data anonymization methods. These limits have to be acknowledged and blanket anonymization or de-identiﬁcation statements are not acceptable anymore. However, as recent crises have SCIENTIFIC DATA \| 5:180286 \| DOI: 10.1038/sdata.2018.286 www.nature.com/sdata/ made abundantly clear, having qualiﬁed researchers being barred from accessing and using valuable mobile phone data is not acceptable either8. We have here proposed four models for the privacy-conscientious use of mobile phone data which we hope, moving forward, will help properly balance technically the need to use this data for good and the legitimate privacy concerns of individuals and societies. made abundantly clear, having qualiﬁed researchers being barred from accessing and using valuable mobile phone data is not acceptable either8. Comment: On the privacy- conscientious use of mobile phone data We have here proposed four models for the privacy-conscientious use of mobile phone data which we hope, moving forward, will help properly balance technically the need to use this data for good and the legitimate privacy concerns of individuals and societies. References 1. International Telecommunication Union. The World in 2014: ICT Facts and Figures (2014). © The Author(s) 2018 Additional Information h Competing interests: The authors declare no competing interests. How to cite this article: de Montjoye, Y.-A. et al. On the privacy-conscientious use of mobile phone data. Sci. Data. 5:180286 doi: 10.1038/sdata.2018.286 (2018). SCIENTIFIC DATA \| 5:180286 \| DOI: 10.1038/sdata.2018.286 www.nature.com/sdata/ 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 Interna- tional 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons. org/licenses/by/4.0/ © The Author(s) 2018 SCIENTIFIC DATA \| 5:180286 \| DOI: 10.1038/sdata.2018.286
https://openalex.org/W3034550020	https://europepmc.org/articles/pmc7352674?pdf=render	English	null	TP53-Deficient Angiosarcoma Expression Profiling in Rat Model	Cancers	2,020	cc-by	12,899	Received: 27 February 2020; Accepted: 3 June 2020; Published: 10 June 2020 Abstract: Sarcomas are a heterogeneous group of malignant tumors, that develop from mesenchymal cells. Sarcomas are tumors associated with poor prognosis and expected short overall survival. Eﬀorts to improve treatment eﬃcacy and treatment outcomes of advanced and metastatic sarcoma patients have not led to signiﬁcant improvements in the last decades. In the Tp53C273X/C273X rat model we therefore aimed to characterize speciﬁc gene expression pattern of angiosarcomas with a loss of TP53 function. The presence of metabolically active tumors in several locations including the brain, head and neck, extremities and abdomen was conﬁrmed by magnetic resonance imaging (MRI) and positron emission tomography (PET) examinations. Limb angiosarcoma tumors were selected for microarray expression analysis. The most upregulated pathways in angiosarcoma vs all other tissues were related to cell cycle with mitosis and meiosis, chromosome, nucleosome and telomere maintenance as well as DNA replication and recombination. The downregulated genes were responsible for metabolism, including respiratory chain electron transport, tricarboxylic acid (TCA) cycle, fatty acid metabolism and amino-acid catabolism. Our ﬁndings demonstrated that the type of developing sarcoma depends on genetic background, underscoring the importance of developing more malignancy susceptibility models in various strains and species to simulate the study of the diverse genetics of human sarcomas. Keywords: angiosarcoma; TP53; p53 TGEM Rat; microarray analysis Article TP53-Deﬁcient Angiosarcoma Expression Proﬁling in Rat Model Urszula Smyczy´nska 1,†, Damian Strzemecki 2,†, Anna M. Czarnecka 2,3,* , Wojciech Fendler 1,4, Michał Fiedorowicz 2,5 , Marlena Wełniak-Kami´nska 2,5, Magdalena Guzowska 2,6, Kamil Synoradzki 2, Łukasz Cheda 7, Zbigniew Rogulski 7 and Paweł Grieb 2 1 Department of Biostatistics and Translational Medicine, Medical University of Lodz, 92-215 Lodz, Poland; ulasmyczynska@gmail.com (U.S.); wojciech_fendler@dfci.harvard.edu (W.F.) 1 Department of Biostatistics and Translational Medicine, Medical University of Lodz, 92-215 Lodz, Poland; ulasmyczynska@gmail.com (U.S.); wojciech_fendler@dfci.harvard.edu (W.F.) y y g j 2 Department of Experimental Pharmacology, Mossakowski Medical Research Centre, Polish Academy of Sciences, 02-106 Warsaw, Poland; damian.strzemecki@gmail.com (D.S.); mﬁedorowicz@imdik.pan.pl (M.F.); marlenak@imdik.pan.pl (M.W.-K.); magdalena_guzowska@sggw.pl (M.G.); ksynoradzki@imdik.pan.pl (K.S.); pgrieb@imdik.pan.pl (P.G.) 3 Department of Soft Tissue, Bone Sarcoma and Melanoma, Maria Sklodowska-Curie National Research Institute of Oncology, 02-781 Warsaw, Poland gy 4 Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA 02284-9168, USA 5 5 Small Animal Magnetic Resonance Imaging Laboratory, Mossakowski Medical Research Centre, Polish Academy of Sciences, 02-106 Warsaw, Poland 6 Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences 02-776 Warsaw, Poland Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 02-093 Warsaw, 7 Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 02-093 Wars Poland; lcheda@chem.uw.edu.pl (Ł.C.); rogul@chem.uw.edu.pl (Z.R.) 7 Faculty of Chemistry, Biological and Chemical Research Centre, Univer Poland; lcheda@chem.uw.edu.pl (Ł.C.); rogul@chem.uw.edu.pl (Z.R.) * Correspondence: anna.czarnecka@gmail.com; Tel.: +48-22-608-6474 † These authors contributed equally to this paper. cancers cancers cancers cancers cancers 1. Introduction Sarcomas, typically divided into soft tissue sarcomas (STS) and bone sarcoma (BS), are a heterogeneous group of malignant tumors, that develop from mesenchymal cells. Globally more Cancers 2020, 12, 1525; doi:10.3390/cancers12061525 www.mdpi.com/journal/cancers www.mdpi.com/journal/cancers 2 of 17 Cancers 2020, 12, 1525 than 130,000 new cases of sarcoma are diagnosed annually, and those make up 1% to 3% of the total number of all tumors in general. Sarcomas are burdened by poor prognosis with expected short overall survival. Only approximately 15% of patients with metastatic STS survive longer than 5 years. With combined treatment (neo-adjuvant chemotherapy and/or radiation therapy, radical surgery, and adjuvant chemotherapy), the ﬁve-year survival rate for patients with localized disease at diagnosis is within a range of 60–80% (depending on STS/BS histology). However, for poor chemotherapy responders and patients with initially metastatic disease, survival times are much shorter, with <50–30% and <10% ﬁve-year survival rates, respectively. Therefore, improvement of sarcoma treatment still represents an important medical challenge. Clinical outcomes of sarcomas have plateaued for the last 10 years and currently, anthracycline-based regimens, cyclophosphamide, vincristine, actinomycin-D, ifosfamide, etoposide and trabectidin are routinely used in treatment of metastatic disease. The only molecular discovery-driven targeted drug that is currently used is pazopanib—a tyrosine kinase inhibitor eﬀective in selected STS, including angiosarcoma. Novel discoveries in sarcoma cell biology, genetics and genomics may contribute to identiﬁcation of novel drug targets and eventually change management paradigms of STS, as was the case for some solid tumors, including breast or colon cancers [1–4]. Eﬀorts to improve treatment eﬃcacy and treatment outcomes of advanced and metastatic sarcoma patients have not led to signiﬁcant improvements in the last several decades [3]. Currently there is still an urgent need to improve basic knowledge about sarcoma molecular biology and oncogenesis in order to accelerate the development of new therapeutic compounds that could potentially address this neglected area. A better understanding of the gene expression landscape observed in sarcomas is indispensable to the development of new eﬀective therapeutic regimens or molecular proﬁling of individual cases in order to implement available drugs based on molecular tumor board decision [5,6]. TP53 is the most frequently altered gene in cancers, with TP53 mutations observed in approximately half of all tumors and more cases exhibiting epigenetic deregulation of TP53 [7–9]. Inactivation of p53 plays a critical role in sarcomagenesis. 1. Introduction As it was previously shown in a mouse model, p53 +/− animals are susceptible to oncogenesis and tumor development—due to a reduction in p53 dosage in cells [16]. All these data suggest that tumor development in Tp53+/− and Tp53−/− organisms can be driven by different mechanisms. Thus, we aimed to define specific gene expression patterns, related with Tp53 loss of function (LOF)-driven sarcomatogenesis in a rat model. Actually, most LOF muta- tions in Tp53 adhere to the two-hit hypothesis, as proposed by [17], and the most common cause of Tp53 LOF is an inactivating missense mutation in one allele and simultaneous deletions in the regions of the 17p chromosome where the Tp53 is located [18]. This animal model seems in fact clinically relevant as a high proportion (58%) of radiation-induced sarcomas exhibit a somatic inactivating mu- tation for one Tp53 allele and a loss of the other. A high frequency (52%) of short deletions is observed in the mutation pattern of radiation-induced sarcomas [19]; Tp53+/C273X rats were recently used as model in the study on such tumors [20]. As we have shown before, Tp53 knockout rats (p53 TGEM® Rat) develop multiple tumors with angiosarcomas as the main tumor histotype [21]. Our experi- ments, therefore, aimed to characterize specific gene expression pattern of angiosarcomas with TP53 LOF and represents a model that could be further used for pre-clinical drug testing and development, as well as imaging studies with novel agents. Briefly, we analyzed four types of samples: tumors (1) excised from Tp53 homozygous knockout rats (‘Sarcoma’ group) or muscles excised from (2) from Tp53 homozygous knockout rats (‘Ko’), (3) Tp53 heterozygous knockout rats (‘Het’) and (4) wild-type healthy controls (‘Healthy’). The roadmap of the experiment is summarized in Figure 1. Figure 1. Roadmap for microarray gene expression analysis. Modified images from smart.servier.com [22] were used (available under Creative Commons license). Figure 1. Roadmap for microarray gene expression analysis. Modified images from smart.servier.com [22] were used (available under Creative Commons license). Figure 1. Roadmap for microarray gene expression analysis. Modified images from smart.servier.com [22] were used (available under Creative Commons license). Figure 1. Roadmap for microarray gene expression analysis. Modified images from smart.servier.com [22] were used (available under Creative Commons license). 1. Introduction It was shown that new germline mutations of the TP53 gene, although rare among patients with sporadic STS, are, however, reported in patients with family history of sarcoma. A high rate of point mutations in TP53 is reported not only in childhood sarcomas and families with the Li-Fraumeni syndrome, but also in adult-onset sarcomas, including leiomyosarcoma, osteosarcoma, and undiﬀerentiated pleomorphic sarcomas [10,11]. It has also been described that alterations of TP53 in rhabdomyosarcoma include a complete deletion of both TP53 alleles, complete deletions of one allele with or without point mutation of the other allele, and absence of detectable transcript (mRNA). On the other hand, osteosarcomas are characterized with homozygous deletions of TP53, lack of TP53 RNA expression or aberrant expression of p53 protein. The most recent genomic analysis has conﬁrmed that, in angiosarcoma, the most common alterations are denoted as TP53 mutation, TP53 c.217-c.1178 missense substitution, and TP53 missense (besides MYC ampliﬁcation, and KDR mutation) [12]. All these data conﬁrm that TP53 functional inactivation by either dominant or recessive manner plays a signiﬁcant role in human sarcomatogenesis including angiosarcoma development [4,13]. We used Wistar strain Tp53 knockout rats (p53 TGEM® Rat; TP53-deﬁcient Wistar rat) Tp53C273X/C273X colony as a sarcoma development model. A single T to A point mutation in the Tp53 DNA-binding domain introduces a premature C to X stop codon in position 273aa and is responsible for a loss of function of this tumor suppressor with no p53 protein detectable in the cells of knockout animals. Due to this nonsense mutation in the sixth exon, no full-length p53 is detectable in homozygous knockout rats and at the same time also no truncated protein is detectable in these homozygous rats, probably due to nonsense-mediated decay of its mRNA. In fact, complete absence of functional p53 protein in homozygous mutant animals was demonstrated already in embryonic ﬁbroblasts [14]. Knockout rats develop angiosarcomas at four months of age at the latest. Surprisingly, it was shown that tumors from homozygous animals show very limited aneuploidy and low degrees of somatic copy number variation in comparison to the tumors that develop in heterozygous Tp53C273X/WT animals. 3 of 17 matic Cancers 2020, 12, 1525 that tumors from h In the tumors from knockout animals the complex structural rearrangements such as chromothripsis and breakage–fusion–bridge cycles were never found, despite being detectable in greater numbers in tumors from heterozygous animals. 1. Introduction At the same time, in comparison to heterozygous tumors, tumors of knockout animals have longer telomeres but do not show clear telomerase activity or alternative lengthening of telomeres [15]. As it was previously shown in a mouse model, p53+/−animals are susceptible to oncogenesis and tumor development—due to a reduction in p53 dosage in cells [16]. All these data suggest that tumor development in Tp53+/−and Tp53−/−organisms can be driven by diﬀerent mechanisms. Thus, we aimed to deﬁne speciﬁc gene expression patterns, related with Tp53 loss of function (LOF)-driven sarcomatogenesis in a rat model. Actually, most LOF mutations in Tp53 adhere to the two-hit hypothesis, as proposed by [17], and the most common cause of Tp53 LOF is an inactivating missense mutation in one allele and simultaneous deletions in the regions of the 17p chromosome where the Tp53 is located [18]. This animal model seems in fact clinically relevant as a high proportion (58%) of radiation-induced sarcomas exhibit a somatic inactivating mutation for one Tp53 allele and a loss of the other. A high frequency (52%) of short deletions is observed in the mutation pattern of radiation-induced sarcomas [19]; Tp53+/C273X rats were recently used as model in the study on such tumors [20]. As we have shown before, Tp53 knockout rats (p53 TGEM® Rat) develop multiple tumors with angiosarcomas as the main tumor histotype [21]. Our experiments, therefore, aimed to characterize speciﬁc gene expression pattern of angiosarcomas with TP53 LOF and represents a model that could be further used for pre-clinical drug testing and development, as well as imaging studies with novel agents. Brieﬂy, we analyzed four types of samples: tumors (1) excised from Tp53 homozygous knockout rats (‘Sarcoma’ group) or muscles excised from (2) from Tp53 homozygous knockout rats (‘Ko’), (3) Tp53 heterozygous knockout rats (‘Het’) and (4) wild-type healthy controls (‘Healthy’). The roadmap of the experiment is summarized in Figure 1. copy number variation in comparison to the tumors that develop in heterozygous Tp53 ani mals. In the tumors from knockout animals the complex structural rearrangements such as chro- mothripsis and breakage–fusion–bridge cycles were never found, despite being detectable in greater numbers in tumors from heterozygous animals. At the same time, in comparison to heterozygous tumors, tumors of knockout animals have longer telomeres but do not show clear telomerase activity or alternative lengthening of telomeres [15]. 2. Results 2. Results 2.1. Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active 2.1. Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active 2.1. Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active 2.1. Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active 2 1 Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active 2.1. Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active Arrows indicate the tumors. Figure 2. MR images depicting typical localization of tumors in Tp53 homozygous knockout rats. Tumors were observed inter alia in the brain (a), skull muscles (b) as well as in the abdomen (c,d). Arrows indicate the tumors. Figure 2. MR images depicting typical localization of tumors in Tp53 homozygous knockout rats. Tumors were observed inter alia in the brain (a), skull muscles (b) as well as in the abdomen (c,d). Arrows indicate the tumors. Figure 2. MR images depicting typical localization of tumors in Tp53 homozygous knockout rats. Tumors were observed inter alia in the brain (a), skull muscles (b) as well as in the abdomen (c,d). Arrows indicate the tumors. Figure 3. Representative FDG PET images (b,d) with reference CT scans (a,c) of tumor-bearing homo- zygous Tp53 knockout rats. Panels a and b illustrate a single tumor localized on the forelimb of the rat. Panels c and d illustrate a rat bearing several tumors: within the head, on the forelimb and within the abdomen. Tumors on PET images are indicated by arrows. Figure 3. Representative FDG PET images (b,d) with reference CT scans (a,c) of tumor-bearing homo- zygous Tp53 knockout rats. Panels a and b illustrate a single tumor localized on the forelimb of the rat. Panels c and d illustrate a rat bearing several tumors: within the head, on the forelimb and within the abdomen. Tumors on PET images are indicated by arrows. Figure 3. Representative FDG PET images (b,d) with reference CT scans (a,c) of tumor-bearing homozygous Tp53 knockout rats. Panels a and b illustrate a single tumor localized on the forelimb of the rat. Panels c and d illustrate a rat bearing several tumors: within the head, on the forelimb and within the abdomen. Tumors on PET images are indicated by arrows. f tumor-bearing homo- h f li b f h Figure 3. Representativ FDG PET images (b,d) wi yg p g rat. Panels c and d illustrate a rat bearing several tumors: within the head, on the forelimb and within the abdomen. Tumors on PET images are indicated by arrows. Figure 3. Representative FDG PET images (b,d) with reference CT scans (a,c) of tumor-bearing homo- zygous Tp53 knockout rats. Panels a and b illustrate a single tumor localized on the forelimb of the rat. 2 1 Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active 2.1. Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active , y Solid tumors appearing in Tp53 homozygous knockout rats (sarcoma group) were visualized by magnetic resonance imaging (MRI) and positron emission tomography (PET). The tumors were hy- perintensive in T2-weighted magnetic resonance images (MRI, Figure 2). They were localized within Solid tumors appearing in Tp53 homozygous knockout rats (sarcoma group) were visualized by magnetic resonance imaging (MRI) and positron emission tomography (PET). The tumors were hyperintensive in T2-weighted magnetic resonance images (MRI, Figure 2). They were localized 4 of 17 re 2b), Cancers 2020, 12, 1525 the central nervou within the central nervous system, (Figure 2a), as well as within the muscles of head and neck (Figure 2b), soft tissues of extremities, abdominal cavity (Figure 2d) and dorsal muscles. The tumors were characterized by elevated 18F[FDG] uptake in comparison to adjacent tissues and clearly visible in positron emission tomography scans (Figure 3). Limb angiosarcoma tumors were selected for a genomic analysis. These tumor tissue samples presented upregulation of some genes typical for sarcoma (Figure S7). Cancers 2020, 12 4 of 18 the central nervous system, (Figure 2a), as well as within the muscles of head and neck (Figure 2b), soft tissues of extremities, abdominal cavity (Figure 2d) and dorsal muscles. The tumors were char- acterized by elevated 18F[FDG] uptake in comparison to adjacent tissues and clearly visible in posi- tron emission tomography scans (Figure 3). Limb angiosarcoma tumors were selected for a genomic analysis. These tumor tissue samples presented upregulation of some genes typical for sarcoma (Figure S7). acterized by elevated 18F[FDG] uptake in comparison to adjacent tissues and clearly visible in posi- tron emission tomography scans (Figure 3). Limb angiosarcoma tumors were selected for a genomic analysis. These tumor tissue samples presented upregulation of some genes typical for sarcoma (Figure S7). Figure 2. MR images depicting typical localization of tumors in Tp53 homozygous knockout rats. Tumors were observed inter alia in the brain (a), skull muscles (b) as well as in the abdomen (c,d). Arrows indicate the tumors. Figure 2. MR images depicting typical localization of tumors in Tp53 homozygous knockout rats. Tumors were observed inter alia in the brain (a), skull muscles (b) as well as in the abdomen (c,d). Arrows indicate the tumors. Figure 2. MR images depicting typical localization of tumors in Tp53 homozygous knockout rats. Tumors were observed inter alia in the brain (a), skull muscles (b) as well as in the abdomen (c,d). 2 1 Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active 2.1. Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active Panels c and d illustrate a rat bearing several tumors: within the head, on the forelimb and within the abdomen. Tumors on PET images are indicated by arrows. Figure 3. Representative FDG PET images (b,d) with reference CT scans (a,c) of tumor-bearing homozygous Tp53 knockout rats. Panels a and b illustrate a single tumor localized on the forelimb of the rat. Panels c and d illustrate a rat bearing several tumors: within the head, on the forelimb and within the abdomen. Tumors on PET images are indicated by arrows. Cancers 2020, 12, 1525 Cancers 2020, 12 5 of 17 5 of 18 2.2. Angiosarcoma Gene Expression Diﬀer from Normal and Non-Sarcoma Tp53 Knockout Tissues 2.2. Angiosarcoma Gene Expression Differ from Normal and Non-Sarcoma Tp53 Knockout Tissues 2.2. Angiosarcoma Gene Expression Diﬀer from Normal and Non-Sarcoma Tp53 Knockout Tissues 2.2. Angiosarcoma Gene Expression Differ from Normal and Non-Sarcoma Tp53 Knockout Tissues In total, 3052 genes were diﬀerentially expressed in tissues of various groups of rats with the false discovery rate corrected p < 0.05 (referred as to FDR < 0.05), as shown by analysis of variance (ANOVA). Within this subset of genes, 1245 genes discriminated angiosarcoma and normal tissue. At the same time, expression of 496 gene-diﬀerentiated Tp53 heterozygotes +/−and angiosarcoma while angiosarcoma and normal tissues of Tp53 knockout −/−were discriminated by expression of 699 genes, both with FDR < 0.05. A large number of genes were diﬀerentially expressed in pairwise comparisons between angiosarcoma and all other groups (Figure 4a–c and Table 1). In total, 3052 genes were differentially expressed in tissues of various groups of rats with the false discovery rate corrected p < 0.05 (referred as to FDR < 0.05), as shown by analysis of variance (ANOVA). Within this subset of genes, 1245 genes discriminated angiosarcoma and normal tissue. At the same time, expression of 496 gene-differentiated Tp53 heterozygotes +/− and angiosarcoma while angiosarcoma and normal tissues of Tp53 knockout −/− were discriminated by expression of 699 genes, both with FDR < 0.05. A large number of genes were differentially expressed in pairwise comparisons between angiosarcoma and all other groups (Figure 4a–c and Table 1). Figure 4. Comparison of gene expression and its overlap in four groups of rats. Volcano plots of gene expression in rats with sarcoma and all other groups: healthy (a), heterozygous knockout (d), total knockout (c). 2 1 Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active 2.1. Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active In panels a, b, and c, the horizontal axis presents log2-transformed fold change of gene expression, while the vertical axis presents log2-transformed p-value (after FDR correction). Venn di- agrams of genes upregulated in sarcoma rats versus healthy controls and rats with heterozygous and total knockout (d), genes downregulated in sarcoma rats versus healthy controls and rats with heter- ozygous and total knockout (e). Hierarchical clustering of the top 50 differentially expressed genes (lowest FDR in ANOVA), Euclidean distance, linkage method: average, row-standardized (f). Figure 4. Comparison of gene expression and its overlap in four groups of rats. Volcano plots of gene expression in rats with sarcoma and all other groups: healthy (a), heterozygous knockout (d), total knockout (c). In panels (a–c), the horizontal axis presents log2-transformed fold change of gene expression, while the vertical axis presents log2-transformed p-value (after FDR correction). Venn diagrams of genes upregulated in sarcoma rats versus healthy controls and rats with heterozygous and total knockout (d), genes downregulated in sarcoma rats versus healthy controls and rats with heterozygous and total knockout (e). Hierarchical clustering of the top 50 diﬀerentially expressed genes (lowest FDR in ANOVA), Euclidean distance, linkage method: average, row-standardized (f). Figure 4. Comparison of gene expression and its overlap in four groups of rats. Volcano plots of gene expression in rats with sarcoma and all other groups: healthy (a), heterozygous knockout (d), total knockout (c). In panels a, b, and c, the horizontal axis presents log2-transformed fold change of gene expression, while the vertical axis presents log2-transformed p-value (after FDR correction). Venn di- agrams of genes upregulated in sarcoma rats versus healthy controls and rats with heterozygous and total knockout (d), genes downregulated in sarcoma rats versus healthy controls and rats with heter- ozygous and total knockout (e). Hierarchical clustering of the top 50 differentially expressed genes (lowest FDR in ANOVA), Euclidean distance, linkage method: average, row-standardized (f). Figure 4. Comparison of gene expression and its overlap in four groups of rats. Volcano plots of gene expression in rats with sarcoma and all other groups: healthy (a), heterozygous knockout (d), total knockout (c). In panels (a–c), the horizontal axis presents log2-transformed fold change of gene expression, while the vertical axis presents log2-transformed p-value (after FDR correction). 2 1 Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active 2.1. Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active Venn diagrams of genes upregulated in sarcoma rats versus healthy controls and rats with heterozygous and total knockout (d), genes downregulated in sarcoma rats versus healthy controls and rats with heterozygous and total knockout (e). Hierarchical clustering of the top 50 diﬀerentially expressed genes (lowest FDR in ANOVA), Euclidean distance, linkage method: average, row-standardized (f). Table 1 Number of genes differentially expressed (FDR < 0 05) in pairwise comparisons between groups Table 1. Number of genes diﬀerentially expressed (FDR < 0.05) in pairwise comparisons between groups. able 1 Number of genes differentially expressed (FDR < 0 05) in pairwise comparisons between groups ble 1. Number of genes diﬀerentially expressed (FDR < 0.05) in pairwise comparisons between groups. Table 1. Number of genes differentially expressed (FDR 0.05) in pairwise comparisons between groups. Genes Upregulated In: Healthy Het Ko Sarcoma Genes Downregu- lated In: Healthy 0 0 554 Het 0 0 273 Ko 0 0 341 Sarcoma 691 223 358 The most extreme differences in gene expression (FC > 10 and FDR < 0.025, marked in red Genes Upregulated In: Healthy Het Ko Sarcoma Genes Downregulated In: Healthy 0 0 554 Het 0 0 273 Ko 0 0 341 Sarcoma 691 223 358 Figure 4a–c) were observed between wild-type (Tp53+/+) normal tissue and angiosarcoma tissue in Tp53−/− rats. The 30 genes most strongly upregulated in angiosacroma were mostly involved in binding, structural and catalytic activities. Functions of 56 genes that showed the strongest downregulation in angiosarcoma included again binding and catalytic activity, and additionally transporter activity. The most extreme diﬀerences in gene expression (FC > 10 and FDR < 0.025, marked in red in Figure 4a–c) were observed between wild-type (Tp53+/+) normal tissue and angiosarcoma tissue in Tp53−/−rats. The 30 genes most strongly upregulated in angiosacroma were mostly involved 6 of 17 Cancers 2020, 12, 1525 in binding, structural and catalytic activities. Functions of 56 genes that showed the strongest downregulation in angiosarcoma included again binding and catalytic activity, and additionally transporter activity. 2 1 Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active 2.1. Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active Among the genes downregulated in sarcoma versus wild-type tissue, seven were identiﬁed as downregulated also in comparison of angiosarcoma tissue to all other groups, namely: muscle contraction and relaxation regulator; muscle chloride channel 1 (Clcn1), protein involved in ubiquitination; Kelch Like Family Member 33 (Klhl33), phosphohydrolase of triglyceride synthesis; Lipin 1 (Lpin1), myogenesis inhibiting myokine; Myostatin (Mstn), the enzyme responsible for dephosphorylation of nucleotides to nucleosides that regulates adenosine levels in muscles during ischemia and hypoxia-5′-Nucleotidase 1A (Nt5c1a), activator of the glycolysis pathway and an inhibitor of the gluconeogenesis pathway; 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 1 (Pfkfb1), responsible for lactic acid and pyruvate transport across plasma membranes; Solute Carrier Family 16 Member 3 (Slc16a3) (Table S2). Overlap of genes signiﬁcantly dysregulated in angiosarcoma versus all other tissues revealed 181 genes consistently upregulated in sarcoma and 184 downregulated ones (Figure 4d,e). At the same time, no genes were identiﬁed that diﬀered in the inconsistent way, i.e., there were no genes signiﬁcantly upregulated in sarcoma versus one group that were at the same time downregulated in sarcoma when compared with other ones (Table S3). Finally, all multiple comparisons between normal healthy tissue (Tp53+/+), with normal heterozygous Tp53 knockout tissue (Tp53+/−), and normal knockout tissue (Tp53−/−), did not indicate any genes with signiﬁcantly diﬀerent expression. Only the angiosarcoma samples diﬀered signiﬁcantly from all other (normal) tissues. Moreover, typical angiosarcoma-related genes have shown an overexpression trend conﬁrming the histopathologic proﬁle of the analyzed tumors—tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (Tie1), Fms-related tyrosine kinase 4, (Flt4), Fli-1 Proto-Oncogene (Fli1), ephrin type-A receptor 2 (Epha2), placental growth factor (Pgf), BHLH transcription factor (Myc) and Endothelin receptor type B (Ednrb) (Figure S8). y g The results of principal component analysis (PCA) (Figure S1) indicated that angiosarcoma tissue samples can be separated from all other tissues on the basis of gene expression, while normal Tp53+/+, Tp53+/−and Tp53−/−tissues were very similar. Hierarchical clustering of the 50 top diﬀerentially expressed genes (lowest FDR) enabled separation of four tissue groups with a predominant diﬀerence between angiosarcoma and all other samples (Figure 4f), and indicating major changes in gene expression upon development of malignancy. In angiosarcoma cells, the most downregulated genes were: DNA binding protein; Endonuclease/Exonuclease/Phosphatase Family Domain-Containing Protein 1 (Eepd1), micro-satellite locus Atplb2, muscle chloride channel (Clcn1), channel regulating excitability of muscle tissues; potassium voltage-gated channel subfamily J member 2 (Kcnjll) and basic helix-loop-helix-leucine zipper transcriptional activator; Transcriptional Activator MondoA (Mlxip). 2 1 Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active 2.1. Tumors Localize in the Head and Neck, Extremities and Abdomen and Are Metabolically Active At the same time, the most upregulated genes in angiosarcoma were DNA helicase, essential for genomic DNA replication: Minichromosome Maintenance Complex Component 2 (Mcm2), intracellular transport of proteins coat complex II component (Sec23b), sub-apical actin network organizing protein; Rho GTPase Activating Protein 42 (Arhgap42), glycogen metabolism regulator; Acetylglucosamine Phosphomutase (Pgm3) and promoting cell motility with stress ﬁbers; Calponin 3 (Cnn3) (Figure 4f). 2.3. Angiosarcoma Presents Deregulated Metabolism (b) Enrichment plots of the Reactome Kinesins gene set that showed signiﬁcant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. (c) Enrichment plots of the Reactome TCA cycle and respiratory electron transport gene set that showed signiﬁcant downregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. g p y g p g p T bl 2 N b f t i ifi tl i h d i i i i i GSEA Table 2. Number of gene sets signiﬁcantly enriched in pairwise comparisons in GSEA. Table 2. Number of gene sets significantly enriched in pairwise comparisons in GSEA. Pathways Upregulated In: Healthy Het Ko Sarcoma Pathways Downregu- lated in: Healthy 1 1 253 Het 61 0 362 Ko 5 0 325 Sarcoma 44 43 45 Next we identified 237 up and 40 downregulated pathways that were repeatedly deregulat Table 2. Number of gene sets signiﬁcantly enriched in pairwise comparisons in GSEA. Pathways Upregulated In: Healthy Het Ko Sarcoma Pathways Downregulated in: Healthy 1 1 253 Het 61 0 362 Ko 5 0 325 Sarcoma 44 43 45 in comparisons of angiosarcoma tissue versus all other normal tissues (Figure 6a,b and list in Table S5). The most upregulated pathways in angiosarcoma vs all other normal tissues were responsible for cell cycle including mitosis and meiosis, chromosome, nucleosome and telomere maintenance, as well as DNA replication and recombination (Figure 7a). As expected, sarcoma-related pathway genes have also been shown as upregulated including coagulation factor VIII (F8), Platelet endothelial cell adhesion molecule (CD31, Pecam1), marker of proliferation Ki-67 (Mki67), Fli1, thrombomodulin (Thbd), Cd34, tyrosine-protein kinase Kit, ETS transcription factor (Erg), podoplanin (Pdpn), mucin 1 (Muc1) cytokeratin Cam5 2 anion exchanger 1 (Slc4a1) and Slc4a3 (Figure S7) Conversely downreg- Next, we identiﬁed 237 up- and 40 downregulated pathways that were repeatedly deregulated in comparisons of angiosarcoma tissue versus all other normal tissues (Figure 6a,b and list in Table S5). The most upregulated pathways in angiosarcoma vs all other normal tissues were responsible for cell cycle including mitosis and meiosis, chromosome, nucleosome and telomere maintenance, as well as DNA replication and recombination (Figure 7a). 2.3. Angiosarcoma Presents Deregulated Metabolism Gene set enrichment analysis yielded results which corresponded to those obtained in a single gene analysis. The gene expression patterns of sarcoma samples diﬀered strongly from all normal tissues with a number of gene sets which signiﬁcantly diﬀered in enrichment analysis. Cell cycle machinery genes (Figure 5a,b) were strongly upregulated in angiosarcoma samples. Conversely, citric acid cycle genes were strongly downregulated in angiosarcomas in comparison to all normal tissues (Figure 5c) as did glucose metabolism, pyruvate degradation, amino-acid catabolism and multiple other metabolic pathway gene sets (Table 2, Table S4). 7 of 17 7 f 18 Cancers 2020, 12, 1525 C 2020 12 cers 2020, 12 7 Figure 5. Gene set enrichment results of comparisons between the sarcoma group, healthy, heterozy- gous and homozygous Tp53 knockout groups. (a) Enrichment plots of the Reactome cell cycle gene set that showed significant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. (b) Enrichment plots of the Reactome Kinesins gene set that showed significant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. (c) Enrichment plots of the Reactome TCA cycle and respiratory electron transport gene set that showed significant downregulation in the sarcoma group in comparison to the homozygous (left panel), het- erozygous (middle panel) and healthy (right panel) groups. Figure 5. Gene set enrichment results of comparisons between the sarcoma group, healthy, heterozygous and homozygous Tp53 knockout groups. (a) Enrichment plots of the Reactome cell cycle gene set that showed signiﬁcant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. (b) Enrichment plots of the Reactome Kinesins gene set that showed signiﬁcant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. (c) Enrichment plots of the Reactome TCA cycle and respiratory electron transport gene set that showed signiﬁcant downregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. Figure 5. Gene set enrichment results of comparisons between the sarcoma group, healthy, heterozy- gous and homozygous Tp53 knockout groups. 2.3. Angiosarcoma Presents Deregulated Metabolism (a) Enrichment plots of the Reactome cell cycle gene set that showed significant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. (b) Enrichment plots of the Reactome Kinesins gene set that showed significant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. (c) Enrichment plots of the Reactome TCA cycle and respiratory electron transport gene set that showed significant downregulation in the sarcoma group in comparison to the homozygous (left panel), het- erozygous (middle panel) and healthy (right panel) groups. Figure 5. Gene set enrichment results of comparisons between the sarcoma group, healthy, heterozygous and homozygous Tp53 knockout groups. (a) Enrichment plots of the Reactome cell cycle gene set that showed signiﬁcant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. (b) Enrichment plots of the Reactome Kinesins gene set that showed signiﬁcant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. (c) Enrichment plots of the Reactome TCA cycle and respiratory electron transport gene set that showed signiﬁcant downregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. Figure 5. Gene set enrichment results of comparisons between the sarcoma group, healthy, heterozy- gous and homozygous Tp53 knockout groups. (a) Enrichment plots of the Reactome cell cycle gene set that showed significant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. (b) Enrichment plots of the Reactome Kinesins gene set that showed significant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. (c) Enrichment plots of the Reactome TCA cycle and respiratory electron transport gene set that showed significant downregulation in the sarcoma group in comparison to the homozygous (left panel), het- erozygous (middle panel) and healthy (right panel) groups. Figure 5. Gene set enrichment results of comparisons between the sarcoma group, healthy, heterozygous and homozygous Tp53 knockout groups. (a) Enrichment plots of the Reactome cell cycle gene set that showed signiﬁcant upregulation in the sarcoma group in comparison to the homozygous (left panel), heterozygous (middle panel) and healthy (right panel) groups. 2.3. Angiosarcoma Presents Deregulated Metabolism As expected, sarcoma-related pathway genes have also been shown as upregulated including coagulation factor VIII (F8), Platelet endothelial cell adhesion molecule (CD31, Pecam1), marker of proliferation Ki-67 (Mki67), Fli1, thrombomodulin (Thbd), 8 of 17 t- re Cancers 2020, 12, 1525 enrichment sco tern observed in Cd34, tyrosine-protein kinase Kit, ETS transcription factor (Erg), podoplanin (Pdpn), mucin 1 (Muc1), cytokeratin Cam5.2, anion exchanger 1 (Slc4a1) and Slc4a3 (Figure S7). Conversely, downregulated genes were responsible for metabolism, including respiratory chain electron transport, TCA cycle, fatty acid metabolism and amino-acid catabolism (Figure 7b). Curiously, a relatively low number of pathways were strongly dysregulated in angiosarcoma versus only one of the comparative groups (Figure 6a,b, Figure S2). Interestingly, angiosarcoma showed suppressed circadian regulation of selected biological processes in comparison to healthy and total Tp53 (−/−) knockout tissues. hibitors (−92.60) and HIF activators (−91.06) (Table S7). The drugs predicted to have the highest ac- tivity were panobinostat (trade name Farydak by Novartis), ellipticine (currently not used in humans due to high toxicity), scriptaid (not yet developed for clinical applications), inducing transcription of p53 protein and blocking p53 ubiquitination—amsacrine (FDA approved in acute adult leukemia), PHA-793887 (currently not used in clinics due to severe dose-related hepatotoxicity), PF-562271 JNK- 9L (a defined promising therapeutic target in phase 1 trial), bisindolylmaleimide-ix (drug candidate by Roche) and mitoxantrone trade name Novantrone and generics, FDA approved in hormone-re- fractory prostate cancer, acute myelogenous leukemia, breast cancer and non-Hodgkin's lymphoma). Figure 6. (a) Overlap of gene sets significantly upregulated in the sarcoma group in comparison to the healthy, heterozygous and total knockout groups. (b) Overlap of gene sets significantly downreg- ulated in the sarcoma group in comparison to the healthy, heterozygous and total knockout groups. Figure 6. (a) Overlap of gene sets signiﬁcantly upregulated in the sarcoma group in comparison to the healthy, heterozygous and total knockout groups. (b) Overlap of gene sets signiﬁcantly downregulated in the sarcoma group in comparison to the healthy, heterozygous and total knockout groups. Cancers 2020, 12 9 of 18 Figure 6. (a) Overlap of gene sets significantly upregulated in the sarcoma group in comparison to the healthy, heterozygous and total knockout groups. (b) Overlap of gene sets significantly downreg- ulated in the sarcoma group in comparison to the healthy, heterozygous and total knockout groups. Figure 6. 2.3. Angiosarcoma Presents Deregulated Metabolism (a) Overlap of gene sets signiﬁcantly upregulated in the sarcoma group in comparison to the healthy, heterozygous and total knockout groups. (b) Overlap of gene sets signiﬁcantly downregulated in the sarcoma group in comparison to the healthy, heterozygous and total knockout groups. Cancers 2020, 12 9 of 18 Figure 7. (a) Enrichment map of pathways significantly up or (b) downregulated in sarcomas versus the other three compared groups. Nodes (pathways) fulfilling filtration criteria, but without connec- tions to other pathways are not shown (39 up- and 6 downregulated in sarcoma). 2 4 A ti it f 53 Si li P th I D l t d i T 53 K k t Ti Figure 7. (a) Enrichment map of pathways signiﬁcantly up or (b) downregulated in sarcomas versus the other three compared groups. Nodes (pathways) fulﬁlling ﬁltration criteria, but without connections to other pathways are not shown (39 up- and 6 downregulated in sarcoma). Figure 7. (a) Enrichment map of pathways significantly up or (b) downregulated in sarcomas versus the other three compared groups. Nodes (pathways) fulfilling filtration criteria, but without connec- tions to other pathways are not shown (39 up- and 6 downregulated in sarcoma). Figure 7. (a) Enrichment map of pathways signiﬁcantly up or (b) downregulated in sarcomas versus the other three compared groups. Nodes (pathways) fulﬁlling ﬁltration criteria, but without connections to other pathways are not shown (39 up- and 6 downregulated in sarcoma). Cancers 2020, 12, 1525 9 of 17 Among genes, signiﬁcantly dysregulated in angiosarcoma (intersections of three circles in Venn diagrams in Figure 4d,e), 96 up- and 128 downregulated ones fulﬁlled the criteria of inclusion in drug discovery analysis. Finally, CMAP used 81 up- and 85 downregulated genes, while the rest were either identiﬁed but unused or unrecognized under any symbol available in NCBI gene database (list in Table S6). CMap analysis revealed six classes of pharmaceutical compounds (CMap classes) with enrichment score below −90.0, which means that their eﬀect is highly opposite to the expression pattern observed in angiosarcoma samples. Those classes are: topoisomerase inhibitor (enrichment score −97.84), HDAC inhibitor (−95.95), CDK inhibitor (−94.38), DNA synthesis inhibitors (−92.69), JAK inhibitors (−92.60) and HIF activators (−91.06) (Table S7). 2.4. Activity of p53 Signaling Pathway Is Dysregulated in Tp53 Knockout Tissues Ingenuity Pathway Analysis (IPA) revealed diﬀerences in expression of genes involved in P53 signaling between wild-type and Tp53 knockout rats. In all knockout rats (sarcoma, ko and het groups) the Tp53 gene itself is slightly, but insigniﬁcantly, downregulated (Figures S3–S5 and Table S2). The presence of Tp53 transcripts, detectable by microarray assay, can be expected in this model, since loss of functional p53 is caused by point mutation, which results in the lack of p53 protein, but not necessarily lack of its mRNA. In the Het group majority, some—but not all—of the genes directly downstream of Tp53 were downregulated, especially the ones responsible for DNA repair, which indicates that their activation by p53 was indeed suppressed. A similar transcriptomic proﬁle was observed in Ko rats, in which, again, a substantial number of genes activated by p53 was downregulated in comparison to wild-type rats. In the sarcoma group, the activity of some of these genes seemed to be restored despite the presence of mutated Tp53. On the other hand, some genes responsible for cell cycle arrest were downregulated even in rats, which was consistent with our GSEA result that showed activation of cell cycle-associated pathways. 2.3. Angiosarcoma Presents Deregulated Metabolism The drugs predicted to have the highest activity were panobinostat (trade name Farydak by Novartis), ellipticine (currently not used in humans due to high toxicity), scriptaid (not yet developed for clinical applications), inducing transcription of p53 protein and blocking p53 ubiquitination—amsacrine (FDA approved in acute adult leukemia), PHA-793887 (currently not used in clinics due to severe dose-related hepatotoxicity), PF-562271 JNK-9L (a deﬁned promising therapeutic target in phase 1 trial), bisindolylmaleimide-ix (drug candidate by Roche) and mitoxantrone trade name Novantrone and generics, FDA approved in hormone-refractory prostate cancer, acute myelogenous leukemia, breast cancer and non-Hodgkin’s lymphoma). 3. Discussion Adult male rats were administered intraperitoneal ENU injections that target spermatogonial stem cells (SSCs) in rat gonads. Animals with a nonsense mutation at amino acid position 273 (Cys to stop) within the DNA binding domain of the p53 protein were selected. This mutation functionally resulted in a full knockout Tp53 mutation. For model development systematic generation of these knockout rats was carried out by random mutagenesis of Wistar rats followed by PCR ampliﬁcation and capillary sequencing check-up, referred to as TGEM® technology by Transposagen. ENU-driven target-selected mutagenesis is an eﬀective approach for artiﬁcial introduction of point mutations. ENU, as an alkylating agent, transfers its ethyl group to oxygen or nitrogen in nucleophilic groups of nucleobases, which results in nucleotide substitutions such as A–T base transversions [28]. Wistar background homozygous mutant Tp53C273X/C273X rats predominantly develop sarcomas with an onset at four months of age and with a high frequency of pulmonary metastases. In our study, MRI and PET examinations revealed metabolically active tumors in several locations, including the brain, head and neck, extremities and abdomen. These sites were consistently similar to those previously described in this model [21]. Heterozygous rats develop sarcomas starting at eight months of age. Tp53C273X/+ rats predominantly develop osteosarcomas, therefore this model may be generally used for soft tissue and bone sarcoma research and should be referred in appropriate papers, including imaging/PET-oriented manuals [28–30]. In the TGEM Rat model the introduced DNA mutation ﬁnally truncates the protein at the DNA binding domain, eliminating functionally essential domains including the nuclear localization domain and the homo-oligomerization domain of the translated protein, which results in rapid degradation of residual non-functional peptide. A genetic analysis conﬁrmed that developed sarcomas in heterozygotes exhibit a loss-of-heterozygosity of the wild-type Tp53 allele [14]. With regard to sarcoma-oriented studies, it must be pointed out that sporadic angiosarcomas (SA) and radiation-associated angiosarcomas (RAA) are similar in histology, immunohistochemical markers, and DNA mutation proﬁles and share a similar prognosis [31]. In angiosarcomas, most of abnormalities are found in the p53 and MAPK pathways. More than 50% of angiosarcomas presented MAPK pathway activation. Simultaneously, angiosarcoma genome analyses revealed mutations and ampliﬁcations of VEGF, MDM2, TP53, CDKN2A, KRAS and MYC. TP53 was reported as mutated in 35% of the lesions and CDKN2A lost in 26%. Activating mutations were found in KRAS, HRAS, NRAS, BRAF, MAPK1, while inactivating mutations in NF1 and PTPRB1. 3. Discussion Several p53-deﬁcient animal models have been developed to mimic sarcomagenesis. In particular, mice with Tp53, inactivated by Cre-loxP-mediated recombination, develop spindle cell sarcomas and pleomorphic sarcomas. Additionally, mesenchymal sarcoma stem cells (Sca-1low) have been isolated from these animals [23]. The rat however is also a feasible model for imaging studies, easier in surgical handling and imaging than mouse. Unlike the Tp53 knockout mouse that often develop lymphomas ﬁrst, the Tp53-knockout rats most often develop sarcomas, which favors the use of the rat model in preclinical studies of sarcoma, including novel drug testing [14,24]. The ﬁrst Tp53-deﬁcient rat—Dark Agouti rat (subsequently referred to as the Tp53-deﬁcient DA rat)—was created via homologous recombination in the rat embryonic stem cells. Homozygous Tp53-deﬁcient DA rats live no longer than six months and develop angiosarcomas and lymphomas. Heterozygous Tp53-deﬁcient DA rats survive up to 12 months of age and demonstrate a wide variety of sarcomas in both males and females, and also develop mammary carcinomas in about 20% of female rats [25,26]. On the contrary, in Fischer-344 (F344)’s rat-based 344-Tp53tm1(EGFP-Pac)Qly/Rrrc (F344-Tp53) model, the tumor spectrum is shifted towards the primary tumor types—osteosarcomas and meningeal sarcomas. The incidence of osteosarcomas is 57% and 36% in F344-Tp53 homozygous and heterozygous animals, respectively. In this model, tumors are highly representative of human disease radiographically and histologically. They typically localize on long bones and are characterized 10 of 17 Cancers 2020, 12, 1525 with frequent pulmonary metastases [7]. At the same time Tp53 knockout rat in the Sprague Dawley background was generated using Zinc Finger Nuclease (ZFN) technology with target site located in the 22-bp exon 3 of the gene. A homozygous null rat—Tp53∆11/∆11—with a complete loss of p53 protein has a shortened disease-free lifespan due to early onset of cancers. The tumor spectrum in these null mutant rats includes both sarcomas and carcinomas, with a predominance of nervous system tumors. The Tp53∆11/+ rats experience a later onset of tumorigenesis and develop skin and endocrine cancers in addition to the cancer types recognized in the null homozygote [27]. Finally, the p53 TGEM Rat model that we used in this study was developed in a Wistar Han background (referred to subsequently as the Tp53-deﬁcient Wistar rat). At the Hubrecht Institute, after N-ethyl-N-nitrosourea (ENU)-driven mutagenesis, a target-selected screen was performed in the outbred Wistar background. 4.1. Animals Tp53 knockout rats (p53 TGEM® Rat) with a Wistar strain genetic background (Charles River, Wilmington, MA, USA—transferred under exclusive license from Transposagen) were maintained as we described before [21]. A single T to A point mutation in the Tp53 DNA-binding domain that introduces a premature C to X stop codon in position 273aa was present in Tp53 gene with complete lack of p53 in homozygous mutant cells [14]. Genotyping of these animals was conducted as we described in detail previously [21]. In brief, DNA was extracted from a tail snip and extracted with a GeneMATRIX EURx kit (EURx Ltd., Gdansk, Poland). Genotypes of the rats were determined using simple allele-discriminating PCR (initial denaturation 94 ◦C for 6 min, 35 cycles of 94 ◦C for 20 s, 52 ◦C for 20 s, 72 ◦C for 20 s, ﬁnal elongation 72 ◦C for 7 min) with two primer sets: (1) Tp53 wild-type reverse primer (5′-GTCTCTCCCAGGACAGGTA-3′), and Tp53 common forward primer (5′-GAAGACTCCAGGTAGGAAGC-3′) or (2) Tp53 mutant reverse primer (5′-GTCTCTCCCAGGACAGGTT -3′) and Tp53 common forward primer. An example of genotyping results is shown in Figure S6. Expression of p53 and CD31 was evaluated with Western blot (Figures S9 and S10). The tissue was pulverized by cryogenic grinding with liquid nitrogen and then lysed in RIPA buﬀer containing PMSF and cocktail of protease inhibitors (Sigma, St. Louis, MO, USA). Lysates were centrifuged at 14,000 rpm for 15 min. Protein concentration was measured using BCA assay (Pierce). In total, 20 µg of protein per lane was loaded on the gel. The antibodies used for Western blot analysis were as follows: mouse monoclonal anti-CD31 antibody (Merck MAB 1393Z, Darmstadt, Germany) and mouse anti-GAPDH (Chemicon MMAB374, Fisher Scientiﬁc, Waltham, MA, USA) and mouse anti-p53 (SantaCruz sc-126, Dallas, TX, USA). Secondary antibody was used as a goat anti-mouse IgG (Chemicon AP124P, Fisher Scientiﬁc, Waltham, MA, USA). For the purpose of gene expression analysis, three rats in each of the four groups (Figure 1) were selected. The groups were the following: (1) Ko with sarcoma already developed (referred to as Sarcoma in ﬁgures and tables), (2) with total knockout of the Tp53 (Ko), (3) with heterozygous knockout of the Tp53 (Het), (4) healthy controls (Healthy). 4.1. Animals Animal research followed internationally accepted guidance for the care and use of laboratory animals, including the National Institute of Public Health—National Institute of Hygiene (NIPH–NIH) guidelines, and was approved by the IV Local Ethics Committee in Warsaw. All animal experiments were carried out in strict accordance with guidelines of IV Animal Ethical Committee in Warsaw (approval number 88/2015). 3. Discussion In particular, MYC gene ampliﬁcations are more common in RAA [32]. In our study, the most upregulated genes included Rho GTPase Activating Protein 42 (Arhgap42), mitochondrial Propionyl-CoA Carboxylase Subunit Alpha (Pcca), LOC294154 (similar to chromosome 6 open reading frame 106 isoform a) with ubiquitin binding activity, Spindle and kinetochore-associated protein 3 (Ska3) and Solute Carrier Family 16, Member 3 (Monocarboxylic Acid Transporter 4—Slc16a3). At the same time, the most upregulated pathways in angiosarcoma vs all other tissues were related to cell cycle with mitosis and meiosis; chromosome, nucleosome and telomere maintenance; as well as DNA replication and recombination. On the other hand, downregulated genes were responsible for metabolism, including respiratory chain electron transport, TCA cycle, fatty acid metabolism and amino-acid catabolism. Thus, the rat model that we studied here represents a novel interesting pre-clinical model that may easily be used for novel Cancers 2020, 12, 1525 11 of 17 drug testing applications that surpasses a lack of tumor–host interactions and no immune response. This model has native microenvironment of an animal and also enables to conduct studies on an intact immune system response, including immunotherapy and cancer vaccines [33–35]. Our study analyzed the transcriptomic proﬁle of Tp53 knockout rats with either homo- or heterozygous point mutation in codon 273, both before and after tumor development. Future research can be aimed at veriﬁcation if similar characteristics are observed at the proteomic level. The main limitations of our study are a relatively small number of animals and the fact that transcriptomic characteristics can change with time while we investigated them once in groups of rats of the same age. In our studies, Ko or Het rats without sarcoma were more similar in terms of transcriptome to wild-type animals than to those with the same mutation and developed tumor. We suspect that some changes may appear if such rats are observed for a longer time, which can be an interesting area of future research. 4.2. Magnetic Resonance Imaging Magnetic resonance imaging (MRI) was performed to monitor the tumor growth in Tp53 knockout rats. 7T Bruker Biospec scanner (70/30 USR, Bruker Biospin, Ettlingen, Germany) was used for 12 of 17 Cancers 2020, 12, 1525 the imaging and was equipped with: (1) a combination of a transmit cylindrical radiofrequency volume coil (8.6 cm inner diameter) with a rat brain dedicated receive-only array surface coil (2 × 2 elements)—for head and neck imaging or (2) a cylindrical radiofrequency volume coil (8.6 cm inner diameter) alone serving as a transmit-receive coil—for imaging of the abdomen. Animals were anesthetized with 1.5–2% isoﬂurane in oxygen, and positioned head ﬁrst and prone in the MR-compatible animal bed. Respiration rate and body temperature were monitored throughout the experiment with a small animal monitoring system. Positioning tripilot scans were performed, followed by T2-weighted anatomical imaging. The head and neck region was imaged with TurboRARE T2 protocol (eﬀective echo time, TEeﬀ= 30 ms; repetition time, TR = 2500 ms; RARE factor = 8; ﬂip angle, FA = 90◦; 16 slices without gaps; ﬁeld of view, FOV= 32 mm × 32 mm; spatial resolution=125 µm × 125 µm × 800 µm; number of acquisitions, NA = 4; time of acquisition, TA = 4 min). For abdomen imaging, the SNAP protocol was employed (TE = 2.6 ms, TR = 14 ms, FA = 25◦, 25 slices, FOV = 80 mm × 100 mm, spatial resolution = 417 µm × 521 µm × 1000 µm, NA = 6, TA = 14 min). 4.4. RNA Isolation and Microarray Analysis In healthy rats, biceps femoris was used for RNA isolation, and in the case of rats with sarcoma a tumor located within this muscle was used. Samples were excised and immediately frozen in liquid nitrogen and further stored at −80 ◦C (Revco Ultra Low–Temperature Freezer). RNA isolation was carried in a mortar bowl with RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany) as per manufacturer protocol with frozen muscle or tumor tissues. The GeneChip™Rat Gene 2.1 ST Arrays and GeneChip WT PLUS Reagent Kit (Thermo Fisher Scientiﬁc, Waltham, MA, USA) were used to obtain expression data. 4.3. [18F]FDG PET/CT PET and CT scans were conducted with Albira PET/SPECT/CT Preclinical Imaging System (Bruker, Billerica, MA, USA) with spatial resolution of 1.5 mm for PET and 90 µm for CT. Animals were anesthetized with isoﬂurane (induction 4%, maintenance 1.5–2%) and 8–13 MBq of [18F]FDG (18F-Fluorodeoxyglucose, Gluscan 500, Advanced Accelerator Applications Sp. z o. o., Warsaw, Poland) was injected intravenously (in a total volume of 100–150 µL). PET/CT scans were started 60 min after [18F]FDG injection and reference CT scans were acquired (tube voltage 45 kVp, tube current 400 µA, number of frames 2, number of projections 600). PET and CT scans were fused using PMOD software, version 3.307, module Fusion Tool (PMOD Technologies LLC, Zurich, Switzerland). 4.5. Data Analysis The experimental data, obtained for GeneChip™Rat Gene 2.1 ST, were originally stored in 12 CEL ﬁles, each containing data about gene expression in a particular rat. AﬀySTExpressionFileCreator from GenePattern [36] was used to convert CEL ﬁles to numeric gene expression values. Additionally, background correction, log2-transform and quantile normalization were applied in conversion module. The gct ﬁle with expression data for 36,685 Probe IDs was created. Then, probe IDs were mapped to genes by ReannotateGCT module of GenePattern, according to probeset annotation ﬁle (release 36) downloaded from manufacturer website (Thermo Fisher Scientiﬁc, Waltham, MA, USA; www.thermoﬁsher.com). Among 36,685 identiﬁed probesets, 21,527 were mapped to 20,740 unique rat genes. In cases when more than one probeset was mapped to a single gene, the highest expression among all those probes was selected for further analysis. Log2-transformed, quantile normalized gene expression data are available in Table S1. Pre-processed data were used in all further analyses. First, gene expression in all groups of rats was compared by ANOVA, then obtained p-values were corrected by the False Discovery Rate (FDR) method [37] in order to ﬁnd diﬀerentially expressed genes. Next, pairwise comparisons between particular groups were performed with the use of the t-test and FDR correction was applied for each pair of groups separately. Corrected p-value lower than 0.05 was considered signiﬁcant. The PANTHER database was used for batch identiﬁcation of molecular Cancers 2020, 12, 1525 13 of 17 functions of diﬀerentially expressed genes [38]. Principal component analysis and hierarchical clustering (with Euclidian distance) were applied in identiﬁcation and visualization of diﬀerences in gene expression proﬁles between the samples. Then, gene expression proﬁles were subjected to Gene Set Enrichment Analysis (GSEA) [39]. The pathways from gene sets of C2CP (canonical pathways) from MSigDB [40] were analyzed as potentially aﬀected by mutation and presence of sarcoma in rats. Since GSEA can compare only 2 phenotypes simultaneously, the 4 groups of rats were analyzed in pairs, giving 6 pairwise comparisons in total. GSEA allows to observe which pathways, not only single genes, are up- and downregulated in one group with respect to the other. Due to a low number of samples, gene set permutation method was used to estimate signiﬁcance levels, which means that statistical signiﬁcance of enrichment of particular gene set is calculated on the basis of gene expression in artiﬁcially created random gene sets of the same size. 4.5. Data Analysis If the expression of the analyzed gene set is more extreme than in the majority (chosen fraction) of artiﬁcial gene sets, its enrichment is considered signiﬁcant. In our analysis, we decided to use 1000 permutations to evaluate the enrichment in each gene set. Gene set size ﬁlter minimum and maximum were set to 15 and 1000, respectively. Additionally, GSEA was applied to examine expression of genes that are typically upregulated in sarcoma tissue [41]. The results obtained from GSEA were ﬁnally loaded into Cytoscape [42] in order to create Enrichment Maps (EM) that show the interconnectedness between the processes identiﬁed as abnormally active or suppressed. For comparisons between rats with sarcoma and any of the other 3 groups the p-value cut-oﬀfor EM was set to 0.001, FDR cut-oﬀto 0.005 and overlap to 0.7, which means than only most signiﬁcantly enriched pathways were retained in EM and the edges were drawn between pathways’ nodes when the number of overlapping genes was equal at least to 70% of size of the smaller of 2 gene sets. For clarity, gene sets without such overlap were excluded from ﬁgures. The P53 signaling pathway was examined in Ingenuity Pathway Analysis (IPA). Our expression data were overlaid on the pathway scheme. This allowed us to compare expression of genes involved in P53 signaling between in 3 groups of knockout rats with wild-type animals. Moreover, angiosarcoma diagnostic genes have been evaluated to conﬁrm histopathologic proﬁle of the tumors including TIE1, FLT4, FLI1, EPHA2, PGF, MYC and EDNRB [43,44]. 4.6. CMap Drug Discovery CMap [45] was used in order to identify compounds that could potentially reverse gene expression changes induced by angiosarcoma. The lists of genes most up- and downregulated in angiosarcoma versus all other groups were loaded into online CMap. Only genes signiﬁcantly dysregulated (FDR < 0.05) in all pairwise comparisons between angiosarcoma and any of the other groups were taken into consideration. They were ﬁltered by fold changed (FC > 4.0 for upregulation and FC < 0.25 for downregulation in all three comparisons of sarcoma rats vs others) so that the length of uploaded list did not exceed the CMap’s functional limit of 150 genes. In the cases when CMap did not recognize a gene symbol, all alternative symbols from the NCBI gene database were tested and the gene was marked as unrecognized when all of them were not identiﬁed by CMap. A list of perturbagens with their enrichment scores was retrieved from CMap. The scores can take values from −100 to 100 with positive sign meaning that particular perturbagen changes gene expression in similar manner to that observed in the analyzed experiment, and the negative sign indicating opposite action and absolute value showing the strength of the eﬀect. In our experiment, the generated list of perturbagens was ﬁltered to leave only compounds and CMap classes, while gene knockouts and over-expressions were omitted, since the study was aimed at in silico drug discovery. A detailed list of compounds from those groups with their individual enrichment scores below −90.0 was selected (Table 3). 14 of 17 Cancers 2020, 12, 1525 Table 3. Compounds belonging to classes identiﬁed by CMap as most opposing to gene expression changes resulting from angiosarcoma. Table 3. Compounds belonging to classes identiﬁed by CMap as most opposing to gene expression changes resulting from angiosarcoma. resulting from angiosarcoma. Compounds Groups Enrichment Scores topoisomerase inhibitor −97.84 ellipticine −98.37 amsacrine −98.28 mitoxantrone −97.89 amonaﬁde −97.85 topotecan −97.72 teniposide −96.72 SN-38 −96.44 irinotecan −96.11 camptothecin −95.88 doxorubicin −95.42 pidorubicine −95.28 daunorubicin −95.14 pirarubicin −94.78 HDAC inhibitor −95.95 panobinostat −98.45 scriptaid −98.31 THM-I-94 −97.57 vorinostat −96.79 belinostat −96.72 apicidin −95.00 trichostatin-a −94.85 ISOX −94.79 HC-toxin −94.26 dacinostat −92.65 givinostat −91.73 CDK inhibitor −94.38 PHA-793887 −97.96 JNJ-7706621 −97.43 AT-7519 −95.84 aminopurvalanol-a −93.70 DNA synthesis inhibitor −92.69 mitomycin-c −94.37 JAK inhibitor −92.60 JAK3-inhibitor-VI −93.52 TG-101348 −92.57 Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/6/1525/s1, Table S1: Gene expression data (after log2-transform and quantile normalization), Table S2: Results of ANOVA and pairwise comparisons between diﬀerent group of rats, Table S3: Lists of genes up- and -downregulated in angiosarcoma shown in Figure 3d,e, Table S4: GSEA results, Table S5: Lists of pathways up- and downregulated in angiosarcoma as shown in Figure 5, Table S6: List of genes used in CMap drug discovery analysis, Table S7: Results of CMap analysis for compounds, Figure S1: Principal component analysis of the microarray expression proﬁles with all 20740 genes included, Figure S2: Enrichment maps of pathways signiﬁcantly change (a) in sarcoma 5. Conclusions SAR—sarcoma; CTL—positive controls, for CD31 rat white blood cells lysate (left lane) and homogenate from rat aorta (right lane); for p53 lysate from Caki1 cells (left lane) and MDA-MB-231 cells (right lane), Figure S10: Uncropped combined immunoblotting corresponding to Figure S9 with molecular weight markers and densitometry readings. Author Contributions: Conceptualization, U.S., A.M.C., W.F. and P.G.; Data curation, A.M.C., W.F. and M.F.; Formal analysis, U.S., A.M.C. and M.F.; Funding acquisition, P.G.; Investigation, U.S., D.S., M.F., M.W.-K., M.G., K.S. and Ł.C.; Methodology, A.M.C., M.F. and Z.R.; Project administration, P.G.; Resources, M.F., Z.R. and P.G.; Software, U.S. and W.F.; Supervision, A.M.C., W.F., Z.R. and P.G.; Visualization, U.S., M.F., K.S. and Ł.C.; Writing—original draft, U.S., A.M.C., M.F. and Ł.C.; Writing—review & editing, W.F., Z.R. and P.G. All authors have read and agreed to the published version of the manuscript. Funding: The study was supported by the National Scientiﬁc Leading Centre KNOW: “The search for new biomarkers of civilization diseases using high-throughput techniques and modern diagnostic imaging”. The study was also supported with statutory funds from Mossakowski Medical Research Centre, PAS. Acknowledgments: The project has been carried out with the use of CePT infrastructure ﬁnanced by the European Union—the European Regional Development Fund in the Operational Programme ”Innovative Economy” for 2007-2013. The authors acknowledge the support of Mrs. Dorota Wawrzyniak, MSci and Foreign Language Teaching Centre, Medical University of Lodz for professional editing and proofreading of this manuscript. The authors would also like to thank Dawid Walerych for providing anti-p53 antibody. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Yang, J.; Ren, Z.; Du, X.; Hao, M.; Zhou, W. The role of mesenchymal stem/progenitor cells in sarcoma: Update and dispute. Stem Cell Investig. 2014, 1, 18. [CrossRef] [PubMed] 1. Yang, J.; Ren, Z.; Du, X.; Hao, M.; Zhou, W. The role of mesenchymal stem/progenitor cells in sarcoma: Update and dispute. Stem Cell Investig. 2014, 1, 18. [CrossRef] [PubMed] 2. Badalamenti, G.; Messina, C.; De Luca, I.; Musso, E.; Casarin, A.; Incorvaia, L. Soft tissue sarcomas in the precision medicine era: New advances in clinical practice and future perspectives. Radiol. Med. 2018. [CrossRef] [PubMed] 2. Badalamenti, G.; Messina, C.; De Luca, I.; Musso, E.; Casarin, A.; Incorvaia, L. Soft tissue sarcomas in the precision medicine era: New advances in clinical practice and future perspectives. Radiol. Med. 2018. [CrossRef] [PubMed] 3. Casali, P.G.; Abecassis, N.; Aro, H.T.; Bauer, S.; Biagini, R.; Bielack, S.; Bonvalot, S.; Boukovinas, I.; Bovee, J.; Brodowicz, T.; et al. Soft tissue and visceral sarcomas: ESMO-EURACAN Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 2018, 29, iv268–iv269. [CrossRef] [PubMed] 3. Casali, P.G.; Abecassis, N.; Aro, H.T.; Bauer, S.; Biagini, R.; Bielack, S.; Bonvalot, S.; Boukovinas, I.; Bovee, J.; Brodowicz, T.; et al. Soft tissue and visceral sarcomas: ESMO-EURACAN Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 2018, 29, iv268–iv269. [CrossRef] [PubMed] 4. Hoang, N.T.; Acevedo, L.A.; Mann, M.J.; Tolani, B. A review of soft-tissue sarcomas: Translation of biological advances into treatment measures. Cancer Manag. Res. 2018, 10, 1089–1114. [CrossRef] [PubMed] 5. Gao, P.; Seebacher, N.A.; Hornicek, F.; Guo, Z.; Duan, Z. Advances in sarcoma gene mutations and therapeutic 4. Hoang, N.T.; Acevedo, L.A.; Mann, M.J.; Tolani, B. A review of soft-tissue sarcomas: Translation of biological advances into treatment measures. Cancer Manag. Res. 2018, 10, 1089–1114. [CrossRef] [PubMed] advances into treatment measures. Cancer Manag. Res. 2018, 10, 1089–1114. [CrossRef] [PubMed] 5. Gao, P.; Seebacher, N.A.; Hornicek, F.; Guo, Z.; Duan, Z. Advances in sarcoma gene mutations and therapeutic targets. Cancer Treat. Rev. 2018, 62, 98–109. [CrossRef] [PubMed] 5. Gao, P.; Seebacher, N.A.; Hornicek, F.; Guo, Z.; Duan, Z. Advances in sarcoma gene mutations and therapeutic targets. Cancer Treat. Rev. 2018, 62, 98–109. [CrossRef] [PubMed] 6. Patel, M.; Kato, S.M.; Kurzrock, R. Molecular Tumor Boards: Realizing Precision Oncology Therapy. Clin. Pharmacol. Ther. 2018, 103, 206–209. [CrossRef] [PubMed] 6. Patel, M.; Kato, S.M.; Kurzrock, R. Molecular Tumor Boards: Realizing Precision Oncology Therapy. Clin. Pharmacol. Ther. 5. Conclusions In conclusion, the Tp53C273X/C273X rat model can be used for studying sarcoma biology, in particular angiosarcoma and osteosarcoma. It may be employed for evaluating new drug candidates and for screening novel compounds for their potential to induce sarcoma cell death, angiogenesis inhibition or anti-sarcoma immune response. Findings from this sarcoma model demonstrate that the developed type of sarcoma depends on genetic background, underscoring the importance of developing more new models in various strains and species to simulate the study of diverse genetics of human sarcomas [27,29,33,35]. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/6/1525/s1, Table S1: Gene expression data (after log2-transform and quantile normalization), Table S2: Results of ANOVA and pairwise comparisons between diﬀerent group of rats, Table S3: Lists of genes up- and -downregulated in angiosarcoma shown in Figure 3d,e, Table S4: GSEA results, Table S5: Lists of pathways up- and downregulated in angiosarcoma as shown in Figure 5, Table S6: List of genes used in CMap drug discovery analysis, Table S7: Results of CMap analysis for compounds, Figure S1: Principal component analysis of the microarray expression proﬁles with all 20740 genes included, Figure S2: Enrichment maps of pathways signiﬁcantly change (a) in sarcoma 15 of 17 15 of 17 Cancers 2020, 12, 1525 Cancers 2020, 12, 1525 versus healthy tissue only (unchanged both versus heterozygous and total knockout) (b) in sarcoma versus total knockout only, (c) in sarcoma versus heterozygous knockout only. Those maps were created as diﬀerences of enrichment maps for each pairwise comparison with nodes ﬁltrated as in Figure 6 (that shows intersection of those same pairwise maps), Figure S3: Comparison of gene expression in P53 signaling pathway between rats with heterozygous knockout and wild-type animals. Green marks genes downregulated in Het group, red genes upregulated in Het rats, white genes not present in our dataset, Figure S4: Comparison of gene expression in P53 signaling pathway between rats with homozygous knockout and wild-type animals. Green marks genes downregulated in Ko group, red genes upregulated in Ko rats, white genes not present in our dataset, Figure S5: Comparison of gene expression in P53 signaling pathway between rats with sarcoma and wild-type animals. Green marks genes downregulated in sarcoma group, red genes upregulated in sarcoma rats, white genes not present in our dataset, Figure S6: An example of genotyping results. 5. Conclusions Product of PCR reaction with primer set designed to detect wild-type Tp53 (wt) and mutated Tp53 (mut) were run for each animal. Single wt band was interpreted as WT, single mut band—Tp53−/−and both bands were interpreted as Tp53+/−, Figure S7: Analysis of expression of genes related to sarcoma, that are typically upregulated, Figure S8: Analysis of expression of genes related to angiosarcoma, that are typically upregulated, Figure S9: Western blot analysis—p53 and CD31 expression. GAPDH expression was used as a control for protein load. SAR—sarcoma; CTL—positive controls, for CD31 rat white blood cells lysate (left lane) and homogenate from rat aorta (right lane); for p53 lysate from Caki1 cells (left lane) and MDA-MB-231 cells (right lane), Figure S10: Uncropped combined immunoblotting corresponding to Figure S9 with molecular weight markers and densitometry readings. versus healthy tissue only (unchanged both versus heterozygous and total knockout) (b) in sarcoma versus total knockout only, (c) in sarcoma versus heterozygous knockout only. Those maps were created as diﬀerences of enrichment maps for each pairwise comparison with nodes ﬁltrated as in Figure 6 (that shows intersection of those same pairwise maps), Figure S3: Comparison of gene expression in P53 signaling pathway between rats with heterozygous knockout and wild-type animals. Green marks genes downregulated in Het group, red genes upregulated in Het rats, white genes not present in our dataset, Figure S4: Comparison of gene expression in P53 signaling pathway between rats with homozygous knockout and wild-type animals. Green marks genes downregulated in Ko group, red genes upregulated in Ko rats, white genes not present in our dataset, Figure S5: Comparison of gene expression in P53 signaling pathway between rats with sarcoma and wild-type animals. Green marks genes downregulated in sarcoma group, red genes upregulated in sarcoma rats, white genes not present in our dataset, Figure S6: An example of genotyping results. Product of PCR reaction with primer set designed to detect wild-type Tp53 (wt) and mutated Tp53 (mut) were run for each animal. Single wt band was interpreted as WT, single mut band—Tp53−/−and both bands were interpreted as Tp53+/−, Figure S7: Analysis of expression of genes related to sarcoma, that are typically upregulated, Figure S8: Analysis of expression of genes related to angiosarcoma, that are typically upregulated, Figure S9: Western blot analysis—p53 and CD31 expression. GAPDH expression was used as a control for protein load. References 2018, 103, 206–209. [CrossRef] [PubMed] 7. Hansen, S.A.; Hart, M.L.; Busi, S.; Parker, T.; Goerndt, A.; Jones, K.; Amos-Landgraf, J.M.; Bryda, E.C. Fischer-344 Tp53-knockout rats exhibit a high rate of bone and brain neoplasia with frequent metastasis. Dis. Model. Mech. 2016, 9, 1139–1146. [CrossRef] [PubMed] 7. Hansen, S.A.; Hart, M.L.; Busi, S.; Parker, T.; Goerndt, A.; Jones, K.; Amos-Landgraf, J.M.; Bryda, E.C. Fischer-344 Tp53-knockout rats exhibit a high rate of bone and brain neoplasia with frequent metastasis. Dis. Model. Mech. 2016, 9, 1139–1146. 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https://openalex.org/W2513798760	https://europepmc.org/articles/pmc5011787?pdf=render	English	null	MiR-137 and miR-34a directly target Snail and inhibit EMT, invasion and sphere-forming ability of ovarian cancer cells	Journal of experimental & clinical cancer research	2,016	cc-by	6,730	Abstract Background: In ovarian cancer (OC) cells, Snail was reported to induce the epithelial-to-mesenchymal transition (EMT), which is a critical step in OC metastasis. At present little is known about controlling Snail expression in OC cells by using specific microRNAs (miRNAs). Methods: We first used a computational target prediction analysis to identify 6 candidate miRNAs that bind to the 3′-untranslated region (3′-UTR) region of the Snail mRNA. Among these miRNAs, two miRNAs (miR-137 and miR-34a) with a potential to regulate Snail were validated by quantitative real-time PCR, Western blot analysis, and Snail 3′-UTR reporter assays. We assessed the effects of miR-137 and miR-34a on EMT, invasion and sphere formation in OC cells. We also evaluated the expression of miR-137 and miR-34a in OC tissues and adjacent normal tissues and analyzed the relationship between their expression and patient survival. Results: We report that OC tissues possess significantly decreased levels of miR-137 and miR-34a and increased expression of Snail when compared to their adjacent normal tissues, and lower miR-137 and miR-34a expression correlates with worse patient survival. Using luciferase constructs containing the 3′-UTR region of Snail mRNA combined with miRNA overexpression and mutagenesis, we identified miR-137 and miR-34a as direct suppressors of Snail in OC cells. The introduction of miR-137 and miR-34a resulted in the suppression of Snail at both the transcript and protein levels, and effectively suppressed the EMT phenotype and sphere formation of OC cells. However, the inhibition of miR-137 and miR-34a with antisense oligonucleotides promoted EMT and OC cell invasion. Moreover, ectopic expression of Snail significantly reversed the inhibitory effects of miR-137 and miR-34a on OC cell invasion and sphere formation. Conclusions: These findings suggest that both miR-137 and miR-34a act as Snail suppressors to negatively regulate EMT, invasive and sphere-forming properties of OC cells. Keywords: microRNA-137, microRNA-34a, Snail, EMT, Cancer stemness, Ovarian cancer Keywords: microRNA-137, microRNA-34a, Snail, EMT, Cancer stemness, Ovarian cancer Abbreviations: EMT, Epithelial-to-mesenchymal transition; miRNAs, microRNAs * Correspondence: dpx1cn@gmail.com; watarih@med.hokudai.ac.jp; jyue@uthsc.edu †Equal contributors 1Department of Women’s Health Educational System, Hokkaido University School of Medicine, Hokkaido University, N15, W7, Sapporo 0608638, Japan 3Department of Gynecology, Hokkaido University School of Medicine, Hokkaido University, N15, W7, Sapporo 0608638, Japan 4Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USA Full list of author information is available at the end of the article © 2016 The Author(s). MiR-137 and miR-34a directly target Snail and inhibit EMT, invasion and sphere-forming ability of ovarian cancer cells Peixin Dong1†, Ying Xiong2†, Hidemichi Watari3, Sharon J. B. Hanley1, Yosuke Konno3, Kei Ihira3, Takahiro Yamada1, Masataka Kudo3, Junming Yue4,5* and Noriaki Sakuragi1,3 * Correspondence: dpx1cn@gmail.com; watarih@med.hokudai.ac.jp; jyue@uthsc.edu †Equal contributors 1Department of Women’s Health Educational System, Hokkaido University School of Medicine, Hokkaido University, N15, W7, Sapporo 0608638, Japan 3Department of Gynecology, Hokkaido University School of Medicine, Hokkaido University, N15, W7, Sapporo 0608638, Japan 4Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USA Full list of author information is available at the end of the article Dong et al. Journal of Experimental & Clinical Cancer Research (2016) 35:132 DOI 10.1186/s13046-016-0415-y Dong et al. Journal of Experimental & Clinical Cancer Research (2016) 35:132 DOI 10.1186/s13046-016-0415-y Dong et al. Journal of Experimental & Clinical Cancer Research (2016) 35:132 DOI 10.1186/s13046-016-0415-y Open Access Sphere formation assay Single cells (1000 cells per well) were plated onto a 24- well ultra-low attachment plate (Corning) in serum- free DMEM/F12 medium supplemented with N2 plus media supplement (Invitrogen), 20 ng/ml epidermal growth factor (Invitrogen), 20 ng/ml basic fibroblast growth factor (Invitrogen) and 4 mg/ml heparin (Sigma-Aldrich). After 10 days of culture, the number of spheres larger than 50 μm was counted under an inverted microscope. In this study, we provide evidence that miR-137 and miR-34a directly bind to and down-regulate Snail levels to suppress EMT, invasion and sphere-forming ability of OC cells, and that the repression of these two miRNAs is significantly correlated with worse patient survival in OC. Cell viability assay and cell apoptosis assay Cell viability assay and cell apoptosis assay Cell counting kit-8 assay (Dojindo) and Caspase-Glo 3/7 assay (Promega) were used to assess cell viability and cell apoptosis as previously reported [16]. For the cell via- bility assay, OC cells and NOEC cells were seeded were seeded at a density of 5 × 103 per well in 96-well plates for 24 h, and then transfected with 30 nM of miR-137 or miR-34a mimic or negative control mimic (Neg mimic). After 72 h, 10 μl of Cell counting kit-8 solution was added into each well and the plates were incubated for additional 4 h at 37 °C. The UV absorbance of each sample was mea- sured in a microplate reader at 450 nm. For the apoptotic assay, caspase-3/7 activity was analyzed in accordance with the manufacturer’s protocol. Briefly, OC and NOEC cells were seeded into 96-well plates and transfected as described above. After 72 h, an equal volume of Caspase- Glo 3/7 reagent was added into each well, and the cells were incubated at room temperature in the dark. 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. Dong et al. Journal of Experimental & Clinical Cancer Research (2016) 35:132 Page 2 of 9 Cell invasion assay OC cells were grown to 50–70 % confluence and trans- fected as indicated. After 24 h, cells were seeded into upper chamber of Boyden chambers coated with Matri- gel as described previously [14, 15]. After incubation for 24 h, the non-invading cells were gently removed with a cotton swab. Invasive cells located on the lower surface of chamber were stained with Giemsa and counted under a microscope. Relative cell invasion activities were expressed as the fold change over res- pective controls. Growing evidence suggests that a number of epigen- etic mechanisms control the expression of genes that facilitate EMT and induce metastasis [6]. For example, microRNAs (miRNAs) have important roles in the regu- lation of cancer cell invasion and motility, by suppress- ing or promoting EMT [7]. In addition, several miRNAs [8–11] have been implicated in the regulation of Snail expression in human cancers other than OC. To date, however, little is known about controlling Snail expres- sion in OC cells by using specific miRNAs. Background performed by using the Takara SYBR Premix Ex Taq II (Takara) and the 7500 Real-Time PCR System (Applied Biosystems). The primer sequences for Snail [12], E- cadherin [12], ZO-1 [12], N-cadherin [12], Vimentin [12], CD44 [13], CD133 [12] and GAPDH [12] have been previously reported. For miRNA analysis, qPCRs were performed using the NCode miRNA qRT-PCR analysis (Invitrogen, CA, USA). Forward primer is the exact sequence of the mature miR-137 and miR-34a. The mRNA and miRNA expression data were normalized to GAPDH and U6, respectively. Results were represented as the fold change relative to respective controls. performed by using the Takara SYBR Premix Ex Taq II (Takara) and the 7500 Real-Time PCR System (Applied Biosystems). The primer sequences for Snail [12], E- cadherin [12], ZO-1 [12], N-cadherin [12], Vimentin [12], CD44 [13], CD133 [12] and GAPDH [12] have been previously reported. For miRNA analysis, qPCRs were performed using the NCode miRNA qRT-PCR analysis (Invitrogen, CA, USA). Forward primer is the exact sequence of the mature miR-137 and miR-34a. The mRNA and miRNA expression data were normalized to GAPDH and U6, respectively. Results were represented as the fold change relative to respective controls. The majority (75 %) of patients with ovarian cancer (OC) present with advanced disease and widely metastatic disease [1, 2]. In OC, the acquisition of invasiveness is ac- companied by a shift from epithelial to mesenchymal phenotype, also called the epithelial-to-mesenchymal tran- sition (EMT), which endows cancer cells with increased motility and invasiveness to seed metastasis and with stem cell-like properties, such as upregulation of stem cell genes (CD44 and CD133) and self-renewal ability [3, 4]. The EMT process can be initiated by a group of transcrip- tion factors including SNAIL (Snail), which repress the ex- pression of epithelial markers (E-cadherin and ZO-1), and induce the levels of mesenchymal markers (Vimentin and N-cadherin) [5]. Therefore, identification of key actors regulating Snail expression and EMT in OC cells would have tremendous clinical utility. Reagents and cell culture Human OC cell lines (SKOV-3 and ES-2) were ob- tained from the American Type Culture Collection (Manassas, VA), and were cultured in DMEM/F12 medium (Invitrogen) supplemented with 10 % fetal bovine serum (FBS, Invitrogen). Normal ovarian epi- thelial cells (NOEC, Pricells, Wuhan, China) were cul- tured in Ham’s F-12 (Gibco) supplemented with 20 % FBS (Gibco). MiRNA mimic and miRNA inhibitor for miR- 137 or miR-34a (30 nM, Ambion), Snail siRNA (5 nM, Ambion) and Snail cDNA plasmids (OriGene) were transfected using Lipofectamine 2000 (Invitrogen) ac- cording to the manufacturer’s protocol. Real-time quantitative RT-PCR (qPCR) Total RNA was extracted using TRIzol reagents (Invitrogen) according to the manufacturer’s instructions. For mRNA and miRNA analysis, cDNAs were synthesized using the PrimeScript RT reagent kit (Takara). Then, qPCRs were Dong et al. Journal of Experimental & Clinical Cancer Research (2016) 35:132 Page 3 of 9 Luminescence was measured after 3 h of incubation with the caspase substrate. Luminescence was measured after 3 h of incubation with the caspase substrate. a Dual Luciferase Reporter Assay (Promega) was per- formed as previously reported [17]. The firefly luciferase activity was normalized to the Renilla luciferase activity. a Dual Luciferase Reporter Assay (Promega) was per- formed as previously reported [17]. The firefly luciferase activity was normalized to the Renilla luciferase activity. Clinical samples p Matched serous OC and corresponding adjacent normal ovarian tissues were obtained from 50 patients undergoing resection at the Department of Gynecology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center (Guangzhou, China). Tumor and non-cancerous tissues were confirmed histologically by Hematoxylin and Eosin staining. All samples were collected from consenting individuals according to the protocols approved by the Ethics Review Board at Sun Yat-sen Uni- versity Cancer Center. All tissue samples were immediately snap-frozen in liquid nitrogen. They were kept in a -80 °C freezer and total RNA was isolated using TRIzol reagents. Dual luciferase reporter assay The Snail 3′-UTR luciferase vector was purchased from OriGene. Mutations in the miR-137 or miR-34a-binding sequence were generated by using the QuickChange Mu- tagenesis Kit (Stratagene). For luciferase assay, OC cells were seeded onto 24-well plates and transfected after 24 h with 100 ng of firefly luciferase reporter plasmid, 10 ng of Renilla report plasmid as normalization control, together with miR-137 or miR-34a mimic or Neg mimic. After 24 h, Western blot analysis Cells were harvested 24 h after transfections. Equal amounts of protein lysates (30 μg) were separated by 10 % SDS-PAGE for immunoblots with antibodies to Snail (Abcam), E-cadherin (GenScript), N-cadherin (BD Biosciences), Vimentin (GenScript) and GAPDH (Santa Cruz). Primary antibodies were used at a dilution of 1:1000. A horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin-G antibody was used as the secondary antibody (1:5000; Santa Cruz). Signals were detected using enhanced chemiluminescence reagents (Amersham Biosciences). Results miR-137 or miR-34a expression and patient prognosis. OC patients population were divided into two groups based on high (n = 25) or low (n = 25) expression of miR-137 or miR-34a according to the median expression levels among OC specimens, and Kaplan–Meier curves for overall survival was plotted. Patients with lower miR- 137 or miR-34a expression had significantly shorter overall survival (Fig. 1d and e). Consistent with these re- sults, both miR-137 and miR-34a were clearly reduced in two OC cell lines (SKOV-3 and ES-2) compared with normal ovarian epithelial NOEC cells (Fig. 2a). MiR-137 and miR-34a are downregulated in OC tissues and OC cell lines, and decreased expression of miR-137 and miR-34a is associated with poor survival in OC patients To investigate miRNA regulation of Snail, we first employed multiple algorithms, including TargetScan, miRSystem and DIANA-MicroT-CDS, to screen the specific miRNAs that can target the 3′-untranslated re- gion (3′-UTR) region of the Snail mRNA. These analyses revealed six common miRNAs predicted to bind the 3′- UTR of the Snail transcript (Fig. 1a). For example, target prediction algorithms predicted one putative binding site for miR-137 in the 3′-UTR of Snail (nt 444-450) and recognized one binding site for miR-34a in Snail 3′- UTR (nt 86–93) [10]. Snail is a direct target of miR-137 and miR-34a in OC cells To determine whether Snail is a direct target of miR-137 and miR-34a, we transfected a human OC ES-2 cell line (which expresses very high levels of endogenous Snail and lacks the expression of miR-137 and miR-34a) with pre-miRNA-137 or pre-miRNA-34a, together with a firefly luciferase reporter vector containing the Snail 3′-UTR. We found significant repression of luciferase activities by either miR-137 or miR-34a (Fig. 2b and c). In addition, these inhibitory effects of miR-137 or miR-34a on lucife- rase activities were eliminated following mutations tar- geting the predicted-binding sites of miR-137 or miR-34a within the Snail 3′-UTR (Fig. 2b and c), demonstrating that they bind directly to the 3′-UTR of Snail. Next, we sought to determine whether their expression is altered in human OC by measuring the expression level of these 6 miRNAs in 50 pairs of human OC sam- ples and adjacent normal ovarian tissues. Interestingly, the levels of all 6 miRNAs were significantly decreased in OC tissues compared with adjacent normal ovarian tissues (data not shown). Among them, miR-137 and miR-34a were the most significantly down-regulated miRNAs (Fig. Statistical analysis Results are expressed as mean ± s.e.m. from at least three independent experiments performed in triplicate. 2-tailed Student’s t-test was used for statistical analysis. The log-rank test was used for survival analysis. The value of P < 0.05 were considered as significant. Fig. 1 MiR-137 and miR-34a are downregulated in OC tissues and decreased expressions of miR-137 and miR-34a are associated with poor survival in OC patients. a Venn diagram showing the overlap of miRNAs that were predicted to bind to the Snail 3′-UTR by alternative algorithms (TargetScan, miRSystem and DIANA-MicroT-CDS). The 6 predicted miRNAs were common to these three algorithms. b, c qPCR analysis of miR-137 (b) and miR-34a (c) levels in 50 paired cancerous and normal tissue samples from OC patients. d, e Kaplan-Meier analysis of overall survival in 50 OC patients with high median (n = 25) or low median (n = 25) expression levels of miR-137 (d) or miR-34a (e) Fig. 1 MiR-137 and miR-34a are downregulated in OC tissues and decreased expressions of miR-137 and miR-34a are associated with poor survival in OC patients. a Venn diagram showing the overlap of miRNAs that were predicted to bind to the Snail 3′-UTR by alternative algorithms (TargetScan, miRSystem and DIANA-MicroT-CDS). The 6 predicted miRNAs were common to these three algorithms. b, c qPCR analysis of miR-137 (b) and miR-34a (c) levels in 50 paired cancerous and normal tissue samples from OC patients. d, e Kaplan-Meier analysis of overall survival in 50 OC patients with high median (n = 25) or low median (n = 25) expression levels of miR-137 (d) or miR-34a (e) Page 4 of 9 Dong et al. Journal of Experimental & Clinical Cancer Research (2016) 35:132 Page 4 of 9 Page 4 of 9 Results 1b and c), and selected to examine their effects on Snail expression. To examine whether the downregulation of miR-137 or miR-34a has any clinical significance in OC, we analyzed the association between Fig. 2 Snail is a direct target of miR-137 and miR-34a in OC cells. a Relative miR-34a expression in OC cell lines (SKOV-3 and ES-2) and normal ovarian epithelial NOEC cells. b, c ES-2 cells were transfected with reporter constructs containing either wild-type (WT) Snail, or Snail 3′-UTR with mutation (MUT), along with miR-137 mimic (b), miR-34a mimic (c), or negative control mimic (Neg mimic), respectively. Relative luciferase activity was measured. d, e qPCR analysis of Snail expression in OC cells after overexpression (d) or knockdown (e) of miR-137 and miR-34a. f Western blotting analysis of Snail expression in OC cells after overexpression or knockdown of miR-137 and miR-34a. g, h qPCR analysis of indicated mRNAs in OC cells after transient overexpression or knockdown of miR-137 (g) and miR-34a (h). i Relative mRNA expression of Snail in OC tissues and matched normal tissues. j Analysis of Snail mRNA expression using microarray (Oncomine) on normal ovary versus OC tissue. P < 0.01 Fig. 2 Snail is a direct target of miR-137 and miR-34a in OC cells. a Relative miR-34a expression in OC cell lines (SKOV-3 and ES-2) and normal ovarian epithelial NOEC cells. b, c ES-2 cells were transfected with reporter constructs containing either wild-type (WT) Snail, or Snail 3′-UTR with mutation (MUT), along with miR-137 mimic (b), miR-34a mimic (c), or negative control mimic (Neg mimic), respectively. Relative luciferase activity was measured. d, e qPCR analysis of Snail expression in OC cells after overexpression (d) or knockdown (e) of miR-137 and miR-34a. f Western blotting analysis of Snail expression in OC cells after overexpression or knockdown of miR-137 and miR-34a. g, h qPCR analysis of indicated mRNAs in OC cells after transient overexpression or knockdown of miR-137 (g) and miR-34a (h). i Relative mRNA expression of Snail in OC tissues and matched normal tissues. j Analysis of Snail mRNA expression using microarray (Oncomine) on normal ovary versus OC tissue. P < 0.01 Page 5 of 9 Page 5 of 9 Dong et al. Results Journal of Experimental & Clinical Cancer Research (2016) 35:132 To directly assess if these miRNAs had a biological effect on Snail, mRNA and protein expression of Snail was examined in ES-2 and SKOV-3 cells following trans- fection of miRNA mimics or inhibitors, respectively. As expected, Snail mRNA and protein were down-regulated in ES-2 cells transfected with miR-137 or miR-34a mimic (Fig. 2d and f). In contrast, transfection with miR-137 or miR-34a inhibitor into SKOV-3 cells (which has relatively higher levels of miR-34a and miR-137) up-regulated Snail expression at mRNA and protein levels (Fig. 2e and f). Then we examined the expression of epithelial marker E- cadherin, mesenchymal marker N-cadherin and Vimentin in OC cells after the overexpression or knockdown of miR-137 and miR-34a, using qPCR analysis. ES-2 cells transfected with miR-137 or miR-34a mimic showed in- creased levels of E-cadherin and decreased expression of N-cadherin and Vimentin (Fig. 2g). However, transfection of SKOV-3 cells with miR-137 or miR-34a inhibitor re- versed these effects (Fig. 2h). database on OC. Importantly, our analysis of the TCGA OC samples revealed significantly higher Snail tran- script levels in OC samples, relative to normal ovaries (Fig. 2j), supporting a negative correlation between miR- 137 or miR-34a and Snail expression in human OCs. Collectively, these results suggest that both miR-137 and miR-34a suppress Snail by targeting specific sites within the 3′-UTR of Snail. MiR-137 and miR-34a inhibit EMT, invasion and sphere-forming ability of OC cells We also examined the potential effects of miR-137 and miR-34a on EMT features, invasive and sphere-forming abilities of OC cells. For this, we transiently reduced or overexpressed miR-137 and miR-34a in SKOV-3 and ES-2 cells, which express relatively higher or lower ex- pression of miR-137 and miR-34a, respectively (Fig. 2a). The down-regulation of miR-137 or miR-34a in SKOV-3 cells induced a spindle-like mesenchymal morphology (Fig. 3a), and the overexpression of miR-137 or miR-34a in ES-2 cells resulted in an epithelial morphology change (Fig. 3b). We further investigated the effects of knock- down and overexpression of these two miRNAs on EMT, OC cell invasion and sphere formation ability. Matrigel invasion assay and sphere formation assay Next, we examined the correlation between the ex- pression of miR-137, miR-34a and Snail mRNA expres- sion. We found that Snail mRNA level was significantly increased in OC samples compared with their non- tumor counterparts (Fig. 2i). Next, we chose to com- pare our data with existing published gene expression Fig. 3 MiR-137 and miR-34a inhibit EMT, invasion and sphere-forming ability of OC cells. a, b Cellular morphology of OC cells after transient knockdown (a) or overexpression (b) of miR-137 and miR-34a. Scale bar = 200 μm (a) and 100 μm (b). c, d Cell invasion (c) and sphere formation (d) of OC cells after transient knockdown or overexpression of miR-137 and miR-34a. e, f qPCR analysis of indicated mRNAs in SKOV-3 (e) and ES-2 (f) cells after transient knockdown or overexpression of miR-137 and miR-34a. g Representative images of invaded SKOV-3 cells after transient transfection with miR-137 inhibitor or miR-34a inhibitor or negative control inhibitor (Neg inhibitor). h Representative images of spheres formed from ES-2 cells transfected with miR-137 or miR-34a mimic or Neg mimic. Scale bar =100 μm. P < 0.01 Fig. 3 MiR-137 and miR-34a inhibit EMT, invasion and sphere-forming ability of OC cells. a, b Cellular morphology of OC cells after transient knockdown (a) or overexpression (b) of miR-137 and miR-34a. Scale bar = 200 μm (a) and 100 μm (b). c, d Cell invasion (c) and sphere formation (d) of OC cells after transient knockdown or overexpression of miR-137 and miR-34a. e, f qPCR analysis of indicated mRNAs in SKOV-3 (e) and ES-2 (f) cells after transient knockdown or overexpression of miR-137 and miR-34a. MiR-137 and miR-34a inhibit EMT, invasion and sphere-forming ability of OC cells g Representative images of invaded SKOV-3 cells after transient transfection with miR-137 inhibitor or miR-34a inhibitor or negative control inhibitor (Neg inhibitor). h Representative images of spheres formed from ES-2 cells transfected with miR-137 or miR-34a mimic or Neg mimic. Scale bar =100 μm. P < 0.01 Dong et al. Journal of Experimental & Clinical Cancer Research (2016) 35:132 Page 6 of 9 Page 6 of 9 could repress the miR-137 or miR-34a inhibitor-induced OC cell invasion and sphere formation, or whether transient over-expression of a Snail open reading frame (ORF) could reverse the inhibitory effects of miR-137 or miR-34a mimic on OC cell invasion and sphere forma- tion. The miR-137 or miR-34a inhibitor-induced SKOV-3 cell invasion and sphere formation were significantly re- duced by Snail siRNA (Fig. 4a and b). The overexpression of Snail ORF in ES-2 cells partially rescued miR-137 or miR-34a mimic-suppressed invasion and sphere formation (Fig. 4a and b). Consistent with these data, inhibiting Snail expression using siRNA in SKOV-3 cells trans- fected with miR-137 or miR-34a inhibitor elevated the expression of E-cadherin but reduced the expression of N-cadherin and Vimentin (Fig. 4c). Moreover, rescuing Snail expression with Snail ORF in the presence of miR-137 or miR-34a mimic resulted in down-regulation of E-cadherin and up-regulation of N-cadherin and Vimentin (Fig. 4c). These data suggest that miR-137 and miR-34a inhibits invasive and stem cell-like proper- ties of OC cells by suppressing Snail expression via its 3′-UTR. demonstrated that knockdown of miR-137 or miR-34a using antisense oligonucleotides in SKOV-3 cells sig- nificantly promoted cell invasion and sphere formation, whereas overexpression of miR-137 or miR-34a with miRNA mimics in ES-2 cells inhibited these malignant features (Fig. 3c and d). Then we examined the expression of epithelial marker ZO-1 and cancer stemness markers (CD44 and CD133) in OC cells after the knockdown and overexpression of miR-137 and miR-34a, using qPCR as- says. SKOV-3 cells transfected with miR-137 or miR-34a inhibitor showed decreased levels of ZO-1 and increased expression of CD44 and CD133 (Fig. 3e). However, trans- fection of ES-2 cells with miR-137 or miR-34a mimic in- creased the mRNA expression of ZO-1 and suppressed that of CD44 and CD133 (Fig. 3f). These data suggest that miR-137 and miR-34a inhibit mesenchymal characteristics and reduces self-renewal properties of OC cells. MiR-137 and miR-34a inhibit EMT, invasion and sphere-forming ability of OC cells MiR-137 and miR-34a modulate EMT, invasion and sphere-forming ability of OC cells through targeting Snail To further corroborate the above observations, we inves- tigated whether silencing of Snail with specific siRNA Fig. 4 MiR-137 and miR-34a modulate EMT, invasion and sphere-forming ability of OC cells through targeting Snail. MiR-137 or miR-34a inhibitor or Neg inhibitor was co-transfected into SKOV-3 cells, together with (or without) Snail siRNA. MiR-137 or miR-34a mimic or Neg mimic was co-transfected into ES-2 cells, together with (or without) Snail cDNA vector lacking the 3′-UTR region. Cell invasion assay (a), sphere formation assay (b) and Western blotting analysis of indicated proteins (c) in OC cells treated as described above were performed. P < 0.01 Fig. 4 MiR-137 and miR-34a modulate EMT, invasion and sphere-forming ability of OC cells through targeting Snail. MiR-137 or miR-34a inhibitor or Neg inhibitor was co-transfected into SKOV-3 cells, together with (or without) Snail siRNA. MiR-137 or miR-34a mimic or Neg mimic was co-transfected into ES-2 cells, together with (or without) Snail cDNA vector lacking the 3′-UTR region. Cell invasion assay (a), sphere formation assay (b) and Western blotting analysis of indicated proteins (c) in OC cells treated as described above were performed. P < 0.01 Page 7 of 9 Page 7 of 9 Dong et al. Journal of Experimental & Clinical Cancer Research (2016) 35:132 Overexpression of miR-137 and miR-34a decreases cell viability and induces apoptosis in OC cells, without significant cytotoxicity to normal NOEC cells Next, we investigated if overexpressing miR-137 and miR-34a could affect the viability and apoptosis of OC cells and normal NOEC cells using cell viability and cell apoptosis assays. The data demonstrated that the res- toration of miR-137 and miR-34a expression in ES-2 and SKOV-3 cells led to a significant reduction in cell viability and the induction of cell apoptosis, but had no significant toxicity to NOEC cells (Fig. 5), indicating the feasibility of inhibiting the growth of OC cells via the therapeutic delivery of miR-137 or miR-34a mimics, with- out causing significant toxicity to normal ovarian tissues. metastasis, such as EMT, cell invasion, sphere-forming ability and chemoresistance [18–23]. MiR-137 and miR-34a inhibit EMT, invasion and sphere-forming ability of OC cells Overexpression of miR-137 and miR-34a decreases cell viability and induces apoptosis in OC cells, without significant cytotoxicity to normal NOEC cells We show that miR-137 and miR-34a are downregu- lated in OC samples, and we found a significant associ- ation between decreased miR-137 or miR-34a expression and worse patient prognosis. Furthermore, our in vitro data have confirmed that reduced expression of miR-137 and miR-34a is critical for enhanced OC cell invasive- ness and self-renewal, suggesting that these two miRNAs can be potential therapeutic targets. Therefore, attenuating the oncogenic functions of Snail by the use of miR-137 and miR-34a could provide an exciting opportunity for the development of therapy against OC. Since a single mRNA might be targeted by multiple miRNAs, we sought to newly identify crucial miRNAs that reduce the expression of Snail in OC cells. We demonstrate for the first time that miR-137 can directly repress Snail expression through its binding to the spe- cific binding site in the 3′-UTR of the human Snail gene, thereby negatively regulating EMT, invasion and self-renewal of OC cells. It is interesting to note that a tumor-suppressive role for miR-137 has also been shown in a variety of human tumors [24–26]. Thus, these data and our current results reveal that, the anti-invasive References 1. Lengyel E. Ovarian cancer development and metastasis. Am J Pathol. 2010; 177:1053–64. 1. Lengyel E. Ovarian cancer development and metastasis. Am J Pathol. 2010; 177:1053–64. 2. Kim A, Ueda Y, Naka T, Enomoto T. Therapeutic strategies in epithelial ovarian cancer. J Exp Clin Cancer Res. 2012;31:14. 3. Vergara D, Merlot B, Lucot JP, Collinet P, Vinatier D, Fournier I, et al. Epithelial-mesenchymal transition in ovarian cancer. Cancer Lett. 2010;291:59–66. 4. Kaufhold S, Bonavida B. Central role of Snail1 in the regulation of EMT and resistance in cancer: a target for therapeutic intervention. J Exp Clin Cancer Res. 2014;33:62. 5. Wang Y, Shang Y. Epigenetic control of epithelial-to-mesenchymal transition and cancer metastasis. Exp Cell Res. 2013;319:160–9. 6. Abba ML, Patil N, Leupold JH, Allgayer H. MicroRNA Regulation of Epithelial to Mesenchymal Transition. J Clin Med. 2016;5(1). doi:10.3390/jcm5010008. 7. Bagnato A, Rosanò L. Epithelial-mesenchymal transition in ovarian cancer progression: a crucial role for the endothelin axis. Cells Tissues Organs. 2007; 185:85–94. Consent for publication N li bl Consent for publication Not applicable. Conclusions 8. Zuo QF, Cao LY, Yu T, Gong L, Wang LN, Zhao YL, et al. MicroRNA-22 inhibits tumor growth and metastasis in gastric cancer by directly targeting MMP14 and Snail. Cell Death Dis. 2015;6:e2000. We identified a new mechanism whereby decreased ex- pression of both miR-137 and miR-34a contributes to enhanced Snail levels, which in turn promotes EMT, in- vasion and sphere-forming of OC cells. Our results sug- gest that these two miRNAs might become candidate targets for the treatment of Snail-overexpressing OC. 9. Kumarswamy R, Mudduluru G, Ceppi P, Muppala S, Kozlowski M, Niklinski J, et al. MicroRNA-30a inhibits epithelial-to-mesenchymal transition by targeting Snai1 and is downregulated in non-small cell lung cancer. Int J Cancer. 2012;130:2044–53. 10. Siemens H, Jackstadt R, Hünten S, Kaller M, Menssen A, Götz U, et al. miR-34 and SNAIL form a double-negative feedback loop to regulate epithelial- mesenchymal transitions. Cell Cycle. 2011;10:4256–71. 10. Siemens H, Jackstadt R, Hünten S, Kaller M, Menssen A, Götz U, et al. miR-34 and SNAIL form a double-negative feedback loop to regulate epithelial- mesenchymal transitions. Cell Cycle. 2011;10:4256–71. Acknowledgements We thank Dr. Zhujie Xu for technical assistance. Acknowledgements We thank Dr. Zhujie Xu for technical assistance. Authors’ contributions PD, HW and JY designed experiments. PD, YX, HW and YK performed experiments. PD, HW and JY wrote the manuscript. SJH, KI, TY, MK and NS contributed to data analysis and discussed the results. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. As an important tumor suppressor, miR-34a controls the expression of a host of target proteins involved in cell proliferation, apoptosis, cancer stemness, metastasis and chemoresistance [27], and it is often down-regulated in numerous tumor types [28–32]. A previous study has shown that miR-34a acts as a suppressor of Snail in colon cancer [10], but to our knowledge, there are also opposite findings showing its tumor-promoting roles in other cancer type [33]. Whether miR-34a affects Snail expression in OC cells have remained elusive. Here, we have demonstrated that miR-34a is a direct inhibitor of Snail in OC cells. Thus, the down-regulation of miR-34a may be essential for Snail to induce EMT and OC metastasis. Ethics approval and consent to participate This work has been approved by the ethical committees at Sun Yat-sen University Cancer Center, and we have obtained written informed consent from all participants involved in the study. Ethics approval for animal research: not applicable. Discussion Despite advances in our understanding of the mecha- nisms underlying Snail up-regulation, little is known about the endogenous miRNA suppressors of Snail in OC cells. We report in this study that in OC cells, both miR-137 and miR-34a act as novel tumor repressors that directly target Snail, which plays a pivotal role in con- trolling the various cellular functions during cancer Fig. 5 Overexpression of miR-137 and miR-34a decreases cell viability and induces apoptosis in OC cells, without significant cytotoxicity to normal NOEC cells. OC cells or normal NOEC cells were transfected with miR-137 mimic, miR-34a mimic or Neg mimic for 72 h, respectively. Cell viability assay (a) and cell apoptosis assay (b) were performed. P < 0.05. P < 0.01. NS: Not Significant Fig. 5 Overexpression of miR-137 and miR-34a decreases cell viability and induces apoptosis in OC cells, without significant cytotoxicity to normal NOEC cells. OC cells or normal NOEC cells were transfected with miR-137 mimic, miR-34a mimic or Neg mimic for 72 h, respectively. Cell viability assay (a) and cell apoptosis assay (b) were performed. P < 0.05. **P < 0.01. NS: Not Significant Dong et al. Journal of Experimental & Clinical Cancer Research (2016) 35:132 Page 8 of 9 Page 8 of 9 Page 8 of 9 Page 8 of 9 Page 8 of 9 effects of miR-137 described here for OC—possibly me- diated by Snail suppression—might be relevant in other tumor types. Therefore, we postulate that replacing tumor suppressive miR-137 targeting Snail might be a promising approach for treating metastatic and recur- rent OC. Acknowledgements 11. Bai Z, Sun J, Wang X, Wang H, Pei H, Zhang Z. MicroRNA-153 is a prognostic marker and inhibits cell migration and invasion by targeting SNAI1 in human pancreatic ductal adenocarcinoma. Oncol Rep. 2015;34:595–602. 11. Bai Z, Sun J, Wang X, Wang H, Pei H, Zhang Z. MicroRNA-153 is a prognostic marker and inhibits cell migration and invasion by targeting SNAI1 in human pancreatic ductal adenocarcinoma. Oncol Rep. 2015;34:595–602. Author details 1 1Department of Women’s Health Educational System, Hokkaido University School of Medicine, Hokkaido University, N15, W7, Sapporo 0608638, Japan. 2Department of Gynecology, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou 510060, People’s Republic of China. 3Department of Gynecology, Hokkaido University School of Medicine, Hokkaido University, N15, W7, Sapporo 0608638, Japan. 4Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USA. 5Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN 38163, USA. Our observation that upregulating tumor suppressive miR-137 and miR-34a via miRNA mimics restored tumor suppressor activity, with successful inhibition of OC cell viability and invasiveness, provided a rationale to investigate “miRNA replacement therapy for miR-137 and miR-34a”. However, there are concerns regarding potential toxicity and off-target effects in normal tissues. We showed here that the viability and apoptosis of nor- mal NOEC cells was not altered after transfection with miR-137 and miR-34a mimics, which display a high se- lectivity for killing OC cells. These findings may be ex- plained by several mechanisms [34]. Especially, large differences in miR-137 and miR-34a levels between normal NOEC cells and OC cells might account for the tolerance of NOEC cells to these two miRNAs. The in vivo anti-tumor efficacy and toxicity of miR-137 and miR-34a warrants further investigation. Received: 26 July 2016 Accepted: 1 September 2016 Received: 26 July 2016 Accepted: 1 September 2016 Availability of data and materials Not applicable. 12. Batlle E, Sancho E, Francí C, Domínguez D, Monfar M, Baulida J, et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol. 2000;2:84–9. Acknowledgements We thank Dr. Zhujie Xu for technical assistance. Funding g This work was supported by funding from the Department of Women’s Health Educational System, a Grant-in-Aid for Scientific Research (C) (15 K10697 and 16 K11123) and Science and Technology Planning Project of Guangdong Province, China (2013B021800155). 12. Batlle E, Sancho E, Francí C, Domínguez D, Monfar M, Baulida J, et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol. 2000;2:84–9. 12. Batlle E, Sancho E, Francí C, Domínguez D, Monfar M, Baulida J, et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol. 2000;2:84–9. 13. Kagara N, Huynh KT, Kuo C, Okano H, Sim MS, Elashoff D, et al. Epigenetic regulation of cancer stem cell genes in triple-negative breast cancer. Am J Pathol. 2012;181:257–67. Availability of data and materials Not applicable. Dong et al. Journal of Experimental & Clinical Cancer Research (2016) 35:132 33. Ma Y, Qin H, Cui Y. MiR-34a targets GAS1 to promote cell proliferation and inhibit apoptosis in papillary thyroid carcinoma via PI3K/Akt/Bad pathway. Biochem Biophys Res Commun. 2013;441:958–63. Availability of data and materials Availability of data and materials Not applicable. Not applicable. Page 9 of 9 Dong et al. Journal of Experimental & Clinical Cancer Research (2016) 35:132 14. Dong P, Karaayvaz M, Jia N, Kaneuchi M, et al. Mutant p53 gain-of-function induces epithelial-mesenchymal transition through modulation of the miR- 130b-ZEB1 axis. Oncogene. 2013;32:3286–95. 15. Dong P, Ihira K, Xiong Y, Watari H, Hanley SJ, Yamada T, et al. Reactivation of epigenetically silenced miR-124 reverses the epithelial-to-mesenchymal transition and inhibits invasion in endometrial cancer cells via the direct repression of IQGAP1 expression. Oncotarget. 2016;7:20260–70. repression of IQGAP1 expression. Oncotarget. 2016;7:20260–70. 16. Dong P, Kaneuchi M, Watari H, Sudo S, Sakuragi N. MicroRNA-106b modulates epithelial–mesenchymal transition by targeting TWIST1 in invasive endometrial cancer cell lines. Mol Carcinog. 2014;53:349–59. 17. Konno Y, Dong P, Xiong Y, Suzuki F, Lu J, Cai M, et al. MicroRNA-101 targets EZH2, MCL-1 and FOS to suppress proliferation, invasion and stem cell-like phenotype of aggressive endometrial cancer cells. Oncotarget. 2014;5:6049–62. 18. Zheng H, Kang Y. Multilayer control of the EMT master regulators. Oncogene. 2014;33:1755–63. 18. Zheng H, Kang Y. Multilayer co Oncogene. 2014;33:1755–63. 19. Chen K, Huang YH, Chen JL. Understanding and targeting cancer stem cells: therapeutic implications and challenges. Acta Pharmacol Sin. 2013;34:732–40. 20. Zhu LF, Hu Y, Yang CC, Xu XH, Ning TY, Wang ZL, et al. Snail overexpression induces an epithelial to mesenchymal transition and cancer stem cell-like properties in SCC9 cells. Lab Invest. 2012;92:744–52. 21. Ota I, Masui T, Kurihara M, Yook JI, Mikami S, Kimura T, et al. Snail-induced EMT promotes cancer stem cell-like properties in head and neck cancer cells. Oncol Rep. 2016;35:261–6. 22. Wang YL, Zhao XM, Shuai ZF, Li CY, Bai QY, Yu XW, et al. Snail promotes epithelial-mesenchymal transition and invasiveness in human ovarian cancer cells. Int J Clin Exp Med. 2015;8:7388–93. 23. Haslehurst AM, Koti M, Dharsee M, Nuin P, Evans K, Geraci J, et al. EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer. BMC Cancer. 2012;12:91. 24. Liu M, Lang N, Qiu M, Xu F, Li Q, Tang Q, et al. miR-137 targets Cdc42 expression, induces cell cycle G1 arrest and inhibits invasion in colorectal cancer cells. Int J Cancer. 2011;128:1269–79. 25. Shen H, Wang L, Ge X, Jiang CF, Shi ZM, Li DM, et al. MicroRNA-137 inhibits tumor growth and sensitizes chemosensitivity to paclitaxel and cisplatin in lung cancer. Oncotarget. 2016;7:20728–42. 26. Availability of data and materials Dong S, Jin M, Li Y, Ren P, Liu J. miR-137 acts as a tumor suppre 26. Dong S, Jin M, Li Y, Ren P, Liu J. miR-137 acts as a tumor suppressor in papillary thyroid carcinoma by targeting CXCL12. Oncol Rep. 2016;35:2151–8. 27. Misso G, Di Martino MT, De Rosa G, Farooqi AA, Lombardi A, Campani V, et al. Mir-34: a new weapon against cancer? Mol Ther Nucleic Acids. 2014;3:e194. 28. Li N, Fu H, Tie Y, Hu Z, Kong W, Wu Y, et al. miR-34a inhibits migration and invasion by down-regulation of c-Met expression in human hepatocellular carcinoma cells. Cancer Lett. 2009;275:44–53. 29. Ji Q, Hao X, Zhang M, Tang W, Yang M, Li L, et al. MicroRNA miR-34 inhibits human pancreatic cancer tumor-initiating cells. PLoS One. 2009;4:e6816. 30. Li Y, Guessous F, Zhang Y, Dipierro C, Kefas B, Johnson E, et al. MicroRNA- 34a inhibits glioblastoma growth by targeting multiple oncogenes. Cancer Res. 2009;69:7569–76. 31. Gallardo E, Navarro A, Viñolas N, Marrades RM, Diaz T, Gel B, et al. miR-34a as a prognostic marker of relapse in surgically resected non-small-cell lung cancer. Carcinogenesis. 2009;30:1903–9. 32. Tazawa H, Tsuchiya N, Izumiya M, Nakagama H. Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells. Proc Natl Acad Sci U S A. 2007;104: 15472–7. 33. Ma Y, Qin H, Cui Y. MiR-34a targets GAS1 to promote cell proliferation and inhibit apoptosis in papillary thyroid carcinoma via PI3K/Akt/Bad pathway. Biochem Biophys Res Commun. 2013;441:958–63. Submit your next manuscript to BioMed Central and we will help you at every step: 34. Bader AG, Brown D, Winkler M. The promise of microRNA replacement therapy. Cancer Res. 2010;70:7027–30. 34. Bader AG, Brown D, Winkler M. The promise of microRNA replacement therapy. Cancer Res. 2010;70:7027–30. 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https://openalex.org/W3133263129	https://www.biorxiv.org/content/biorxiv/early/2021/02/17/2021.02.17.431606.full.pdf	English	null	Measuring the unknown: an estimator and simulation study for assessing case reporting during epidemics	bioRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	11,118	. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Measuring the unknown: an estimator and simulation study for assessing case reporting during epidemics Short title: Estimating reporting of cases in disease outbreaks Christopher I Jarvis1,2, Amy Gimma1,2, Flavio Finger1,2,3, Tim P Morris4, Jennifer A Thompson1, Olivier le Polain de Waroux2,5, W John Edmunds1,2, Sebastian Funk1,2, Thibaut Jombart1,2,6,7 1 Department of Infectious Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, United Kingdom 2 Centre for Mathematical Modelling of Infectious Diseases, London School of Hygiene & Tropical Medicine, London, UK. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint 3 Epicentre, Paris, France 4 MRC Clinical Trials Unit at UCL, London, United Kingdom 5 Public Health England, London, United Kingdom 6 MRC Centre for Global Infectious Disease Analysis, Department of Infectious Disease Epidemiology, School of Public Health, Imperial College London, United Kingdom 7 UK Public Health Rapid Support Team, London, United Kingdom Corresponding authors: Christopher I Jarvis Department of Infectious Disease Epidemiology London School of Hygiene & Tropical Medicine Keppel Street London WC1E 7HT United Kingdom christopher.jarvis@lshtm.ac.uk Thibaut Jombart Department of Infectious Disease Epidemiology London School of Hygiene & Tropical Medicine Keppel Street London WC1E 7HT Measuring the unknown: an estimator and simulation study for assessing case reporting during epidemics Short title: Estimating reporting of cases in disease outbreaks Christopher I Jarvis1,2, Amy Gimma1,2, Flavio Finger1,2,3, Tim P Morris4, Jennifer A Thompson1, Olivier le Polain de Waroux2,5, W John Edmunds1,2, Sebastian Funk1,2, Thibaut Jombart1,2,6,7 1 Department of Infectious Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, United Kingdom 2 Centre for Mathematical Modelling of Infectious Diseases, London School of Hygiene & Tropical Medicine, London, UK. 3 Epicentre, Paris, France 4 MRC Clinical Trials Unit at UCL, London, United Kingdom 5 Public Health England, London, United Kingdom 6 MRC Centre for Global Infectious Disease Analysis, Department of Infectious Disease Epidemiology, School of Public Health, Imperial College London, United Kingdom 7 UK Public Health Rapid Support Team, London, United Kingdom Corresponding authors: Christopher I Jarvis Department of Infectious Disease Epidemiology London School of Hygiene & Tropical Medicine Keppel Street London WC1E 7HT United Kingdom christopher.jarvis@lshtm.ac.uk Thibaut Jombart Department of Infectious Disease Epidemiology London School of Hygiene & Tropical Medicine Keppel Street London WC1E 7HT Corresponding authors: Christopher I Jarvis Department of Infectious Disease Epidemiology London School of Hygiene & Tropical Medicine Keppel Street London WC1E 7HT United Kingdom christopher.jarvis@lshtm.ac.uk Thibaut Jombart Department of Infectious Disease Epidemiology London School of Hygiene & Tropical Medicine Keppel Street London WC1E 7HT United Kingdom thibautjombart@gmail.com 1 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . Abstract The fraction of cases reported, known as ‘reporting’, is a key performance indicator in an outbreak response, and an essential factor to consider when modelling epidemics and assessing their impact on populations. Unfortunately, its estimation is inherently difficult, as it relates to the part of an epidemic which is, by definition, not observed. We introduce a simple statistical method for estimating reporting, initially developed for the response to Ebola in Eastern Democratic Republic of the Congo (DRC), 2018-2020. This approach uses transmission chain data typically gathered through case investigation and contact tracing, and uses the proportion of investigated cases with a known, reported infector as a proxy for reporting. Using simulated epidemics, we study how this method performs for different outbreak sizes and reporting levels. Results suggest that our method has low bias, reasonable precision, and despite sub-optimal coverage, usually provides estimates within close range (5-10%) of the true value. Being fast and simple, this method could be useful for estimating reporting in real-time in settings where person-to-person transmission is the main driver of the epidemic, and where case investigation is routinely performed as part of surveillance and contact tracing activities. 2 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Author summary When responding to epidemics of infectious diseases, it is essential to estimate how many cases are not being reported. Unfortunately reporting, the proportion of cases actually observed, is difficult to estimate during an outbreak, as it typically requires large surveys to be conducted on the affected populations. Here, we introduce a method for estimating reporting from case investigation data, using the proportion of cases with a known, reported infector. We used simulations to test the performance of our approach by mimicking features of a recent Ebola epidemic in the Democratic Republic of the Congo. We found that despite some uncertainty in smaller outbreaks, our approach can be used to obtain informative ballpark estimates of reporting under most settings. This method is simple and computationally inexpensive, and can be used to inform the response to any epidemic in which transmission events can be uncovered by case investigation. 3 3 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Introduction The response to infectious disease outbreaks increasingly relies on the analysis of various data sources to inform operation in real time [1,2]. Outbreak analytics can be used to characterise key factors driving epidemics, such as transmissibility, severity, or important delays like the incubation period or the serial interval [2]. Amongst these factors, the amount of infections remaining undetected in the affected populations is a crucial indicator for assessing the state of an epidemic, and yet this quantity is often hard to estimate in real time [3–6]. Indeed, estimation of the overall proportion of individuals infected (attack rates) typically requires time-consuming serological surveys [7–9] which may not be achievable in resource-limited, large-scale emergencies such as the 2014-2016 Ebola virus disease (EVD) outbreak in West Africa [10], or the more recent EVD outbreak in Eastern provinces of the Democratic Republic of the Congo (DRC) [11,12]. As an alternative, one may attempt to quantify reporting, i.e. the proportion of all infections which result in notified cases. Unfortunately, this quantity is also hard to estimate, and usually requires the analysis of epidemiological and genomic data through complex methods for reconstructing transmission trees [13–15] or transmission clusters [16]. Such requirements can mean that by the time estimates are available, decisions have already been made, or the outbreak situation has changed [17–19]. Therefore, approaches for timely estimation of reporting to help inform decision making during a response are required. Methods for estimating reporting in real time should ideally exploit data which is routinely collected as part of the outbreak response. In diseases where dynamics are mostly governed by 4 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint person-to-person transmission, case investigation and contact tracing can be powerful tools for understanding past transmission events as well as detecting new cases as early as possible [11,20–23]. Estimating reporting from epidemiological links Our method exploits transmission chains derived from case investigation and contact tracing data. The rationale for the approach is to consider the proportion of cases with a known infector as a proxy for the proportion of infections (including asymptomatic but infectious individuals) reported. The proportion of cases with a known infector is by definition the proportion of infectors who were reported (Figure 1), so that the reporting probability can be estimated as π where is the number of secondary cases (infectees) with a known infector and π ︿= nk n + n k u nk nu is the number of secondary cases without a known infector. Methods We present the analytical derivation of our method of estimating reporting, defined as the proportion of cases actually notified during an outbreak. We then describe the simulation study, using the ADEMP (Aim, Data generating mechanism, Estimand, Methods, Performance measures) framework as described by Morris et al 2019 [24,25], used to evaluate the performance of the methods under various conditions. Introduction For vaccine-preventable diseases, contact tracing can also be used for designing ring vaccination strategies, as seen in recent EVD outbreaks in the DRC [11,20]. These data also contain information about reporting. Intuitively, the frequency of cases whose infector is a known and reported case is indicative of the level of reporting: the more frequently case investigation identifies a known infector, the higher the corresponding case reporting should be. Conversely, cases with no known epidemiological link after investigation are indicative of unobserved infections, and therefore under-reporting. In this article, we introduce a method to estimate case reporting from contact tracing data. This approach, designed during the Ebola outbreak in Eastern DRC [11,12], was originally aimed at assessing case reporting in a context where insecurity made surveillance difficult, and under-reporting likely [12]. The approach utilized transmission chain data and calculated the proportion of cases with a known epidemiological link as a proxy for reporting. We provide a derivation of the estimator and explain the rationale of this approach and assess its performance using simulated outbreaks of different sizes with varying levels of reporting. Based on the simulation results, we make some suggestions regarding the use of this method to inform strategic decision making during an outbreak response. In this article, we introduce a method to estimate case reporting from contact tracing data. This approach, designed during the Ebola outbreak in Eastern DRC [11,12], was originally aimed at assessing case reporting in a context where insecurity made surveillance difficult, and under-reporting likely [12]. The approach utilized transmission chain data and calculated the proportion of cases with a known epidemiological link as a proxy for reporting. We provide a derivation of the estimator and explain the rationale of this approach and assess its performance using simulated outbreaks of different sizes with varying levels of reporting. Based on the simulation results, we make some suggestions regarding the use of this method to inform strategic decision making during an outbreak response. 5 5 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint We define - number of reported infectors mr - number of unreported infectors mu - number of secondary cases (infectees) with known infector nk - number of secondary cases without known infector nu - number of reported infectors mr - number of unreported infectors mu - number of secondary cases (infectees) with known infector nk - number of secondary cases without known infector nu - number of reported infectors mr - number of reported infectors mr - number of unreported infectors mu - number of secondary cases (infectees) with known infector nk - number of secondary cases without known infector nu 6 6 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint - reproduction number, i.e. average number of secondary cases by case R CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m The copyright holder for this prepr this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Uncertainty for reporting The uncertainty associated with this estimation can be estimated using various methods for computing confidence intervals of proportions. Using the standard approach for estimating standard errors for a proportion we have E . S π = n +n k u π×(1 −π) ︿︿ E . S π = n +n k u π×(1 −π) ︿︿ Here, we used exact binomial confidence intervals which can be calculated: Here, we used exact binomial confidence intervals which can be calculated: 1 ) π (1 ) ( + n − n +1 k n F[ ; 2n ,2(n − n +1)] k 2 α k k −1 < < + n − nk n F[1 − ; 2(n +1), 2(n − n )] k 2 α k k −1 Where , total number of secondary cases. is the c quantile from an n n n = k + u (c; , ) F d1 d2 F-distribution with degrees of freedom and is the confidence level. , d1 d2 α 1 − Where , total number of secondary cases. is the c quantile from an n n n = k + u (c; , ) F d1 d2 F distribution with degrees of freedom and is the confidence level d d 1 Where , total number of secondary cases. is the c quantile from an n n n = k + u (c; , ) F d1 d2 F-distribution with degrees of freedom and is the confidence level. , d1 d2 α 1 − F-distribution with degrees of freedom and is the confidence level. , d1 d2 α 1 − F-distribution with degrees of freedom and is the confidence level. , d1 d2 α 1 − - reproduction number, i.e. average number of secondary cases by case R - reproduction number, i.e. average number of secondary cases by case R - reporting probability following some unspecified probability distribution with unknown π probability parameter such that where secondary cases are assumed to follow E(π) = mr m + m r u the same reporting distribution as primary infections. porting probability following some unspecified probability distribution with unknown Let be the total number of secondary cases following an unspecified probability nu + nk distribution so that . R E(n ) (m ) u + nk = r + mu Let be the total number of secondary cases following an unspecified probability nu + nk distribution so that . R E(n ) (m ) u + nk = r + mu Then the expected number of reported infectees with a known infector is Then the expected number of reported infectees with a known infector is . Rπ E(n ) m k = r . Rπ E(n ) m k = r . Rπ E(n ) m k = r Similarly, the expected number of reported infectees without a known infector is Similarly, the expected number of reported infectees without a known infector is Similarly, the expected number of reported infectees without a known infector is Rπ E(n ) m u = u Rπ E(n ) m u = u Rπ E(n ) m u = u Rπ E(n ) m u = u From this we have that m and m r = Rπ E(n ) k u = Rπ E(n ) u By definition π = mr m +m r u Therefore (π) E = Rπ E(n ) k Rπ E(n ) + E(n ) k u = E(n ) k E(n ) + E(n ) k u From this we have that m and m r = Rπ E(n ) k u = Rπ E(n ) u By definition π = mr m +m r u Therefore (π) E = Rπ E(n ) k Rπ E(n ) + E(n ) k u = E(n ) k E(n ) + E(n ) k u and replacing the expectations with their estimates from the data, we get the estimator and replacing the expectations with their estimates from the data, we get the estimator . π ︿= nk n + n k u 7 . Aim We aim to test the performance of the method for different outbreak sizes and actual reporting, in terms of bias, coverage, and precision (in an operational context) using simulated outbreaks. Data generating mechanism We considered twelve data-generating mechanisms (three reporting rates by four reported outbreaks sizes) and performed 4000 repetitions per mechanism. Each repetition corresponded to a hypothetical outbreak with a known transmission tree. To simulate the reporting process, cases were removed randomly from the transmission chains 8 8 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint using a Binomial process with a probability (1 - reporting). We will thus distinguish the total outbreak size, which represents all cases in the outbreak, and the reported outbreak size, which represents the number of cases reported. For simplicity, we assumed that all cases reported were investigated, so that it is known if they had a documented epidemiological link, or not, amongst reported cases. For each outbreak (repetition) we removed observations so that reporting was 25%, 50%, or 75%. Therefore a single simulated outbreak will give three different observed outbreaks. We categorised the simulations into reported outbreak sizes of 1-99, 1-499, 500-999, 1000+. Λt = (ns / n) ∑i λi,t Outbreak simulation We used the R package simulacr [26] to simulate outbreaks, the reporting process, and the subsequently observed transmission chains. simulacr implements and extends individual-based simulations of epidemics previously used to evaluate transmission tree reconstruction methods [13,14,27]. A Poisson branching process is used to generate new cases in time (with daily time-steps), drawing from a distribution for the reproduction number (R) and the infectious period to determine rates of infection. The infectiousness of a given individual i at time t is, noted λi,t, is calculated as: λi,t = Ri w(t - si) where Ri is the reproduction number for individual i, si is their date of symptom onset, and w is the probability mass function of the duration of infectiousness (time interval between onset of symptom and new secondary infections). New cases generated at time t+1 are drawn from a Poisson distribution with a rate Λt summing the infectiousness of all cases: Λt = (ns / n) ∑i λi,t 9 9 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint where ns is the number of susceptible individuals and n the total population size, so that the branching process includes a density-dependence in which rates of infection decrease with the proportion of susceptibles. Estimand: Reporting We considered a single estimand the level of reporting. π We considered a single estimand the level of reporting. π where ns is the number of susceptible individuals and n the total population size, so that the branching process includes a density-dependence in which rates of infection decrease with the proportion of susceptibles. Transmission trees are built by assigning infectors to newly infected individuals according to a multinomial distribution in which potential infectors have a probability λi,t / ∑i λi,t of being drawn. The dates of symptom onset and case notification are generated for each new case using user-provided distributions for the incubation time and reporting delays. Simulations run until any of the set duration of the simulation is reached (here, 365 days). Here, we used parameters values and distributions in line with estimates from the Eastern DRC Ebola outbreak [12,28], the details of which are provided in Table 1. All code used for running these simulations is available from https://github.com/jarvisc1/2020-reporting. Method For each repetition we calculated the proportion of the number of cases with a known infector over the total number of reported cases, that is the estimator We further calculated . π ︿= nk n + n k u the standard error and 95% exact binomial confidence intervals. the standard error and 95% exact binomial confidence intervals. 10 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Performance measures The performance of the method was measured using bias, coverage, and precision. For bias and coverage, the Monte-Carlo standard errors were calculated to quantify uncertainty about the estimates of the performance [29]. The equations used are detailed in Table 2 and were taken from Morris et al [24]. In addition, results were classified according to different ranges of absolute error, for a more operational interpretation of the results. Bias is the difference between the expected value and the true value. It was measured by taking the difference between the average estimate of reporting versus the true reporting. Unbiasedness is a desirable statistical quality but a small amount of bias may be tolerated in exchange for other desirable qualities of an estimator. The estimates of reporting were presented visually by displaying the estimates of all 4000 simulations for each scenario. Coverage is the percentage of CIs containing the true value. In the case of a 95% CI this should contain the true value 95% of the time. We counted the number of repetitions where the true value was contained in the 95% CI and divided by the total number of repetitions. The coverage was visualised through the use of Zip plots. This new visualisation created by Morris et al [24], helps to assess the coverage of a method by viewing the CIs directly. Assessing an expected 95% coverage with a Monte-Carlo standard error of 0.35 requires 3877 simulations [24] which is well within our 4000 simulations . 11 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Precision represents how close the estimates are to each other. The model based and empirical standard error were also calculated to provide an indication of precision. The model based standard error is mean of the square of the bias, and the empirical standard error represents the spread of the estimates. Performance measures This gives an indication of how much the point estimates vary across simulations based on the level of reporting and sample size. Although the method may give unbiased estimates with good coverage under repeated sampling, an imprecise method could lead to large differences from the true value when applied to a single dataset (that is, confidence intervals may cover the true value honestly but are wide). We further explored the impact of bias and precision of the estimator by considering the deviations of the estimates from the true value termed absolute error. The absolute error is defined as the absolute difference between the estimated reporting and its true value, We further explored the impact of bias and precision of the estimator by considering the deviations of the estimates from the true value termed absolute error. The absolute error is defined as the absolute difference between the estimated reporting and its true value, expressed as percentages. For instance, estimates of 43% and 62% for a true reporting of 50% would correspond to absolute errors of 7% and 12%, respectively. During a disease outbreak, decisions are frequently made in the face of large uncertainties, and small absolute differences in the estimated level of reporting are unlikely to result in strategic changes. Therefore, as a perhaps more operationally relevant metric, we categorised results according to how far from the true value estimates were, using an arbitrary scale: very close (≤5% absolute error), close (≤10%), approximate (≤15%) or inaccurate (≤20%). 12 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Bias There was very little bias across all the simulated scenarios (Table 3 and Figure 2). For outbreaks with over 100 cases all estimates of bias were 0 with decreasing Monte Carlo error from 0.04 to 0.01 as the size of the reported outbreak increased. For outbreaks reported as less than 100 cases the bias was -0.1 for reporting of 0.50 and 0.75 and 0 for 0.25 with Monte Carlo error of 0.07. Table 3 presents the bias for each scenario and it can be seen that all of these estimates were within one standard error from zero, suggesting reasonable confidence that this is an overall unbiased estimator. There was very little bias across all the simulated scenarios (Table 3 and Figure 2). For outbreaks with over 100 cases all estimates of bias were 0 with decreasing Monte Carlo error from 0.04 to 0.01 as the size of the reported outbreak increased. For outbreaks reported as less than 100 cases the bias was -0.1 for reporting of 0.50 and 0.75 and 0 for 0.25 with Monte Carlo error of 0.07. Table 3 presents the bias for each scenario and it can be seen that all of these estimates were within one standard error from zero, suggesting reasonable confidence that this is an overall unbiased estimator. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Precision The model based standard error was below 0.07 for all estimates and below 0.04 for reported outbreaks of over 100 cases. Similar patterns are seen for the empirical standard error. Imprecise estimates were most marked when reported outbreaks were less than 100 cases and had 0.75 reporting. The precision increased (model based and empirical standard error decreased) as the reported outbreak size increased (Figure2, Table 3). Overall the precision appears reasonable when outbreaks are larger than 100. Coverage The coverage varied across the simulated scenarios with all but reported outbreak size 10-99 with reporting at 0.25 displaying under-coverage (Figure 3). The coverage was poor with all coverage estimates more than one standard error away from 95%, and most several standard errors away (Table 3). There was some suggestion of the counterintuitive pattern that coverage decreased as the reporting increased and that coverage decreased as the outbreak size increased. 13 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Absolute error Results showed that the estimates were rarely more than 15% away from the true reporting value in all simulation settings (Figure 4, Table 4). The absolute error was negligible in all larger reported outbreaks (500 cases and above), with the nearly all estimates very close (within 5%) to the true reporting value. Performance decreased in smaller outbreaks, but most estimates remained close (within 10%) to the true value. Results were worse in smaller outbreaks (10-99 reported cases), but even there about half of the estimates were very close (within 5%) to the true value, and more than 80% of estimates were within 10% of the target. 14 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Discussion We have presented a new estimator for the levels of reporting in an outbreak based on the proportion of cases with known infectors, which can be derived from case investigation data. Using simulated outbreaks to assess the performance of the method, we found that this approach generally had little bias, reasonable precision, but poor coverage. Across all simulations, estimated reporting was most often within 10% of the true value, suggesting the method will retain operational relevance under different settings. Simulation results indicate a first limitation of the method lies in the analysis of smaller outbreaks. Overall, the approach performed better in larger outbreaks, with all metrics pointing to improved results in outbreaks of more than 100 reported cases. Our approach also assumes a uniform sampling of the transmission tree over the time period on which the analysis is carried. It would in theory be prone to under-estimating reporting when entire branches of the transmission tree remain unobserved. For instance, if an epidemic is spreading in a location where surveillance is totally absent, a substantial number of cases may remain unnoticed, and such under-reporting would not be accounted for in our estimates. As a consequence, our method is best applied to geographic areas over which surveillance and case investigation efforts do not vary drastically. We also assumed that the reproduction number (R) was independent from the reporting process, so that reported source cases cause the same average number of secondary cases as non-reported ones. This condition may not always be met, for instance if unreported individuals 15 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint tend to cause more super-spreading events. In the context of Ebola, this may occur through community deaths, in which funeral exposure of a large number of relatives may give rise to a new cluster of cases from a single, unreported source case. Discussion ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint These methodologies are also much more complex and computer-intensive, as they either involve the reconstruction of transmission trees [13,14] or of outbreak clusters [16]. In contrast, the approach introduced here is fast and simple, and can be used in real time to estimate reporting based on data routinely collected as part of contact tracing activities and surveillance. We evaluated the performance of the method using simulated EVD outbreaks in line with estimates of transmissibility and epidemiological delays of the Eastern DRC Ebola epidemic [12,28], as this was the original context in which the method was developed. Further work should be devoted to investigating the method’s performance for other diseases and different epidemic contexts. In particular, it would be interesting to study the potential impact of correlations between transmissibility and under-reporting, i.e. situations in which non-reported cases may exhibit increased infectiousness and cause super-spreading events. Discussion Under such circumstances, we would expect our method to under-estimate reporting, although this should be further quantified by dedicated simulation studies. Another limitation of our method relates to data availability and quality. Our approach relies on case investigation data, a time-consuming process usually requiring interviews of patients and/or their close relatives. There are several possible outcomes from such investigation: i) a single infector can be identified amongst reported cases ii) a single infector can be identified, but is not amongst reported cases iii) there are several likely infectors, possibly mixing reported and unreported cases iv) likely infector(s) could not be identified. Our approach requires case investigations to fall within the first two categories. In our simulations, we assumed for simplicity that all reported cases were successfully investigated, so that the reported outbreak size effectively corresponds to the number of data points available for the estimation. In practice, the actual sample size will be the number of case investigations which led to identifying a single source case (reported, or not). As our method performs better in larger datasets (e.g. more than 100 cases successfully investigated), the data requirement for estimating reporting from transmission chains will involve substantial field work. Unfortunately, alternative approaches for estimating under-reporting are very demanding in terms of data, typically needing to combine information on dates of onset, location of the cases, full genome sequences of the pathogen for nearly all cases, good prior knowledge on key delays (e.g. incubation period, serial interval) [13,16], and ideally contact tracing data [14]. 16 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. Conclusion In this paper, we provided a derivation of a straightforward and pragmatic estimator to real-time estimation of case reporting in outbreak settings, and tested this approach under a range of simulated conditions. The method exhibited little bias, reasonable precision, and while coverage was suboptimal under some settings (in large outbreaks with higher reporting), most estimates were within reasonable range (10-15%) of the true value. This suggests the method will be useful for informing the response to outbreaks in which person-to-person transmission is the main driver of transmission, and where enough (ideally > 100) chains of transmissions can be retraced through epidemiological investigation. 17 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Acknowledgements TJ, CIJ and WJE receive funding from the Global Challenges Research Fund (GCRF) project ‘RECAP’ managed through RCUK and ESRC (ES/P010873/1). TJ and OPW receive funding from the UK Public Health Rapid Support Team funded by the United Kingdom Department of Health and Social Care. TJ receives funding from the National Institute for Health Research - Health Protection Research Unit for Modelling Methodology. The authors alone are responsible for the views expressed in this article and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated. TPM was funded by the UK MRC (grants MC_UU_12023/21 and MC_UU_12023/29). JAT was jointly funded by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement and is also part of the EDCTP2 programme supported by the European Union (MR/R010161/1) The UK Public Health Rapid Support Team is funded by the United Kingdom Department of Health and Social Care. The views expressed in this publication are those of the authors and not necessarily those of the National Health System, the National Institute for Health Research or the Department of Health and Social Care. We are thankful to the R Epidemics Consortium (RECON) for developing free software tools used in the simulations studies. 18 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint (In alphabetic order) Formal Analysis, software and visualization: AG, CIJ, TJ All authors read and approved the final version of the manuscript. 19 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Figures Figure 1: rationale of the method for estimating reporting. This diagram illustrates e 1: rationale of the method for estimating reporting. This diagram illustrates transmission events inferred by case investigation of reported secondary cases, with arrows pointing from infectors to infectees. Darker shades are used to indicate documented transmission events, while lighter shades show unknown infectors. Numbers of secondary cases with (blue) or without (orange) known infectors are used to estimate the reporting probability. This example uses an approximate reporting of 50%. 20 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Figure 2: comparison of estimated versus actual reporting. This graph shows the results of reporting estimated by the method for 4000 simulated outbreaks, broken down by outbreak size category (y-axis). Each dot corresponds to an independent simulation. The vertical red bars indicate the average within each category. True reporting used in the simulations is indicated by colors. Figure 2: comparison of estimated versus actual reporting. This graph shows the results of reporting estimated by the method for 4000 simulated outbreaks, broken down by outbreak size category (y-axis). Each dot corresponds to an independent simulation. The vertical red bars indicate the average within each category. True reporting used in the simulations is indicated by colors. 21 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. (In alphabetic order) It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Fi 3 Zi l f h i l Thi h h h 9 % fid Figure 3: Zip plot of showing coverage results. This graph shows the 95% confidence Figure 3: Zip plot of showing coverage results. This graph shows the 95% confidence Figure 3: Zip plot of showing coverage results. This graph shows the 95% confidence intervals estimated by the method, broken down by reported outbreak size category and true reporting value. The vertical axis represent the fractional centile of where and Z\| \| Z = SEi (π −π) i π is reporting. The confidence intervals are ranked by their level of coverage and thus the vertical axis can be used to determine the proportion of confidence intervals that contain the true value where 0.95 would represent a coverage of 95%. 22 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Figure 4: Absolute error in reporting estimation. This graph shows, for different simulation settings, the proportion of results within a given margin of absolute error, expressed as the absolute difference between the true and the estimated reporting (in %). Rows correspond to different outbreak size categories (outbreak size as reported) True reporting is indicated in Figure 4: Absolute error in reporting estimation. This graph shows, for different simulation settings, the proportion of results within a given margin of absolute error, expressed as the absolute difference between the true and the estimated reporting (in %). Rows correspond to different outbreak size categories (outbreak size as reported) True reporting is indicated in Figure 4: Absolute error in reporting estimation. (In alphabetic order) This graph shows, for different simulation settings, the proportion of results within a given margin of absolute error, expressed as the absolute difference between the true and the estimated reporting (in %). Rows correspond to different outbreak size categories (outbreak size as reported). True reporting is indicated in color. Figure 4: Absolute error in reporting estimation. This graph shows, for different simulation settings, the proportion of results within a given margin of absolute error, expressed as the absolute difference between the true and the estimated reporting (in %). Rows correspond to different outbreak size categories (outbreak size as reported). True reporting is indicated in color. Figure 4: Absolute error in reporting estimation. This graph shows, for different simulation settings, the proportion of results within a given margin of absolute error, expressed as the absolute difference between the true and the estimated reporting (in %). Rows correspond to different outbreak size categories (outbreak size as reported). True reporting is indicated in color. 23 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Tables P ︿ low ≤ ≤ ︿ upp = . (θ θ θ ) P ︿ low ≤ ≤ ︿ upp Precision Model based standard error √V ar(θ) Empirical based sstandard error E[(θ θ) ] ︿ − 2 Absolute error \| \|θi ˆ −θ Table 2: Metrics used to measure performance in the simulation study. Performance measure Definition Bias where is the true value and is the estimate of value E[θ] θ δ = ︿ − θ θ ︿ Coverage If we define a confidence interval as the θ , θ ) ( ︿ low ︿ upp where then a 95% CI is when (θ θ θ ) ψ P ︿ low ≤ ≤ ︿ upp = 0, 1] ψ ∈[ It follows that coverage is the (θ θ θ ) 0.95. P ︿ low ≤ ≤ ︿ upp = . (θ θ θ ) P ︿ low ≤ ≤ ︿ upp Tables Table 1: Parameters used for simulating outbreaks. This table details input parameters used for simulating outbreaks using the R package simulacr. Fixed values were used for all simulations, and reflect the natural history of the 2018-2020 Eastern DRC Ebola outbreak. Variable values changed across simulations. Population size is controlled in each simulation, the outbreak sizes are determined after the outbreaks have been simulated and the proportion of cases not reported have been removed. Fixed values Maximum duration of the outbreak 365 days Incubation time distribution Discretised gamma distribution mean of 9.7 days, sd = 5.5 days. Infectious period distribution Discretised gamma distribution mean = 5 days, sd = 4.7 days. Reproduction number distribution Gamma distribution: rate of 1.2 shape of 2. Variable values Population size 200, 500, 1000, 2000, 5000, 7500, 10000, 15000, 20000 Outbreak size* 10-99, 100-499, 500-999, 1000+ Proportion of cases not reported 0.25, 0.50, 0.75 Table 1: Parameters used for simulating outbreaks. This table details input parameters used for simulating outbreaks using the R package simulacr. Fixed values were used for all simulations, and reflect the natural history of the 2018-2020 Eastern DRC Ebola outbreak. Variable values changed across simulations. outbreaks have been simulated and the proportion of cases not reported have been removed. 24 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Table 2: Metrics used to measure performance in the simulation study. Performance measure Definition Bias where is the true value and is the estimate of value E[θ] θ δ = ︿ − θ θ ︿ Coverage If we define a confidence interval as the θ , θ ) ( ︿ low ︿ upp where then a 95% CI is when (θ θ θ ) ψ P ︿ low ≤ ≤ ︿ upp = 0, 1] ψ ∈[ It follows that coverage is the (θ θ θ ) 0.95. Table 2: Metrics used to measure performance in the simulation study. If we define a confidence interval as the θ , θ ) ( ︿ low ︿ upp where then a 95% CI is when (θ θ θ ) ψ P ︿ low ≤ ≤ ︿ upp = 0, 1] ψ ∈[ It follows that coverage is the (θ θ θ ) 0.95. P ︿ low ≤ ≤ ︿ upp = . (θ θ θ ) P ︿ low ≤ ≤ ︿ upp 25 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Table 3: Performance measures from 4000 simulation by reported outbreak size and true reporting level. Estimate (Monte-carlo standard error) Reported outbreak size Performance measures (MCSE) Proportion reported 10-99 100-499 500-999 1000 or more Bias 0.25 0 (0.07) 0 (0.03) 0 (0.02) 0 (0.01) 0.5 -0.01 (0.07) 0 (0.04) 0 (0.02) 0 (0.01) 0.75 -0.01 (0.07) 0 (0.04) 0 (0.02) 0 (0.01) Coverage 0.25 95.7% (0.3) 94.1% (0.4) 94.4% (0.4) 93% (0.4) 0.5 92.6% (0.4) 92.4% (0.4) 91.3% (0.4) 91.2% (0.4) 0.75 92.3% (0.4) 91.5% (0.4) 89.2% (0.5) 88.6% (0.5) Model standard error 0.25 0.065 (0) 0.024 (0) 0.015 (0) 0.01 (0) 0.5 0.061 (0) 0.038 (0) 0.019 (0) 0.011 (0) 0.75 0.059 (0.001) 0.036 (0) 0.014 (0) 0.011 (0) Empirical standard error 0.25 0.071 (0.001) 0.025 (0) 0.016 (0) 0.01 (0) 0.5 0.07 (0.001) 0.044 (0) 0.022 (0) 0.012 (0) 0.75 0.068 (0.001) 0.043 (0) 0.017 (0) 0.013 (0) Table 3: Performance measures from 4000 simulation by reported outbreak size and true reporting level. Estimate (Monte-carlo standard error) 26 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . Table 2: Metrics used to measure performance in the simulation study. CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint Table 4: Comparison of absolute error from 4000 simulations between true reporting levels and estimate of reporting by reported outbreak size and true reporting level Absolute error from true value Proportion reported Reported outbreak size ≤ 5% ≤ 10% ≤ 15% ≤ 20% 0.25 10-99 2213 (55.3%) 3376 (84.4%) 3849 (96.2%) 3973 (99.3%) 100-499 3817 (95.4%) 4000 4000 4000 500-999 3995 (99.9%) 4000 4000 4000 1000+ 3999 (100%) 4000 4000 4000 0.5 10-99 2110 (52.8%) 3430 (85.8%) 3860 (96.5%) 3978 (99.4%) 100-499 2981 (74.5%) 3899 (97.5%) 3998 (100%) 4000 500-999 3905 (97.6%) 4000 4000 4000 1000+ 4000 4000 4000 4000 0.75 10-99 2400 (60%) 3575 (89.4%) 3835 (95.9%) 3942 (98.6%) 100-499 3067 (76.7%) 3890 (97.2%) 3991 (99.8%) 4000 500-999 3988 (99.7%) 4000 4000 4000 1000+ 3992 (99.8%) 4000 4000 4000 Table 4: Comparison of absolute error from 4000 simulations between true re omparison of absolute error from 4000 simulations between true reporting Table 4: Comparison of absolute error from 4000 simulations between true reporting levels and estimate of reporting by reported outbreak size and true reporting level 27 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 17, 2021. ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint References ; https://doi.org/10.1101/2021.02.17.431606 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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https://openalex.org/W4310827943	https://repository.library.noaa.gov/view/noaa/48925/noaa_48925_DS1.pdf	English	null	Assessing the consistency of satellite-derived upper tropospheric humidity measurements	Atmospheric measurement techniques	2,022	cc-by	12,187	Correspondence: Lei Shi (lei.shi@noaa.gov) Correspondence: Lei Shi (lei.shi@noaa.gov) Received: 1 July 2022 – Discussion started: 20 July 2022 Received: 1 July 2022 – Discussion started: 20 July 2022 Revised: 19 October 2022 – Accepted: 24 October 2022 – Published: 2 December 2022 Revised: 19 October 2022 – Accepted: 24 October 2022 – Published: 2 December 2022 Abstract. Four upper tropospheric humidity (UTH) datasets derived from satellite sounders are evaluated to assess their consistency as part of the activities for the Global Energy and Water Exchanges (GEWEX) water vapor assessment project. The datasets include UTH computed from bright- ness temperature measurements of the 183.31±1 GHz chan- nel of the Special Sensor Microwave – Humidity (SSM/T- 2), Advanced Microwave Sounding Unit-B (AMSU-B), and Microwave Humidity Sounder (MHS) and from channel 12 of the High-resolution Infrared Radiation Sounder (HIRS). The four datasets are generally consistent in the interannual temporal and spatial variability of the tropics. Large positive anomalies peaked over the central equatorial Paciﬁc region during El Niño events in the same phase with the increase of sea surface temperature (SST). Conversely, large nega- tive anomalies were obtained during El Niño events when the tropical-domain average is taken. The weakened ascend- ing branch of the Paciﬁc Walker circulation in the western Paciﬁc and the enhanced descending branches of the local Hadley circulation along the Paciﬁc subtropics largely con- tributed to widespread drying areas and thus negative anoma- lies in the upper troposphere during El Niño events as shown in all four datasets. During a major El Niño event, UTH had higher correlations with the coincident precipitation (0.60 to 0.75) and with 200 hPa velocity potential (−0.42 to −0.64) than with SST (0.37 to 0.49). Due to differences in retrieval deﬁnitions and gridding procedures, there can be a difference of 3 %–5 % UTH between datasets on average, and larger magnitudes of anomaly values are usually observed in spa- tial maps of microwave UTH data. Nevertheless, the tropical- domain averaged anomalies of the datasets are close to each other with their differences being mostly less than 0.5 %, and more importantly the phases of the time series are generally consistent for variability studies. than with SST (0.37 to 0.49). Correspondence: Lei Shi (lei.shi@noaa.gov) Due to differences in retrieval deﬁnitions and gridding procedures, there can be a difference of 3 %–5 % UTH between datasets on average, and larger magnitudes of anomaly values are usually observed in spa- tial maps of microwave UTH data. Nevertheless, the tropical- domain averaged anomalies of the datasets are close to each other with their differences being mostly less than 0.5 %, and more importantly the phases of the time series are generally consistent for variability studies. Atmos. Meas. Tech., 15, 6949–6963, 2022 https://doi.org/10.5194/amt-15-6949-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Atmos. Meas. Tech., 15, 6949–6963, 2022 https://doi.org/10.5194/amt-15-6949-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Assessing the consistency of satellite-derived upper tropospheric humidity measurements Lei Shi1, Carl J. Schreck III2, Viju O. John3, Eui-Seok Chung4, Theresa Lang5,6, Stefan A. Buehler5, and Brian J Soden7 Lei Shi1, Carl J. Schreck III2, Viju O. John3, Eui-Seok Chung4, Theresa Lang5,6, Stefan A. Buehler5, and Brian J. Soden7 1NOAA NESDIS National Centers for Environmental Information (NCEI), Asheville, NC, USA 2Cooperative Institute for Satellite Earth System Studies (CISESS), North Carolina State University, Asheville, NC, USA 3EUMETSAT and Met Ofﬁce Hadley Centre, Exeter, UK 4Korea Polar Research Institute, Incheon, South Korea 1NOAA NESDIS National Centers for Environmental Information (NCEI), Asheville, NC, USA 2Cooperative Institute for Satellite Earth System Studies (CISESS), North Carolina State University, Asheville, NC, USA 3EUMETSAT and Met Ofﬁce Hadley Centre, Exeter, UK 4Korea Polar Research Institute, Incheon, South Korea 5 g g g y 6International Max Planck Research School on Earth System Modelling, Max Planck Institute for Meteorology, Hamburg, Germany 7Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL, USA 7Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL, USA 1 Introduction The Global Energy and Water Exchanges (GEWEX) project’s water vapor assessment (G-VAP) is organized by the GEWEX Data and Analysis Panel. Three Global Cli- mate Observing System (GCOS) essential climate variables on water vapor are assessed in the G-VAP project, includ- ing total column water vapor, upper tropospheric humidity (UTH), and water vapor and associated temperature proﬁles. The present study is part of the G-VAP activities, focusing on the consistency assessment among satellite-derived UTH measurements. Measurement of UTH has traditionally been obtained from global radiosonde observations as part of atmospheric water L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity 6950 vapor proﬁles (e.g., Durre et al., 2018; Ferreira et al., 2019; Brogniez et al., 2015). In the satellite era, operational rou- tine satellite infrared measurements of UTH started with the High-resolution Infrared Radiation Sounder (HIRS) in- strument on board Television InfraRed Observation Satel- lite N (TIROS-N), which was launched in 1978, and the measurement has been continuously produced from the Na- tional Oceanic and Atmospheric Administration (NOAA) and Meteorological Operational satellite (Metop) polar- orbiting satellites to the present. UTH measurements from geostationary observations have been generated since 1983. Then microwave sounding measurements have been added to the suite of UTH observations since 1991. UTH can also be derived from the new-generation hyper-spectral sounders including the Atmospheric Infrared Sounder (AIRS), In- frared Atmospheric Sounding Interferometer (IASI), and Cross-track Infrared Sounder (CrIS), as well as other satel- lite instruments such as Sondeur Atmosphérique du Proﬁl d’Humidité Intertropicale par Radiométrie (SAPHIR). These satellite sounder measurements complement each other in providing a long-term full picture of the UTH ﬁeld. both microwave and infrared sounders are used together with ground-based observations and climate model simulations to examine global-scale changes in water vapor and response to surface temperature variability (Allan et al., 2022). Water vapor is an important greenhouse gas. Its concen- tration in the free troposphere is controlled by condensation at the cold point and subsequent advection. This leads to a roughly constant relative humidity, which implies a strong increase in absolute humidity content with warming (So- den et al., 2005; Chung et al., 2014). This well-understood overall picture is modulated by subtle changes in the dis- tribution of humidity, as measured by the UTH, linked to changes in atmospheric dynamics with warming (Held and Soden, 2000). Intercomparison of independently generated UTH datasets provides veriﬁcation of the datasets’ credibility for their uses in research and long-term monitoring. An earlier consis- tency study (Chung et al., 2016) analyzed UTH derived from HIRS, Advanced Microwave Sounding Unit-B (AMSU- B)/Microwave Humidity Sounder (MHS), and AIRS and showed that all three products exhibit consistent spatial and temporal patterns of interannual variability. The ﬁrst phase of the GEWEX UTH assessment (Schröder et al., 2017) included UTH derived from polar-orbiting HIRS, AMSU- B/MHS, and the geostationary Meteosat visible and infrared imager (MVIRI) and Spinning Enhanced Visible and In- fraRed Imager (SEVIRI). L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity Since then, two new polar-orbiting satellite microwave UTH datasets have been developed, and there are now new versions and extended records available for the HIRS and the microwave dataset examined previ- ously. In this study we provide an update on the intercompar- ison of the polar-orbiting satellite UTH datasets by includ- ing four participating datasets, two of which are new datasets and two of which have updated versions and extended time series. The development of UTH datasets and the examination of temporal and spatial variabilities of UTH have been pre- sented in numerous studies, including both infrared datasets (Soden and Bretherton, 1993; Jackson and Bates, 2001; Brogniez et al., 2006; Shi and Bates, 2011; Iacono et al., 2003; Chung et al., 2007; Gierens et al., 2014; Schröder et al., 2014; Gierens et al., 2018) and microwave datasets (Brogniez and Pierrehumbert, 2006; Chung et al., 2013; Sohn et al., 2000; Buehler et al., 2008; Lang et al., 2020b; Brog- niez et al., 2015; Moradi et al., 2016). The variability of UTH is regulated by the large-scale atmospheric circulation. The spatial patterns of UTH measurement are highly correlated with widely used climate indices such as the Niño 3.4, Paciﬁc Decadal Oscillation (PDO), Paciﬁc–North American (PNA), and North Atlantic Oscillation (NAO) indices (Shi and Bates, 2011; Shi et al., 2018). The measurements have been applied in various atmospheric variability studies. For example, UTH datasets facilitated studies that showed a strong relationship between UTH and El Niño–Southern Oscillation (ENSO) (Mccarthy and Toumi, 2004; Bates et al., 1996; Soden and Lanzante, 1996). UTH was closely associated with deep con- vection and the evolution of large-scale weather systems (So- den and Fu, 1995; Brogniez et al., 2009; Zelinka and Hart- mann, 2009; Luo et al., 2007; Schreck et al., 2013) and in- teracting with the tropical cirrus life cycle (Luo and Rossow, 2004). The measurements have been used in studies on the strengthening of the Hadley and Walker circulations (Sohn and Park, 2010), the widening of the tropical width (Mantsis et al., 2017), and a possible expansion of the subtropical dry zones (Tivig et al., 2020). The UTH datasets facilitated the evaluation of climate models and contributed to a better un- derstanding of large-scale atmospheric processes (Allan et al., 2003; Soden et al., 2005; Chung et al., 2016; Allan et al., 2022; John et al., 2021). The UTH measurements from Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 2.1 CMSAF UTH individual pixels before gridding. Only pixels close to the nadir view of the satellite are selected. The microwave sounder UTH data (version 1.0) are derived from AMSU-B and MHS from the 183.31 ± 1 GHz chan- nel. The dataset is based on a microwave humidity sounder dataset record generated by EUMETSAT within the frame- work of the ERA-CLIM2 project. A combination of methods was used to estimate inter-satellite biases for the microwave humidity sounders (John et al., 2013; Saunders et al., 2013). There is a simple linear relationship between brightness tem- perature (Tb) emanating from water vapor emissions in the upper troposphere and the natural logarithm of UTH (e.g., see Buehler and John, 2005), which is the Jacobian-weighted relative humidity in the upper troposphere: L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity ln(UTH) = a + b × Tb. ln(UTH) = a + b × Tb. UTH = cosθ p0 e(a+b×T6.7), (2) (2) The coefﬁcients a and b are determined by linear regres- sion, using a training dataset of atmospheric temperature and humidity proﬁles, in which a = 23.467520 and b = −0.099240916. in which θ is the viewing angle. p0 is the pres- sure of the 240 K isotherm divided by 300 hPa (p0 = p[T =240 K]/300 hPa), which is determined from a training set of ECMWF proﬁles for 1986–1989 as a function of month, latitude, and longitude. The coefﬁcients a and b are deter- mined based on the training proﬁles and radiative transfer model simulation of T6.7, in which a = 31.5 and b = −0.115. The HIRS UTH dataset has a monthly coverage based on clear-sky observations with a spatial resolution of 2.5◦×2.5◦. The UTH is computed from gridded brightness temperature data. The data analyzed in this report cover the period of November 1978–December 2020. The CMSAF UTH is derived for individual pixels and then gridded. The product is provided to users on a global, daily 1.0◦× 1.0◦latitude–longitude grid. UTH is retrieved for all cloud- and surface-cleared and limb-corrected bright- ness temperatures for each day. These are then separated for ascending and descending passes and binned into each 1.0◦ grid cell. The time series analyzed in this report covers 1999– 2019 for the CMSAF data. 2 Datasets The four datasets analyzed in this study include UTH gen- erated by the Satellite Application Facility on Climate Mon- itoring (CMSAF), the Fidelity and Uncertainty in Climate data records from Earth Observations (FIDUCEO) project, the National Centers for Environmental Information (NCEI), and the University of Miami (UMIAMI). Three of these are based on microwave sounder measurements, and one is based on infrared sounder measurements. The CMSAF and UMIAMI datasets are derived from AMSU-B/MHS mea- surements. The FIDUCEO dataset adds Special Sensor Mi- crowave Humidity (SSM/T-2) to the microwave measure- ments that extends the time series back to 1994. The NCEI UTH data are derived from HIRS channel 12 measurements. The following provides details of the four datasets. https://doi.org/10.5194/amt-15-6949-2022 Atmos. Meas. Tech., 15, 6949–6963, 2022 6951 2.3 NCEI UTH The NCEI UTH dataset is based on version 3.2 of HIRS channel 12 brightness temperature data (Shi and Bates, 2011). Because an infrared sounder cannot sense through clouds, cloudy pixels are removed from the dataset. The cloud-ﬁltered and limb-corrected channel brightness temper- atures are calibrated using derived adjustment coefﬁcients from matched overlapping HIRS data between satellites. In this study the UTH is calculated based on the relationship between UTH and HIRS channel 12 brightness temperatures centered at 6.7 µm (T6.7) derived by Soden and Bretherton (1996): ln(UTH) = a + b × Tb. (1) (1) 3.1 Intercomparison of time series The UTH datasets are most often used to monitor tropi- cal atmospheric activities (e.g., Brogniez et al., 2015; Tivig et al., 2020; and John et al., 2021). Therefore, the as- sessment focuses on the consistency of the tropical data. Figure 1 plots the time series of UTH datasets aver- aged over the domain 20◦S–20◦N. These include UTH derived from both microwave 183.31 ± 1 GHz brightness temperatures and infrared 6.7 µm brightness temperatures. Figure 1a displays domain-averaged monthly mean val- ues of UTH; Fig. 1b shows the corresponding anoma- lies, and Fig. 1c and d show the differences in UTH and in anomaly values, respectively, relative to the val- ues of UMIAMI. In the anomaly calculation, the period 2000–2015 is used for climatology. Figure 1e displays the time series of the Oceanic Niño Index (ONI) (avail- able at https://origin.cpc.ncep.noaa.gov/products/analysis_ monitoring/ensostuff/ONI_v5.php, last access: 16 June 2022). The ONI is constructed using the 3-month running- average sea surface temperature (SST) anomalies in the Niño 3.4 region (5◦S–5◦N, 120–170◦W) (originally presented by Trenberth, 1997). During an El Niño event (such as the 2015–2016 and 2009–2010 events as displayed in Fig. 1e) the infrared dataset tends to have a smaller value of averaged UTH compared to microwave UTH values, and the opposite oc- curs during a La Niña event (such as the 2010–2011 and 2007–2008 events). This indicates that the infrared clear-sky dataset may not fully capture the increase of water vapor dur- ing El Niño events due to the exclusion of very humid pixels associated with clouds and tends to have better sampling of the dry regions. Figure 1d also shows that the tropical mean UTH has a larger moistening trend in CMSAF than the other datasets. Allan et al. (2022) presented tropical (30◦S–30◦N) ocean and land averaged anomaly time series of ERA5 rela- tive humidity (RH), AIRS RH, HIRS UTH, and MW UTH (Figs. 6 and 7 of their study). The HIRS and MW UTH are the NCEI and UMIAMI UTH datasets analyzed in the present study, and the features of these two datasets are sim- ilar to the NCEI and UMIAMI UTH time series in Fig. 1b. In Fig. 1a, the four datasets appear to be separated with two groups of similar UTH values. The values of CMSAF and FIDUCEO UTH are larger than the values of NCEI and UMIAMI UTH. 3.1 Intercomparison of time series Among the datasets, the UTH of CMSAF and FIDUCEO is ﬁrst computed for each pixel before taking grid averages. For the UMIAMI and HIRS dataset, the grid- ding of the brightness temperature is done ﬁrst and then UTH is computed from averaged brightness temperatures. Based on a study by John et al. (2006), such different ways of com- puting UTH can lead to a difference of up to 6 % UTH due to the non-linearities in the formula that calculates UTH from brightness temperature values. Figure 1c shows that there is a difference of approximately 3 %–5 % UTH between two groups of UTH datasets when a tropical average is taken. In spite of this structural discrepancy, the anomaly plot of the UTH in Fig. 1b shows consistency in seasonal and inter- annual variability patterns among the four datasets. All four datasets show major peaks and dips along the time series in the same phases, though there are differences in the magni- tudes of the ﬂuctuations. In the FIDUCEO dataset, SSM/T-2 data before 1998 were at times sparse or missing, causing a few data gaps and some uncertainty in monthly means. De- spite different deﬁnitions and ways of computing UTH, the anomalies of the four datasets are close to each other. During major El Niño events, tropical water vapor ﬁelds exhibit distinct characteristics, and the enhanced signals fa- cilitate the comparison of datasets. Figure 2 shows the time series of UTH over the Niño 4 region (equatorial central Pa- ciﬁc 5◦S–5◦N, 160◦E–150◦W). Figure 2a shows that the interannual variability of UTH is much larger compared to tropical mean values in Fig. 1a, but similar differences be- tween datasets remain. The UTH values of the CMSAF and FIDUCEO datasets are generally larger than the values of NCEI and UMIAMI datasets by approximately 5 % UTH on average (Fig. 2c). In the anomaly plots (Fig. 2b), all datasets depict consistent interannual variations. In Fig. 2d, the in- frared UTH again shows smaller values compared to mi- crowave UTH values during El Niño events and larger values during La Niña events, similar to the features displayed in the tropical average time series in Fig. 1d. Though a moistening trend is shown in the CMSAF UTH time series in Fig. L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity 6952 To quantify the differences between datasets, the relative differences are calculated. Note that any of the four datasets can be used as a reference for this purpose. Among the mi- crowave (MW) UTH datasets, the UMIAMI dataset has the lengthiest time period of AMSU-B and MHS to allow for the longest MW comparison with others, and it is used as the relative reference in the calculation. Figure 1d shows that the anomaly values are mostly within ±0.5 % UTH of each other, with the exceptions during El Niño events when the anomaly differences can be larger. Chung et al. (2016) ana- lyzed the relative differences among the brightness temper- atures of the channels sensing upper tropospheric humidity from HIRS, AMSU-B/MHS, and AIRS. The brightness tem- perature differences between the HIRS and AMSU-B/MHS were mostly within ±0.2 K. 3 Results and discussions The assessment examines several aspects of the UTH datasets, including consistency in time series, spatial feature consistency, and changes during the datasets’ common pe- riod. The following describes the analyses and results. 2.4 UMIAMI UTH The FIDUCEO UTH (version 1.2) is based on the FIDUCEO fundamental climate data record of recalibrated microwave sounder brightness temperatures (Hans et al., 2019), cover- ing the sensors SSM/T-2, AMSU-B, and MHS. It uses a new UTH deﬁnition (Lang et al., 2020b) based on the concept that the atmospheric emission layer for a water vapor chan- nel is bounded by two characteristic amounts of water va- por integrated from the top of the atmosphere downwards. Using this idea, UTH is deﬁned as the mean relative hu- midity in a layer between two altitude levels z(IWV1) and z(IWV2), at which the integrated water vapor (IWV) exceeds two viewing-angle-dependent thresholds IWV1 and IWV2. The thresholds IWV1 and IWV2 play a similar role in cap- turing the atmospheric emission layer to the Jacobian in the traditional deﬁnition. The IWV thresholds were optimized in such a way that the linear relationship between the Tb and the logarithm of UTH is best fulﬁlled for the European Cen- tre for Medium-Range Weather Forecasts (ECMWF) training atmospheres. The data record covers the time between 1994 and 2017 and provides monthly mean brightness tempera- tures and derived UTH along with estimates of measurement uncertainty on a 1◦× 1◦latitude–longitude grid covering the tropical region (30◦S to 30◦N). The UTH is ﬁrst derived for The UMIAMI data (Chung et al., 2013) are available as grid- ded brightness temperatures from AMSU-B and MHS on a 1.5◦× 1.5◦latitude–longitude grid. Biases due to the differ- ence in local observation time between satellites and spu- rious trends associated with satellite orbital drift are diag- nosed and adjusted for using synthetic radiative simulations based on the interim European Centre for Medium-Range Weather Forecasts re-analysis (ERA-Interim) and ERA5. The adjusted, rain-cloud-ﬁltered, and limb-corrected bright- ness temperatures are then intercalibrated using zonal-mean brightness temperature differences. In this study the formula that is used by the CMSAF dataset is applied to compute UTH. However, unlike the computation of the CMSAF UTH in which the UTH is ﬁrst derived for each individual pixel before gridding, the UMIAMI UTH is computed from grid- ded averaged brightness temperature values. The time series for this study covers 1999–2020. Atmos. Meas. Tech., 15, 6949–6963, 2022 https://doi.org/10.5194/amt-15-6949-2022 https://doi.org/10.5194/amt-15-6949-2022 Atmos. Meas. Tech., 15, 6949–6963, 2022 3.1 Intercomparison of time series 1c and d where the tropical average is taken, the moistening trend is not as apparent for the Niño 4 region as displayed in Fig. 2c and d. It is interesting to observe that between Figs. 1b and 2b the phases of the variations are mostly opposite. During the Atmos. Meas. Tech., 15, 6949–6963, 2022 https://doi.org/10.5194/amt-15-6949-2022 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity 6953 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity Figure 1. Time series of UTH (%) averaged over 20◦S–20◦N for (a) the averaged values of UTH, (b) the corresponding anomalie to the 2000–2015 climatology, (c) the differences of UTH values relative to the values of UMIAMI, and (d) the differences of anoma relative to those of UMIAMI. A 5-month moving average is applied to the UTH time series to reduce short-term ﬂuctuations. Panel ( the time series of ONI. Figure 1. Time series of UTH (%) averaged over 20◦S–20◦N for (a) the averaged values of UTH, (b) the corresponding anomalies relative to the 2000–2015 climatology, (c) the differences of UTH values relative to the values of UMIAMI, and (d) the differences of anomaly values relative to those of UMIAMI. A 5-month moving average is applied to the UTH time series to reduce short-term ﬂuctuations. Panel (e) shows the time series of ONI. Figure 1. Time series of UTH (%) averaged over 20◦S–20◦N for (a) the averaged values of UTH, (b) the corresponding anomalies relative to the 2000–2015 climatology, (c) the differences of UTH values relative to the values of UMIAMI, and (d) the differences of anomaly values relative to those of UMIAMI. A 5-month moving average is applied to the UTH time series to reduce short-term ﬂuctuations. Panel (e) shows the time series of ONI. However, the increased water vapor in the upper atmosphere is conﬁned to relatively small areas. The study of Lim et al. (2017) showed that during a major El Niño the rising motion of the Hadley circulation is dominant between 10◦S and the Equator. The branch of sinking motion in the sub- tropics (15–25◦N) is well organized, stretching from the sur- face to the upper troposphere. In the upper troposphere, large positive anomalies of total cloud fraction are formed over 10◦S–5◦N, and negative cloud anomalies occurred over the subtropics. 3.1 Intercomparison of time series A 5-month moving average is applied to the UTH time series to reduce short-term ﬂuctuations. 6954 Figure 2. Time series of UTH (%) averaged over the Niño 4 region for (a) the averaged values of UTH, (b) the corresponding anomalie relative to the 2000–2015 climatology, (c) the differences of UTH values relative to the values of UMIAMI, and (d) the differences o l l l ti t th f UMIAMI A 5 th i i li d t th UTH ti i t d h t t ﬂ t ti Figure 2. Time series of UTH (%) averaged over the Niño 4 region for (a) the averaged values of UTH, (b) the corresponding anomalies relative to the 2000–2015 climatology, (c) the differences of UTH values relative to the values of UMIAMI, and (d) the differences of anomaly values relative to those of UMIAMI. A 5-month moving average is applied to the UTH time series to reduce short-term ﬂuctuations. most signiﬁcant three El Niño events occurred in 1982– 1983, 1997–1998, and 2015–2016 according to ONI shown in Fig. 1e. During these events the UTH ﬁeld is marked by increased anomalies in the central-eastern and correspond- ing decreased UTH in the western equatorial Paciﬁc. All three events can be clearly identiﬁed in the NCEI time se- ries, which has the longest temporal coverage. tropical average is taken, the larger areas of negative anoma- lies overcompensate for the smaller areas of positive anoma- lies and result in mean negative anomalies during El Niño events. As the Niño 4 region is the center of enhanced deep convection during El Niño events, the phase of UTH is con- sistent with that of the water vapor in the lower atmosphere and consistent with the phase of sea surface temperature dur- ing El Niño events as shown in Fig. 1e and as described in, e.g., Trenberth (1997), McPhaden (1999), Wolter and Timlin (2011), Lim et al. (2017), and Santoso et al. (2017). g p g The 1997–1998 and 2015–2016 events are also clearly dis- played in the FIDUCEO time series. However, the sparsity of the SSM/T-2 data before 1998 can be seen in the noisier ap- pearance of the anomalies during that period. 3.1 Intercomparison of time series Beyond the constrained positive UTH anoma- lies around the Equator, the water vapor in the upper tropo- sphere is suppressed in large areas outside the Niño 4 region, which causes large areas of negative UTH anomalies, consis- tent with the sinking motion of the Hadley branch. When a major El Niño events (for example, 1982–1983, 1997–1998, and 2015–2016), the tropical averaged time series exhibited large negative values of anomalies (Fig. 1b), while at the same time, large positive anomalies occurred in the Niño 4 region (Fig. 2b). An earlier study (Shi et al., 2018) showed that unlike UTH, the total column water vapor (TCWV) on the tropical average exhibited large positive anomalies dur- ing El Niño events, having the same phase as the Niño 4 re- gion UTH time series. The TCWV is largely weighted by water vapor in the lower troposphere. During an El Niño event, there are larger areas of water vapor increase in the lower atmosphere as reﬂected in the TCWV ﬁeld, compared to the UTH ﬁeld. The enhanced deep convection provides a conduit to transport more water vapor to the atmosphere. However, the increased water vapor in the upper atmosphere is conﬁned to relatively small areas. The study of Lim et al. (2017) showed that during a major El Niño the rising motion of the Hadley circulation is dominant between 10◦S and the Equator. The branch of sinking motion in the sub- tropics (15–25◦N) is well organized, stretching from the sur- face to the upper troposphere. In the upper troposphere, large positive anomalies of total cloud fraction are formed over 10◦S–5◦N, and negative cloud anomalies occurred over the subtropics. Beyond the constrained positive UTH anoma- lies around the Equator, the water vapor in the upper tropo- sphere is suppressed in large areas outside the Niño 4 region, which causes large areas of negative UTH anomalies, consis- tent with the sinking motion of the Hadley branch. When a https://doi.org/10.5194/amt-15-6949-2022 Atmos. Meas. Tech., 15, 6949–6963, 2022 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity 2. Time series of UTH (%) averaged over the Niño 4 region for (a) the averaged values of UTH, (b) the corresponding anomalies to the 2000–2015 climatology, (c) the differences of UTH values relative to the values of UMIAMI, and (d) the differences of y values relative to those of UMIAMI. L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity The infrared dataset has the smallest proportion of humid anomalies compared to the MW datasets at both levels (> 5 % and > 1 %) due to the exclusion of cloudy pixels. Figure 3. Time versus longitude section of UTH monthly anomaly. The analysis is based on an average of data between 5◦N and 5◦S. Multivariate El Niño–Southern Oscillation Index (MEI) in- dicates similar differences in the strength of these El Niño events. In addition to the commonly used sea surface tem- perature (SST) anomalies, the MEI also incorporates sur- face air temperature, sea-level pressure, zonal and merid- ional components of the surface wind, and total cloudiness fraction of the sky (Wolter and Timlin, 2011). The Multi- variate ENSO Index Version 2 (MEI.v2) values (available at https://psl.noaa.gov/enso/mei/#data, last access: 3 June 2022) show that MEI reached as high as 2.5 and remained at or above 2.0 for 12 consecutive months during the 1997– 1998 El Niño event. During the 2015–2016 El Niño, the MEI was as high as 2.2, and remained above 2.0 for only 2 months. The UMIAMI and CMSAF UTH time series both started in late 1998, and they have similar patterns in the Hovmöller analysis, both distinctively showing the 2015–2016 El Niño event. HIRS UTH also generally has the smallest proportion of the driest anomalies (< −5 %), but the ratios are often close to those of the UMIAMI dataset. Interestingly, when the ma- jority of the negative anomalies are examined (UTH anoma- lies < −1 % in Fig. 4b), the HIRS dataset frequently has the largest ratios of the dry anomalies. This phenomenon is par- ticularly signiﬁcant during both major El Niño and La Niña events. For example, during the 2015–2016 El Niño, the ra- tios of UTH anomalies < −1 % are approximately 51 % for HIRS, 47 % for UMIAMI, 46 % for FIDUCEO, and 45 % for the CMSAF dataset. In other words, the HIRS data iden- tify more dry anomalies than the MW datasets, though the magnitude of the driest HIRS UTH does not usually reach as large values as those of the MW UTH, likely due to the deﬁnition of the UTH formula used. Overall, the FIDUCEO Allan et al. (2022) examined changes in the anomaly char- acteristics in the zonal mean of AMIP 300–500 hPa RH, ERA5 300–500 hPa RH, and HIRS UTH (their Fig. 8a–c). L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity 6955 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity Figure 3. Time versus longitude section of UTH monthly anomaly. The analysis is based on an average of data between 5◦N and 5◦S. also shows subtly stronger UTH amplitudes before 2000, al- beit with only a few years of data available. These changes after 2000 seem to be coincident with the decrease in the strength of El Niño events after 2000 as depicted by the MEI.v2, though such changes are not displayed in SST-only Niño indices such as the ONI. During the common period when data were available from all four datasets, the most signiﬁcant La Niña event occurred in 2010–2011, in which the MEI.v2 value reached −2.4. The UTH ﬁeld was marked by decreased UTH in 120◦E–160◦W and increased UTH in 80–120◦E. The event can be seen from all UTH datasets. In general, the equatorial UTH anomalies in the infrared measurements are relatively weaker than those in the microwave measurements. The deﬁnition used to com- pute the HIRS UTH may be the primary factor for the smaller magnitude. The averaging of pixel-level brightness temper- atures to the grids ﬁrst before the UTH is computed may further smooth out the largest anomalies (both positive and negative). To quantify the changing proportion of dry and humid regions derived from the different datasets, we calculate the percentage of grids with anomaly values greater or less than several selected values over 20◦S–20◦N (Fig. 4). The anomalies are relative to each of the grid points and desea- sonalized before the percentages are calculated. Grids with UTH anomaly values > 5 % represent very humid anoma- lies, while those < −5 % represent very dry anomalies. Among the MW datasets, the SSM/T-2-derived UTH in the FIDUCEO series has the highest proportion of very humid anomalies. For the AMSU-B/MHS series, the FIDUCEO dataset generally has 2 %–4 % more very humid anomalies than those of the UMIAMI dataset. The gridding of UTH after the pixel-level brightness temperature values are aver- aged in the UMIAMI dataset may have smoothed out some of the most humid measurements. The CMSAF UTH has fewer dry anomalies before 2005 than the other datasets, but it has the largest proportion of very humid anomalies in re- cent years. 3.1 Intercomparison of time series Nonetheless, both the NCEI and FIDUCEO datasets show that the 1997– 1998 event was marked with higher anomaly values and ex- tended further east in the Paciﬁc in terms of large positive UTH anomalies compared to the 2015–2016 El Niño. The We use longitude–time Hovmöller diagrams to examine spatiotemporal variability of UTH over the deep tropics. Figure 3 shows longitude–time evolutions of monthly UTH anomalies around the Equator, averaged between 5◦S and 5◦N for the four datasets. During the past 40 years, the Atmos. Meas. Tech., 15, 6949–6963, 2022 https://doi.org/10.5194/amt-15-6949-2022 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity Both the AMIP and the HIRS time series showed a de- tectable decreasing trend in UTH 30–60◦S, and all three datasets showed decreasing amplitudes of anomalies after 2000. More speciﬁcally, AMIP and HIRS showed smaller positive anomalies, while ERA5 exhibited smaller negative anomalies. The FIDUCEO MW UTH in Fig. 3c of our study https://doi.org/10.5194/amt-15-6949-2022 Atmos. Meas. Tech., 15, 6949–6963, 2022 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity 6956 Figure 4. Time series of the proportions of grids over 20◦S–20◦N with anomaly values less than −5 % and −1 % and greater than 1 % and 5 %. Figure 4. Time series of the proportions of grids over 20◦S–20◦N with anomaly values less than −5 % and −1 % and greater than 1 % and 5 %. Figure 4. Time series of the proportions of grids over 20◦S–20◦N with anomaly values less than −5 % and 5 %. Figure 4. Time series of the proportions of grids over 20◦S–20◦N with anomaly values less than −5 % and −1 % and greater than 1 % and 5 %. dataset has the largest amplitude of the ratios for both the most humid and the driest measurements. show the large-scale atmospheric circulation and SST ﬁelds. The GPCP data are generated by combining satellite retrieval and in situ precipitation into a ﬁnal merged gridded prod- uct (Adler et al., 2003). The ERSSTv5 dataset is derived from the International Comprehensive Ocean–Atmosphere Dataset (ICOADS) and is available at gridded monthly global coverage (Huang et al., 2017). Velocity potential anoma- lies at 200 hPa are taken from the Climate Forecast System Reanalysis (CFSR) (Saha et al., 2010) for 2000–2010 and the related Climate Forecast System v2 (CFSv2) operational analyses (Saha et al., 2014) for 2011–2016. 3.2 Spatial anomalies during major El Niño and La Niña events In the NCEI HIRS UTH panel, the magnitudes of both positive anomalies along the central-eastern equatorial Paciﬁc and the negative anomalies in the western Paciﬁc appear smaller than those in the other three microwave UTH panels, consistent with what is seen in the Hovmöller analysis discussed ear- lier. However, over the tropical domain, the HIRS data have larger proportions of dry areas in the subtropics during El Niño events (resulting in larger overall dry area ratios shown in Fig. 4b), leading to deeper dips of UTH during El Niño events displayed in Fig. 1b. Taking an example of the FIDUCEO UTH dataset, there are approximately 34 % of grids in the tropical domain 20◦S– 20◦N that have UTH anomalies greater than 1 %, compared to more than 49 % of grids having UTH anomalies less than −1 % at the peak of the 2015–2016 El Niño as shown in Fig. 4. The other three datasets also show larger portions of dry grids than humid grids during the event. When a tropical- domain average of anomalies is taken, it results in a nega- tive anomaly during an El Niño event as shown in Fig. 1. In the NCEI HIRS UTH panel, the magnitudes of both positive anomalies along the central-eastern equatorial Paciﬁc and the negative anomalies in the western Paciﬁc appear smaller than those in the other three microwave UTH panels, consistent with what is seen in the Hovmöller analysis discussed ear- lier. However, over the tropical domain, the HIRS data have larger proportions of dry areas in the subtropics during El Niño events (resulting in larger overall dry area ratios shown in Fig. 4b), leading to deeper dips of UTH during El Niño events displayed in Fig. 1b. Figure 5. Anomalies of UTH during the peak 6 months of the 2015– 2016 El Niño event. The box shows the Niño 4 domain in the central Paciﬁc (5◦S–5◦N, 160◦E–150◦W). anomalies surrounding the positive anomalies. The positive SST anomalies were centered along the equatorial central- eastern Paciﬁc (Fig. 6b). Anomalous divergence developed over the warmed SST and was balanced by the anomalous convergence over the western Paciﬁc and the Indian Ocean (Fig. 6c). The pattern of positive anomalies of UTH above the Niño 4 region and along 5–10◦N in the eastern Paciﬁc highly resembles the pattern of the positive precipitation anomalies (as shown in Fig. 3.2 Spatial anomalies during major El Niño and La Niña events During the common period of the four datasets, the most sig- niﬁcant El Niño and La Niña events occurred in 2015–2016 and 2010–2011, respectively. The spatial patterns of UTH anomalies for 60◦S–60◦N during the peak 6 months of the 2015–2016 El Niño event are shown in Fig. 5. The anomalies of several environmental variables, including data from the Global Precipitation Climatology Project (GPCP), NOAA Extended Reconstructed SST V5 (ERSSTv5), and modeled 200 hPa velocity potential, for the same peak 6-month pe- riod of the 2015–2016 El Niño are displayed in Fig. 6 to Similarly to that discussed in Shi et al. (2018), during the 2015–2016 El Niño event, UTH developed strong positive anomalies over the equatorial central Paciﬁc, extending to the eastern Paciﬁc in 5–10◦N. The enhanced El Niño convec- tion drove compensating subsidence and thus negative UTH https://doi.org/10.5194/amt-15-6949-2022 Atmos. Meas. Tech., 15, 6949–6963, 2022 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity 6957 Figure 5. Anomalies of UTH during the peak 6 months of the 2015– 2016 El Niño event. The box shows the Niño 4 domain in the central Paciﬁc (5◦S–5◦N, 160◦E–150◦W). Figure 6. Anomalies of GPCP precipitation, ERSSTv5 SST, and CFSR 200 hPa velocity potential during the peak 6 months of the 2015–2016 El Niño event. The box shows the Niño 4 domain in the central Paciﬁc (5◦S–5◦N, 160◦E–150◦W). Figure 6. Anomalies of GPCP precipitation, ERSSTv5 SST, and CFSR 200 hPa velocity potential during the peak 6 months of the 2015–2016 El Niño event. The box shows the Niño 4 domain in the central Paciﬁc (5◦S–5◦N, 160◦E–150◦W). Taking an example of the FIDUCEO UTH dataset, there are approximately 34 % of grids in the tropical domain 20◦S– 20◦N that have UTH anomalies greater than 1 %, compared to more than 49 % of grids having UTH anomalies less than −1 % at the peak of the 2015–2016 El Niño as shown in Fig. 4. The other three datasets also show larger portions of dry grids than humid grids during the event. When a tropical- domain average of anomalies is taken, it results in a nega- tive anomaly during an El Niño event as shown in Fig. 1. 3.2 Spatial anomalies during major El Niño and La Niña events 6a), indicating the strong linkage between the two variables. Similar patterns of precipitation during the 2015–2016 El Niño were also shown in the study of Santoso et al. (2017). To further assess the consistency of UTH datasets with several environmental variables, histograms of UTH anoma- lies vs. anomalies of GPCP precipitation, ERSSTv5 SST, and CFSR 200 hPa velocity potential during the peak 6 months of the 2015–16 El Niño are presented in Figs. 7–9. The cor- relations between the anomalies of UTH and those of the three variables are also calculated, and the correlation val- ues are labeled (as Corr) at the top of each panel. Among Overall, the area of the strong positive UTH anomalies over the equatorial central Paciﬁc is smaller than the sur- rounding areas of strong negative anomalies in the tropics. https://doi.org/10.5194/amt-15-6949-2022 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity 7 except for UTH anomalies vs. ERSSTv5 anomalies. Figure 8. Similar to Fig. 7 except for UTH anomalies vs. ERSSTv5 anomalies. Figure 7. Histograms of UTH anomalies of the four datasets vs. anomalies of GPCP precipitation during the peak 6 months of the 2015–2016 El Niño. The blue line represents the linear regression line. The correlations between UTH anomalies and GPCP precipi- tation anomalies are labeled at the top of the panels. Figure 7. Histograms of UTH anomalies of the four datasets vs. anomalies of GPCP precipitation during the peak 6 months of the 2015–2016 El Niño. The blue line represents the linear regression line. The correlations between UTH anomalies and GPCP precipi- tation anomalies are labeled at the top of the panels. Figure 8. Similar to Fig. 7 except for UTH anomalies vs. ERSSTv5 anomalies. Figure 9. Similar to Fig. 7 except for UTH anomalies vs. anomalies of CFSR 200 hPa velocity potential. the three variables, precipitation has the highest correlations with UTH (Fig. 7), while SST has the lowest (Fig. 8). Both precipitation and velocity potential are proxies for vertical motion, so they are more directly tied to wet/dry UTH than the SST surface forcing. The increases of SST during El Niño events usually occur most signiﬁcantly in the equatorial eastern-central Paciﬁc, while the increases of both UTH and precipitation are more conﬁned over the equatorial central Paciﬁc. The UTH and precipitation ﬁelds both have a more balanced dipole between the central and western equatorial Paciﬁc during a major El Niño, while the decrease of SST in the western equatorial Paciﬁc does not match the strength of positive anomalies in the central-eastern equatorial Paciﬁc. These patterns lead to overall higher correlations between UTH and precipitation than those between UTH and SST. The correlation values also illustrate that an SST-only ENSO index may not be as good an indicator for the strength of UTH compared to an index that includes other environmen- tal variables such as the MEI. Figure 9. Similar to Fig. 7 except for UTH anomalies vs. anomalies of CFSR 200 hPa velocity potential. Fig. 11 displays the anomalies of GPCP, ERSSTv5, and CFSR 200 hPa velocity potential data for the same time pe- riod. L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity 6958 6958 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity Figure 7. Histograms of UTH anomalies of the four datasets vs. anomalies of GPCP precipitation during the peak 6 months of the 2015–2016 El Niño. The blue line represents the linear regression line. The correlations between UTH anomalies and GPCP precipi- tation anomalies are labeled at the top of the panels. the three variables, precipitation has the highest correlations with UTH (Fig. 7), while SST has the lowest (Fig. 8). Both precipitation and velocity potential are proxies for vertical motion, so they are more directly tied to wet/dry UTH than the SST surface forcing. The increases of SST during El Niño events usually occur most signiﬁcantly in the equatorial eastern-central Paciﬁc, while the increases of both UTH and precipitation are more conﬁned over the equatorial central Paciﬁc. The UTH and precipitation ﬁelds both have a more balanced dipole between the central and western equatorial Paciﬁc during a major El Niño, while the decrease of SST in the western equatorial Paciﬁc does not match the strength of positive anomalies in the central-eastern equatorial Paciﬁc. These patterns lead to overall higher correlations between UTH and precipitation than those between UTH and SST. The correlation values also illustrate that an SST-only ENSO index may not be as good an indicator for the strength of UTH compared to an index that includes other environmen- tal variables such as the MEI. Among the UTH datasets, the MW data have higher cor- relations with the three environmental variables. The HIRS UTH correlation values are about 0 1 lower primarily due to Figure 8. Similar to Fig. 7 except for UTH anomalies vs. ERSSTv5 anomalies. Figure 9. Similar to Fig. 7 except for UTH anomalies vs. anomalies of CFSR 200 hPa velocity potential. Fig. 11 displays the anomalies of GPCP, ERSSTv5, and CFSR 200 hPa velocity potential data for the same time pe- riod During a La Niña event the central Paciﬁc and Indone- Figure 8. Similar to Fig. 7 except for UTH anomalies vs. ERSSTv5 anomalies. Figure 7. Histograms of UTH anomalies of the four datasets vs. anomalies of GPCP precipitation during the peak 6 months of the 2015–2016 El Niño. The blue line represents the linear regression line. The correlations between UTH anomalies and GPCP precipi- tation anomalies are labeled at the top of the panels. Figure 8. Similar to Fig. https://doi.org/10.5194/amt-15-6949-2022 Atmos. Meas. Tech., 15, 6949–6963, 2022 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity During a La Niña event, the central Paciﬁc and Indone- sia exhibited mostly opposite signs of anomalies for UTH, SST, precipitation, and 200 hPa velocity potential compared to the El Niño patterns depicted in Figs. 5 and 6, except that the negative anomalies of the 200 hPa velocity potential were more conﬁned to the center over Indonesia and Australia. La Niña events tend to lead to signiﬁcant increases of UTH over Indonesia and the equatorial eastern Indian Ocean and over Paciﬁc subtropics and decreases of UTH over the Niño 4 re- Among the UTH datasets, the MW data have higher cor- relations with the three environmental variables. The HIRS UTH correlation values are about 0.1 lower, primarily due to the lack of very humid anomalies in the infrared dataset. The histograms show that for all UTH datasets, the highest densi- ties of anomalies are consistently centered around zero. The density of HIRS positive anomalies decreases rapidly beyond 5 %, in line with the lowest ratio of large HIRS UTH shown in Fig. 4d. Figure 10 shows the UTH anomaly ﬁelds averaged over 6 months near the peak of the La Niña in 2010–2011, and Atmos. Meas. Tech., 15, 6949–6963, 2022 https://doi.org/10.5194/amt-15-6949-2022 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity 6959 6959 p pp Figure 10. Anomalies of UTH during the peak 6 months of the 2010–2011 La Niña event. The box shows the Niño 4 domain in the central Paciﬁc (5◦S–5◦N, 160◦E–150◦W). Figure 10. Anomalies of UTH during the peak 6 months of the 2010–2011 La Niña event. The box shows the Niño 4 domain in the central Paciﬁc (5◦S–5◦N, 160◦E–150◦W). Figure 11. Anomalies of GPCP precipitation, ERSSTv5 SST, a CFSR 200 hPa velocity potential during the peak 6 months of t 2010–2011 La Niña event. The box shows the Niño 4 domain in t central Paciﬁc (5◦S–5◦N, 160◦E–150◦W). consistency, and the results should not be interpreted as lon term trends. The La Niña event in 1998–2000 at the begi ning of the common period and the strong El Niño event 2015–2016 near the end of the common period can signi cantly impact the resulting trend values. The Mann–Kenda test is used to test the signiﬁcance of the trends at each gri The trends appear to be signiﬁcant at 0.95 only in a fe small places, mainly sparsely spotted along subtropical P Figure 11. L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity Anomalies of GPCP precipitation, ERSSTv5 SST, and CFSR 200 hPa velocity potential during the peak 6 months of the 2010–2011 La Niña event. The box shows the Niño 4 domain in the central Paciﬁc (5◦S–5◦N, 160◦E–150◦W). Figure 11. Anomalies of GPCP precipitation, ERSSTv5 SST, and CFSR 200 hPa velocity potential during the peak 6 months of the 2010–2011 La Niña event. The box shows the Niño 4 domain in the central Paciﬁc (5◦S–5◦N, 160◦E–150◦W). consistency, and the results should not be interpreted as long- term trends. The La Niña event in 1998–2000 at the begin- ning of the common period and the strong El Niño event in 2015–2016 near the end of the common period can signiﬁ- cantly impact the resulting trend values. The Mann–Kendall test is used to test the signiﬁcance of the trends at each grid. The trends appear to be signiﬁcant at 0.95 only in a few small places, mainly sparsely spotted along subtropical Pa- ciﬁc belts of negative change rates (not plotted in Fig. 12), indicating that the time series is too short for a meaningful trend study for the majority of areas. In the present study, the trend results are only used as a consistency evaluation of the datasets. Figure 10. Anomalies of UTH during the peak 6 months of the 2010–2011 La Niña event. The box shows the Niño 4 domain in the central Paciﬁc (5◦S–5◦N, 160◦E–150◦W). gion. Slightly positive UTH anomalies may be found in the equatorial eastern Paciﬁc during a La Niña event. Similar pat- terns of tropical features are shown in all four datasets, al- though the magnitudes are again smaller in the infrared UTH (Fig. 10a). General consistency of the change patterns in the tropics is found among the four datasets. They all show increased UTH over the Niño 4 region (5◦S–5◦N, 160◦E–150◦W) and over the eastern Paciﬁc near 5–10◦N and a decrease of UTH over Peru and surrounding areas. Decreased UTH values along both the northern and the southern Paciﬁc sub- tropics are seen in all datasets. The change rate patterns over the tropical and subtropical Paciﬁc follow the 2015–2016 El Niño UTH patterns (as shown in Fig. 5) to some extent, in- dicating the inﬂuence of the El Niño signals on the change rate calculation. Over the Indian Ocean, decreased UTH cen- tered over the equatorial central Indian Ocean is surrounded 4 Conclusions In this study we assess the consistency of four UTH datasets derived from both microwave and infrared sounders of polar- orbiting satellites as part of the GEWEX water vapor as- sessment activities. These include measurements from the 183.31 ± 1 GHz channel on SSM/T-2, AMSU-B, and MHS and HIRS channel 12 (calibrated to 6.7 µm). The main con- clusions are as follows: 1. The four datasets exhibit consistency in tropical spa- tial patterns and in interannual variability. Large pos- itive anomalies peaked over the Niño 4 region during El Niño events in the same phase with the increase of sea surface temperature as expected. At the same time, opposite phases of anomalies were obtained in the aver- aged tropical anomalies because the compensating dry- ing areas of dissipation are larger than the relatively conﬁned moistening area above deep convection. All four datasets exhibit such similar temporal variability. Figure 12. Change rates of the four UTH datasets during the com- mon period 1999 to 2017. with increased UTH in most datasets, except that the cen- ter of decreasing rates is conﬁned to a smaller area around 15◦S for the CMSAF UTH. The change rates (both positive and negative) in the NCEI HIRS dataset (Fig. 12a) are gen- erally smaller than those in microwave datasets. The largest change rates are found in the CMSAF image, with positive changes covering most of the areas, consistent with the trend in Fig. 1d. An earlier study (Lang et al., 2020b) plotted the time series of individual satellites’ UTH from NOAA-15 to Metop-B for both FIDUCEO and CMSAF datasets (Fig. 6 in that article). Their Fig. 6b showed that offsets between the UTH time series from consecutive satellite missions in the CMSAF record tend to be positive over time. When all the satellites are merged into one time series, this may lead to a positive trend. 2. The infrared UTH dataset exhibits the largest propor- tions of dry areas at the peak of El Niño and La Niña events (more than 4 % larger ratio of dry areas com- pared to those of MW datasets). L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidit 6960 Figure 12. Change rates of the four UTH datasets during the com- mon period 1999 to 2017. water vapor, though. Over high elevations (similarly to over high latitudes) there are contributions of the surface tempera- ture to the radiances measured by satellite UTH sounders. A decrease in calculated UTH values over a high elevation can be caused by either a decrease in water vapor or an increase in the surface temperature. The clear-sky measurement ex- cludes some high-humidity data due to removal of cloudy pixels compared to MW datasets. The Jacobian of less-humid data has a lower peak in the atmosphere, and the lower tail of the Jacobian proﬁle is closer to the surface (e.g., see Fig. 1 in Brogniez et al., 2006). Over a high elevation, increasing surface effect can be included in the observation radiances. A warming at the surface may contribute more to an in- frared dataset due to a larger portion of less-humid data. Over the mid-latitude Paciﬁc, both NCEI and UMIAMI data show negative change rates in 45–60◦N, while the CMSAF dataset shows positive change rates. Over the Southern Hemisphere mid-latitude, the CMSAF dataset displays increased humid- ity, while both positive and negative change rates are found in the NCEI and UMIAMI datasets. 3.3 UTH changes during the common period of the datasets The common period when all four UTH datasets have data spans from 1999 to 2017. To analyze UTH changes of each dataset during the common period, we use the linear trend method to calculate the change rate of each grid, and the re- sults are displayed in Fig. 12. In this study, the linear trend method is employed to show the change rates during a rel- atively short common period as a way to examine dataset https://doi.org/10.5194/amt-15-6949-2022 Atmos. Meas. Tech., 15, 6949–6963, 2022 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity The HIRS dataset is the longest, over 43 years of observations so far, for long- term studies, and its variability, temporal phases, and spatial patterns are generally consistent with MW ob- servations. However, being a clear-sky dataset, it does not capture the most humid regions. The MW datasets have a shorter time series, but they retain almost all sky data, removing only the precipitating pixels, and thus have better sampling for a full spectrum of UTH espe- cially for very humid data. 7. The infrared and MW UTH datasets have their own strengths and weaknesses. The HIRS dataset is the longest, over 43 years of observations so far, for long- term studies, and its variability, temporal phases, and spatial patterns are generally consistent with MW ob- servations. However, being a clear-sky dataset, it does not capture the most humid regions. The MW datasets have a shorter time series, but they retain almost all sky data, removing only the precipitating pixels, and thus have better sampling for a full spectrum of UTH espe- cially for very humid data. 4 Conclusions The MW datasets have a larger proportion of humid measurements during El Niño events, while during a major La Niña such as the 2010–2011 event, the ratios of humid areas are close to each other among three UTH datasets (differences less than 1 %), except the CMSAF dataset which overall has larger humid areas. The three datasets that have mid-latitude coverage (Fig. 12a, b, and d) exhibit negative change rates over the Ti- betan Plateau. This may not necessarily indicate a decrease in 3. Through the common period of 1999 to 2017, differ- ences are observed in the changing rates of the datasets. https://doi.org/10.5194/amt-15-6949-2022 Atmos. Meas. Tech., 15, 6949–6963, 2022 6961 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity A wider spread of UTH moistening is observed in the CMSAF datasets. A wider spread of UTH moistening is observed in the CMSAF datasets. Author contributions. The research was designed by LS and CJS III. CJS III performed data analyses. LS wrote the paper with in- put from co-authors. LS, CJS III, VOJ, and ESC were involved in discussions on datasets and analysis results at various stages of the study. TL, SAB, and BJS provided valuable comments. All of the authors reviewed the manuscript and provided input. Author contributions. The research was designed by LS and CJS III. CJS III performed data analyses. LS wrote the paper with in- put from co-authors. LS, CJS III, VOJ, and ESC were involved in discussions on datasets and analysis results at various stages of the study. TL, SAB, and BJS provided valuable comments. All of the authors reviewed the manuscript and provided input. 4. The four datasets show that during a major El Niño event, there are signiﬁcant increases of UTH over a narrow belt of the equatorial central Paciﬁc consistent with the positive anomalies of the precipitation pattern, though typically the positive anomalies of SST cover a larger latitude span and are more prominent in the east- ern Paciﬁc. Negative anomalies develop over the weak- ened ascending branch of the Paciﬁc Walker circulation in the western Paciﬁc and eastern Indian Ocean where there is a positive anomaly of the 200 hPa velocity po- tential and over the enhanced descending branches of the local Hadley circulation along the Paciﬁc subtrop- ics. Competing interests. The contact author has declared that none of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Special issue statement. This article is part of the special issue “Analysis of atmospheric water vapour observations and their un- certainties for climate applications (ACP/AMT/ESSD/HESS inter- journal SI)”. It is not associated with a conference. 5. During a major El Niño, the spatial correlations between UTH and SST are not high, with the correlation val- ues in the range of 0.37 to 0.49. In the meantime, the spatial correlations between UTH and precipitation are higher, ranging from 0.60 to 0.75. References Adler, R. F., Huffman, G. J., Chang, A., Ferraro, R., Xie, P.-P., Janowiak, J., Rudolf, B., Schneider, U., Curtis, S., Bolvin, D., Gruber, A., Susskind, J., Arkin, P., and Nelkin, E.: The Version-2 Global Precipitation Climatology Project (GPCP) Monthly Precipitation Analysis (1979–Present), J. Hydrometeorol., 4, 1147–1167, https://doi.org/10.1175/1525- 7541(2003)004<1147:Tvgpcp>2.0.Co;2, 2003. Allan, R. P., Ringer, M. A., and Slingo, A.: Evaluation of moisture in the Hadley Centre climate model using simulations of HIRS water-vapour channel radiances, Q. J. Roy. Meteor. Soc., 129, 3371–3389, https://doi.org/10.1256/qj.02.217, 2003. Data availability. The CMSAF UTH dataset can be down- loaded from https://doi.org/10.5676/EUM_SAF_CM/UTH/V001 (Tsamalis et al., 2019). The FIDUCEO UTH data are obtained from https://doi.org/10.5285/2083b33b5c3d4cf0acb9a49226789caa (Lang et al., 2020a). The NCEI HIRS channel 12 brightness tem- perature data are available at https://doi.org/10.25921/zqqa-ks18 (Shi and NOAA CDR Program, 2021). Allan, R. P., Willett, K. M., John, V. O., and Trent, T.: Global Changes in Water Vapor 1979–2020, J. Geophys. Res.-Atmos., 127, e2022JD036728, https://doi.org/10.1029/2022JD036728, 2022. Atmos. Meas. Tech., 15, 6949–6963, 2022 L. Shi et al.: Intercomparison of satellite-derived upper tropospheric humidity The infrared dataset has lower correlation values (about 0.1 smaller) with SST, precipitation, and 200 hPa velocity potential com- pared to those for the MW UTH datasets due to the lack of very humid data in the infrared dataset. 5. During a major El Niño, the spatial correlations between UTH and SST are not high, with the correlation val- ues in the range of 0.37 to 0.49. In the meantime, the spatial correlations between UTH and precipitation are higher, ranging from 0.60 to 0.75. The infrared dataset has lower correlation values (about 0.1 smaller) with SST, precipitation, and 200 hPa velocity potential com- pared to those for the MW UTH datasets due to the lack of very humid data in the infrared dataset. Acknowledgements. This study is part of the GEWEX water vapor assessment (G-VAP) organized by the GEWEX Data and Analy- sis Panel (GDAP). We thank Jessica Matthews for reviewing the preprint. We are grateful to the three anonymous reviewers for con- structive comments and suggestions. 6. Though there are apparent and expected differences in the values of total UTH due to differences in the deﬁni- tion and in the gridding procedure, the tropical averaged anomalies of the datasets are close to each other (mostly within ±0.5 % over tropical-domain average), and more importantly the phases of the time series are generally consistent for variability studies. Financial support. Carl Schreck has been supported by the National Oceanic and Atmospheric Administration (grant no. NA19NES4320002). Financial support. Carl Schreck has been supported by the National Oceanic and Atmospheric Administration (grant no. NA19NES4320002). Review statement. This paper was edited by Shu-Peng Ho and re- viewed by three anonymous referees. 7. The infrared and MW UTH datasets have their own strengths and weaknesses. 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https://openalex.org/W2912436760	https://europepmc.org/articles/pmc6432047?pdf=render	English	null	Coronary artery plaque characteristics and treatment with biologic therapy in severe psoriasis: results from a prospective observational study	Cardiovascular research	2,019	public-domain	7,362	Youssef A. Elnabawi1, Amit K. Dey1, Aditya Goyal1, Jacob W. Groenendyk1, Jonathan H. Chung1, Agastya D. Belur1, Justin Rodante1, Charlotte L. Harrington1, Heather L. Teague1, Yvonne Baumer1, Andrew Keel1, Martin P. Playford1, Veit Sandfort1, Marcus Y. Chen1, Benjamin Lockshin2, Joel M. Gelfand3, David A. Bluemke4, and Nehal N. Mehta1* Youssef A. Elnabawi1, Amit K. Dey1, Aditya Goyal1, Jacob W. Groenendyk1, Jonathan H. Chung1, Agastya D. Belur1, Justin Rodante1, Charlotte L. Harrington1, Heather L. Teague1, Yvonne Baumer1, Andrew Keel1, Martin P. Playford1, Veit Sandfort1, Marcus Y. Chen1, Benjamin Lockshin2, Joel M. Gelfand3, David A. Bluemke4, and Nehal N. Mehta1* Youssef A. Elnabawi1, Amit K. Dey1, Aditya Goyal1, Jacob W. Groenendyk1, Jonathan H. Chung1, Agastya D. Belur1, Justin Rodante1, Charlotte L. Harrington1, Heather L. Teague1, Yvonne Baumer1, Andrew Keel1, Martin P. Playford1, Veit Sandfort1, Marcus Y. Chen1, Benjamin Lockshin2, Joel M. Gelfand3, David A. Bluemke4, and Nehal N. Mehta1* f Inﬂammation and Cardiometabolic Disease, National Heart, Lung, and Blood Institute; National Institutes of Health, Bethesda, MD, USA; 2D partment of Dermatology, University of Pennsylvania, Philadelphia, PA, USA; and 4Department of Radiology, University of Wisconsin, Madiso 4 October 2018; revised 17 December 2018; editorial decision 8 January 2019; accepted 12 January 2019; online publish-ahead-of-print 5 February 20 Received 24 October 2018; revised 17 December 2018; editorial decision 8 January 2019; accepted 12 January 2019; online publish-ahead-of-print Aims The use of biologic therapy has increased over the past decade well beyond primary autoimmune diseases. Indeed, a recent trial using an anti-IL-1beta antibody reduced second myocardial infarction (MI) in those who have had MI. Psoriasis is a chronic inﬂammatory disease often treated with biologics when severe, is associated with increased risk of MI, in part driven by high-risk coronary plaque phenotypes by coronary computed tomography angiography (CCTA). We hypothesized that we would observe a reduction in inﬂammatory-driven phenotypes of coronary pla- que, including non-calciﬁed coronary plaque burden and lipid-rich necrotic core in those treated with biologic ther- apy after one-year compared with non-biologic therapy. ...................................................................................................................................................................... In a prospective, observational study, 290 participants were recruited from 1 January 2013 through 31 October 2018 with 215 completing one-year follow-up. Of the 238, 121 consecutive participants who were biologic treat- ment naı¨ve at baseline were included. A blinded reader (blinded to patient demographics, visit and treatment) quan- tiﬁed total coronary plaque burden and plaque subcomponents (calciﬁed and non-calciﬁed) in the three main coro- nary vessels >2 mm using dedicated software (QAngio, Medis, Netherlands). Published by Oxford University Press on behalf of the European Society of Cardiology 2019. This work is written by US Government employees and is in the public domain in the US. Psoriasis • CCTA • Coronary artery disease • Biologic therapy • Coronary plaque characteristics Coronary artery plaque characteristics and treatment with biologic therapy in severe psoriasis: results from a prospective observational study Youssef A. Elnabawi1, Amit K. Dey1, Aditya Goyal1, Jacob W. Groenendyk1, Jonathan H. Chung1, Agastya D. Belur1, Justin Rodante1, Charlotte L. Harrington1, Heather L. Teague1, Yvonne Baumer1, Andrew Keel1, Martin P. Playford1, Veit Sandfort1, Marcus Y. Chen1, Benjamin Lockshin2, Joel M. Gelfand3, David A. Bluemke4, and Nehal N. Mehta1* 1Section of Inﬂammation and Cardiometabolic Disease, National Heart, Lung, and Blood Institute; National Institutes of Health, Bethesda, MD, USA; 2DermAssociates, Silver Spring, MD, USA; 3Department of Dermatology, University of Pennsylvania, Philadelphia, PA, USA; and 4Department of Radiology, University of Wisconsin, Madison, WI, USA Received 24 October 2018; revised 17 December 2018; editorial decision 8 January 2019; accepted 12 January 2019; online publish-ahead-of-print 5 February 2019 Ti f i i 18 d Cardiovascular Research (2019) 115, 721–728 FAST-TRACK COMMUNICATION doi:10.1093/cvr/cvz009 Coronary artery plaque characteristics and treatment with biologic therapy in severe psoriasis: results from a prospective observational study Youssef A. Elnabawi1, Amit K. Dey1, Aditya Goyal1, Jacob W. Groenendyk1, Jonathan H. Chung1, Agastya D. Belur1, Justin Rodante1, Charlotte L. Harrington1, Heather L. Teague1, Yvonne Baumer1, Andrew Keel1, Martin P. Playford1, Veit Sandfort1, Marcus Y. Chen1, Benjamin Lockshin2, Joel M. Gelfand3, David A. Bluemke4, and Nehal N. Mehta1* 1Section of Inﬂammation and Cardiometabolic Disease, National Heart, Lung, and Blood Institute; National Institutes of Health, Bethesda, MD, USA; 2DermAssociates, Silver Spring, MD, USA; 3Department of Dermatology, University of Pennsylvania, Philadelphia, PA, USA; and 4Department of Radiology, University of Wisconsin, Madison, WI, USA Received 24 October 2018; revised 17 December 2018; editorial decision 8 January 2019; accepted 12 January 2019; online publish-ahead-of-print 5 February 2019 Ti f i i 18 d Cardiovascular Research (2019) 115, 721–728 FAST-TRACK COMMUNICATION doi:10.1093/cvr/cvz009 Cardiovascular Research (2019) 115, 721–728 doi:10.1093/cvr/cvz009 FAST-TRACK COMMUNICATION ity Press on behalf of the European Society of Cardiology 2019. This work is written by US Government employees and is in the public dom 2.1 Study design and population 2.1 Study design and population y g p p In a prospective, observational study, 290 participants participating in an ongoing cohort study to understand the association between psoriasis and cardiometabolic disease under the Psoriasis, Atherosclerosis and Cardiometabolic Disease Initiative were recruited from 1 January 2013 through 31 October 2018 with 238 completing one-year follow-up and 121 consecutive patients meeting the inclusion criteria (Supplementary material online, Figure S1). Detailed inclusion and exclusion criteria of the study have been previously reported.7 A study provider confirmed the onset and duration of psoriasis and assessed psoriasis severity using the Psoriasis Area and Severity Index (PASI) score, which combines the se- verity of lesions and the area affected into a single score, considering ery- thema, induration, and desquamation within each lesion. Previous psoriasis literature has established that the degree of PASI score re- sponse defined as greater than 50% improvement is clinically significant and denotes meaningful improvement.8 For this study, only participants who were naı¨ve to biologic or systemic therapies at baseline were in- cluded and followed for one year with clinical and laboratory data as well as serial coronary computed tomography angiography (CCTA). Biologic therapy initiation was performed one day to one month after the initial visit whereby an independent dermatologist started treatment. Treatment agents included TNF-a inhibitors (adalimumab, etanercept), interleukin-12/23 inhibitor (ustekinumab), and interleukin-17 inhibitors (secukinumab, ixekizumab). Participants who elected to not receive bio- logic therapy at their follow-up visit were used as a referent group and To account for variable coronary artery lengths, plaque volume (in cu- bic millimetres) was divided by the corresponding segment length (in millimetres), yielding a plaque index.9 Total plaque burden was defined as the sum of calcified plaque burden and non-calcified plaque burden. Non-calcified plaque subcomponents were obtained after adaptively correcting for lumen attenuation and depicted based on Hounsfield Units derived by the software. Youssef A. Elnabawi1, Amit K. Dey1, Aditya Goyal1, Jacob W. Groenendyk1, Jonathan H. Chung1, Agastya D. Belur1, Justin Rodante1, Charlotte L. Harrington1, Heather L. Teague1, Yvonne Baumer1, Andrew Keel1, Martin P. Playford1, Veit Sandfort1, Marcus Y. Chen1, Benjamin Lockshin2, Joel M. Gelfand3, David A. Bluemke4, and Nehal N. Mehta1* Psoriasis patients were middle-aged [mean (standard deviation) age, 50.5 (12.1) years], mostly male (n = 70, 58%) with low cardiovascular risk by Framingham score [median (interquartile range, IQR), 3 (1–6)] and had moderate to severe skin disease at baseline [median (IQR) Psoriasis Area Severity Index, PASI, 8.6 (5.3–14.0)]. Biologic therapy was associated with a 6% reduc- tion in non-calciﬁed plaque burden (P = 0.005) reduction in necrotic core (P = 0.03), with no effect on ﬁbrous bur- den (P = 0.71). Decrease in non-calciﬁed plaque burden in the biologic treated group was signiﬁcant compared with slow plaque progression in non-biologic treated (D, -0.07 mm2 vs. 0.06 mm2; P = 0.02) and associated with bi- ologic treatment beyond adjustment for traditional cardiovascular risk factors (b = 0.20, P = 0.02). Conclusion In this observational study, we demonstrate that biologic therapy in severe psoriasis was associated with favourable modulation of coronary plaque indices by CCTA. These ﬁndings highlight the importance of systemic inﬂammation in coronary artery disease and support the conduct of larger, randomized trials. * Corresponding author. Tel: þ1 301 827 0483; fax: þ1 301 827 0915, E-mail: nehal.mehta@nih.gov 722 Y.A. Elnabawi et al. .......................................................................... 2.2 Sample size calculations Modulation of the primary outcome, non-calcified plaque burden, was assessed on a per-artery basis, yielding 363 total arteries in a cohort of 121 study subjects based on prior published methods.10,11 We hypothe- sized a 15% difference in non-calcified plaque burden with a standard de- viation (SD) of 0.5 between treatment groups. Thus, the evaluation of 182 arteries was required for a study with 90% power. ................................................................................................ Psoriasis is a chronic inflammatory skin disease associated with accel- erated atherosclerosis affecting about 3% of the population. Severe pso- riasis is associated with early MI risk by over 50%,4 with rates of coronary artery disease being similar to Type 2 diabetes.5 The inflamma- tory milieu of psoriatic skin harbours cytokines critical to early athero- genesis and plaque rupture, with derangements in pro-inflammatory and pro-atherogenic cytokines, such as IL-1b, interleukin-17 (IL-17), and tu- mour necrosis factor-a (TNF-a). ................................................................................................. 1. Introduction were treated with topical and/or light therapies only. Individuals on sys- temic therapies or started on statin treatment over the course of the one-year study period were excluded. Study protocols were approved by the institutional review board at the National Institutes of Health and all participants provided written informed consent. The study was in ac- cordance with the Declaration of Helsinki. Strengthening the reporting of observational studies in epidemiology guidelines were followed for reporting the findings.9 ................................................................................................. Cardiovascular disease remains the leading cause of death, with residual risk due to inflammation being an emerging critical target.1,2 In a recent study of patients with myocardial infarction (MI) and high residual inflam- matory risk (high sensitivity C-reactive protein >2 mg/L), canakinumab, a monoclonal antibody targeting interleukin-1b (IL-1b), decreased the rate of non-fatal MI, non-fatal stroke, and cardiovascular death without affecting cholesterol levels.3 Findings from the study support the need to expand our understanding of potential effects of biologic therapies on coronary vasculature. 2.3.2 Analysis All scans were read in a blinded fashion to patient characteristics, visit date, and treatment. Coronary plaque characteristics were analysed across each of the main coronary arteries >2 mm using dedicated soft- ware (QAngio CT, Medis; The Netherlands).10,11 Automated longitudi- nal contouring of the inner lumen and outer wall was performed and results were manually adjusted when clear deviations were present.12 Results of the automated contouring were also reviewed on transverse reconstructed cross-sections of the artery on a section-by-section basis at 0.25-mm increments. Lumen attenuation was adaptively corrected on an individual scan basis using gradient filters and intensity values within the artery. Intra-rater reliability was high, with intra-class correlation co- efficient = 0.900, 95% CI (0.903–0.919). 2.3 Coronary computed tomography angiography Psoriasis, when severe, is treated with biologic therapy. This provides a reliable model to study inflammatory atherogenesis and the longitudi- nal impact of modulating specific cytokines on vascular behaviour, while treating the primary skin disease with FDA approved biologic therapies.6 In this context, the aim of this study was to perform coronary artery pla- que characterization before and after biological therapy in an open-label, one-year follow up study. We hypothesized that these inflammatory driven phenotypes of coronary plaque, including lipid-rich, non-calcified coronary plaque burden, and lipid-rich necrotic core, would decrease following biological therapy compared with patients not treated with bi- ologic therapy after one-year. 2.3.1 Acquisition All patients underwent CCTA on the same day as blood draw, using the same CT scanner (320-detector row Aquilion ONE ViSION, Toshiba, Japan). Guidelines implemented by the NIH Radiation Exposure Committee were followed. Scans were performed with prospective EKG gating, 100 or 120 kV tube potential, tube current of 100–850mA adjusted to the patient’s body size, with a gantry rotation time of 275ms. Images were acquired at a slice thickness of 0.5 mm with a slice incre- ment of 0.25 mm. 2.4 Clinical data and laboratory measurements Upon recruitment of participants, initial contact with investigators in- volved a comprehensive medical history, physical examination, medica- tion evaluation, and anthropometric measurements. Blood samples were collected after an overnight fast. Samples were analysed for basic chemistry, complete lipid panel, insulin, and high sensitivity C-reactive protein at the NIH Clinical Center. Cholesterol efflux capacity was 723 Biologic therapy and coronary artery plaque in psoriasis ...................................................................... ...................................................................... .............................................................................................................................................................................................................................. Table 1 Baseline and one-year follow-up characteristics of patients with psoriasis Parameters Biologic treated (n 5 89) Non-biologic treated (n 5 32) At baseline Baseline One-year P-value Baseline One-year P-value P-value Demographics and medical history Age (years) 49.1 ± 12.2 50.2 ± 12.2 – 51.2 ± 12.0 53.1 ± 12.3 – 0.35 Males 50 (56) 50 (56) – 20 (63) 20 (63) – 0.65 Body mass index 29.9 ± 6.0 29.6 ± 6.1 0.18 29.4 ± 5.6 29.0 ± 5.4 0.06 0.57 Hypertension 27 (30) 26 (29) 0.71 10 (31) 9 (28) 0.32 0.63 Hyperlipidaemia 32 (36) 30 (34) 0.76 14 (44) 14 (44) 1.00 0.46 Statin treatment 25 (28) 22 (25) 0.71 10 (31) 9 (28) 0.32 0.92 Type-2 diabetes mellitus 8 (9) 6 (7) 0.16 2 (6) 3 (9) 0.32 0.60 Current smoker 9 (10) 7 (8) 0.18 4 (13) 4 (13) 1.00 0.59 Clinical and laboratory data Total cholesterol (mg/dL) 181.3 ± 33.3 181.2 ± 36.0 0.49 184.4 ± 38.5 183.4 ± 41.2 0.43 0.50 HDL cholesterol (mg/dL) 53.8 ± 14.2 54.7 ± 16.2 0.17 53.4 ± 16.2 55.8 ± 19.5 0.16 0.79 LDL cholesterol (mg/dL) 105.6 ± 28.0 102.4 ± 33.0 0.19 103.1 ± 28.2 98.8 ± 35.9 0.25 0.51 Framingham risk score 3 (1–6) 2 (1–5) 0.15 3 (2–7) 4 (1–7) 0.65 0.42 C-reactive protein 2.0 (0.8–5.0) 1.4 (0.7–3.6) <0.001 2.3 (0.6–4.5) 1.8 (0.7–3.8) 0.21 0.71 HOMA-IR 3.1 (2.0–5.6) 2.9 (1.9–4.9) 0.11 2.6 (1.6–4.9) 2.7 (1.8–5.3) 0.57 0.59 Psoriasis characterization PASI score 9.0 (5.6–15.0) 3.2 (1.8–5.7) <0.001 8.1 (5.0–12.0) 7.0 (4.0–9.9) 0.08 0.64 Disease duration 23.0 ± 14.4 24.0 ± 14.7 – 20.3 ± 14.6 21.3 ± 17.1 – 0.48 Topical therapy 56 (63) 39 (44) 0.03 22 (69) 25 (78) 0.32 0.62 Light therapy 16 (18) 11 (12) 0.25 9 (28) 10 (31) 0.66 0.08 Systemic therapy 0 (0) 0 (0) – 0 (0) 0 (0) – 1.00 Values are reported as mean ± SD or median (IQR) for continuous data and N (%) for categorical data. 2.5 Statistical analysis Skewness and kurtosis measures were considered to assess normality. Data were reported as mean with SD for parametric variables, median with interquartile range (IQR) for non-parametric variables and as per- centages for categorical variables. Parametric variables were compared between two groups using paired t-test. Non-parametric variables were compared using Wilcoxon signed-rank test and Pearson’s v2 test was performed for categorical variables between two groups. We conducted Spearman’s rank order correlation coefficient to evaluate the relation- ship between non-calcified plaque burden and different cardiometabolic risk factors. 2.4 Clinical data and laboratory measurements Two-tailed P-values less than 0.05 deemed signiﬁcant (bold values). HOMA-IR, homeostatic model assessment of insulin resistance; PASI, Psoriasis Area and Severity Index. aComparison between groups at baseline. Table 1 Baseline and one-year follow-up characteristics of patients with psoriasis Values are reported as mean ± SD or median (IQR) for continuous data and N (%) for categorical data. Two-tailed P-values less than 0.05 deemed signiﬁcant (bold values). HOMA-IR, homeostatic model assessment of insulin resistance; PASI, Psoriasis Area and Severity Index. aComparison between groups at baseline. ............................................................................... 3.1 Baseline characteristics of study groups 3.1 Baseline characteristics of study groups Study participants were middle-aged [mean (SD) age, 50.4 (12.1) years], mostly male (n = 70, 58%) with low cardiovascular risk by Framingham score [median (IQR), 3 (1–6)] and had moderate to severe skin disease at baseline [median (IQR) PASI, 8.6 (5.3–14.0)] (Table 1). There were no significant differences in demographic characteristics, in baseline medica- tion use or laboratory values between the two groups along with no bio- logic or systemic therapy use at baseline in either group. 3. Results measured using a validated ex vivo assay of J774 cholesterol-loaded mac- rophages as previously published.11 Blood inflammatory markers includ- ing interferon-c, TNF-a, and cytokines were quantified using multiplex ELISA assays (Mesoscale Diagnostics, Gaithersburg, MD, USA). 3.2 Relationship of non-calcified plaque burden with risk factors Table 2 demonstrates associations between non-calcified plaque burden and cardiometabolic risk factors. Non-calcified plaque burden was cor- related with traditional cardiovascular risk factors, including male gender (b = 0.37; P < 0.001), body mass index, (b = 0.52; P < 0.001), hyperten- sion (b = 0.24; P < 0.001), hyperlipidaemia (b = 0.10; P = 0.02), HDL cho- lesterol (b = -0.30; P < 0.001), Framingham risk score (b = 0.29; P < 0.001), C-reactive protein (b = 0.11, P = 0.005), and homeostatic model assessment insulin resistance score (HOMA-IR, b = 0.19; P < 0.001). Additionally, non-calcified plaque burden was correlated with skin disease severity as assessed by PASI score (b = 0.20; P < 0.001), which remained significant after adjustment for traditional cardiovascular risk factors and high sensitivity C-reactive protein (hsCRP) (b = 0.13; P < 0.001). ................................. To understand the change in various coronary parameters over one- year follow up, we used paired t-test for parametric variables, Wilcoxon signed-rank test for non-parametric variables and Pearson’s v2 test for categorical variables. The change in coronary parameters over one-year was compared between groups using paired t-test. We performed multi-variable linear regression analysis and adjusted for Framingham risk score, body mass index, and statin use. A two-tailed P-value <0.05 was considered statistically significant. Statistical analysis was performed using STATA-12 software (STATA Inc., College Station, TX, USA). 724 Y.A. Elnabawi et al. Y.A. Elnabawi et al. Table 3 summarizes measures of coronary artery disease burden as determined by CCTA. In those receiving biologic therapy, there was a 5% reduction in total coronary plaque burden [mean (SD), 1.30 mm2 (0.60) vs. 1.24 mm2 (0.60); P = 0.009], primarily driven by a reduction in non-calcified plaque burden [mean (SD), 1.22 mm2 (0.59) vs. 1.15 mm2 (0.60); P = 0.005] (Figure 1A and B). We observed no change in fibrous burden (P = 0.71), and there was a significant reduction in both fibro- fatty burden [mean (SD), 0.22 mm2 (0.19) vs. 0.10 mm2 (0.14); P = 0.004] and necrotic burden [mean (SD), 0.07 mm2 (0.19) vs. 0.03 mm2 (0.09); P = 0.03]. On the contrary, in those not receiving systemic or biologic therapy over one-year, there were no significant changes in total plaque burden [mean (SD), 1.28 mm2 (0.53) vs. 1.31 (0.48); P = 0.22] and non- calcified plaque burden [mean (SD), 1.19 mm2 (0.41) vs. 3.3 Modulation of coronary plaque characteristic following treatment Table 2 Multivariable linear regressions for the associa- tions between non-calciﬁed coronary plaque burden and cardiovascular risk factors and psoriasis characterization When comparing change in plaque characteristics between groups over one-year, the decrease in non-calcified plaque burden in the bio- logic treated group was significant compared with non-biologic treated (D, -0.07 mm2 vs. 0.06 mm2; P = 0.03) and associated with biologic ther- apy even after adjustment for traditional cardiovascular risk factors, in- cluding Framingham risk score, body mass index, and statin use (b = 0.20, P = 0.02). We performed an exploratory analysis in the biologic treated group by stratifying by treatment agents (anti-TNF, anti-IL12/23, and anti-IL17). There were no significant differences in demographic characteristics, in baseline medication use, or laboratory values between the three treat- ment groups (Supplementary material online, Table S1). After one-year of therapy, an improvement in hsCRP was observed in the anti-IL12/23 and anti-IL17 treated groups (P = 0.02 and P = 0.01; respectively). An im- provement in HDL cholesterol was observed only in the anti-IL17 treated patients [mean (SD), 54.2 (19.9) vs. 61.2 (28.6); P = 0.03]. At baseline, there were no differences in coronary characteristics between the three groups. After one-year of therapy, we observed the following: a 5% reduction in non-calcified plaque burden on anti-TNF therapy (P = 0.06), a 2% reduction on anti-IL12/23 therapy (P = 0.36), and a 12% reduction on anti-IL17 (P = <0.001) (Table 4, Supplementary material on- line, Table S2). The reduction in coronary plaque burden observed on anti-IL17 therapy was significantly greater than that observed on anti- IL12/23 and no biologic treatment. Reduction in non-calcified coronary ................................................................................ ................................................................................ .............................................................................................................................................................................................................................. 3.3 Modulation of coronary plaque characteristic following treatment At one-year follow-up (Table 1), we observed a significant improvement in psoriasis by PASI score in the biologic treated group (64% improve- ment, P < 0.001) and not in the non-biologic treated group [median (IQR), 8.1 (5.0–12.0) vs. 7 (4.0–9.9); P = 0.08]. There were no significant effects on body mass index, lipids, or glucose. A reduction in hsCRP was seen only in the biologic treated group [median (IQR), 2.0mg/L (0.8–5.0) vs. 1.4mg/L (0.7–3.6); P < 0.001]. No participants in either group started a new lipid lowering therapy during the study period. ...................................................................................................... Table 2 Multivariable linear regressions for the associa- tions between non-calciﬁed coronary plaque burden and cardiovascular risk factors and psoriasis characterization Parameters Non-calcified plaque burden (mm2) (n 5 121) Demographics and medical history b (P-value) Age (years) 0.04 (0.40) Males 0.37 (<0.001) Body mass index 0.52 (<0.001) Hypertension 0.24 (<0.001) Hyperlipidaemia 0.10 (0.02) Type-2 diabetes mellitus 0.05 (0.19) Current smoker 0.10 (0.03) Clinical and laboratory data Total cholesterol (mg/dL) -0.05 (0.45) HDL cholesterol (mg/dL) -0.30 (<0.001) LDL cholesterol (mg/dL) -0.01 (0.72) Framingham risk score 0.29 (<0.001) C-reactive protein 0.11 (0.005) HOMA-IR 0.19 (<0.001) Psoriasis characterization PASI score 0.20 (<0.001) Disease duration 0.05 (0.24) All data in the table is expressed as standardized b (P-value). Two-tailed P-values less than 0.05 deemed signiﬁcant (bold values). HOMA-IR, homeostasis model assessment of insulin resistance; PASI, Psoriasis Area and Severity Index. ...................................................................................................... Table 2 Multivariable linear regressions for the associa- tions between non-calciﬁed coronary plaque burden and cardiovascular risk factors and psoriasis characterization Parameters Non-calcified plaque burden (mm2) (n 5 121) Demographics and medical history b (P-value) Age (years) 0.04 (0.40) Males 0.37 (<0.001) Body mass index 0.52 (<0.001) Hypertension 0.24 (<0.001) Hyperlipidaemia 0.10 (0.02) Type-2 diabetes mellitus 0.05 (0.19) Current smoker 0.10 (0.03) Clinical and laboratory data Total cholesterol (mg/dL) -0.05 (0.45) HDL cholesterol (mg/dL) -0.30 (<0.001) LDL cholesterol (mg/dL) -0.01 (0.72) Framingham risk score 0.29 (<0.001) C-reactive protein 0.11 (0.005) HOMA-IR 0.19 (<0.001) Psoriasis characterization PASI score 0.20 (<0.001) Disease duration 0.05 (0.24) All data in the table is expressed as standardized b (P-value). Two-tailed P-values less than 0.05 deemed signiﬁcant (bold values). HOMA-IR, homeostasis model assessment of insulin resistance; PASI, Psoriasis Area and Severity Index. 3.2 Relationship of non-calcified plaque burden with risk factors 1.25 (0.10); P = 0.17] with no change in fibrous burden (4% decrease, P = 0.22), a sig- nificant increase in fibro-fatty burden (38% increase, P = 0.004) and non- significant increase in necrotic core (33% increase, P = 0.27) (Figure 2A and B). 3.3 Modulation of coronary plaque characteristic following treatment We recently showed that this increase in MI in psoriasis might be due to early, increased coronary artery disease that is equivocal to individuals who are on average 10 years older with diagnosed hyperlipidaemia.11 Whether these burdens modulate with skin disease treatment has been the topic of intense investigation. Furthermore, when a sample of psoriasis patients was followed for one year after any treatment, a 6% decrease in aortic vascular inflammation was observed6; however, the coronary arteries were not analysed in that study. CCTA has long been utilized for characterization of coronary plaque burden beyond X-ray angiography and has been extensively compared with and validated against intravascular ultrasound.14 CCTA provides characterization of not only lumen stenosis and arterial remodelling, but also plaque subcomponents, including calcified, non-calcified, and high- risk features.15 Studies have shown that there is an increase in non- calcified plaque volumes in acute coronary syndrome patients,16 obese diabetics,9 and also undergoes modulation in response to statin ther- apy.17,18 Recently, when CCTA plaque features are accounted for, patients with widespread non-obstructive coronary artery disease had similar event rates when compared with patients with localized obstruc- tive disease,19 suggesting that plaque characteristics are important in de- fining accurate cardiovascular risk beyond obstructive stenosis. Figure 1 Change in coronary plaque burden components over one- year by treatment. (A) Percent change in coronary plaque burden com- ponents over one-year by treatment. (B) Change in non-calcified plaque burden over one-year by treatment. g y The use of biologic therapy for psoriasis has rapidly increased over the past decade given its remarkable success in early clearance of psori- atic plaque. First, we observed reduction in non-calcified plaque across all three classes of biologic agents in the study with varying degrees, sug- gesting that clearance of psoriasis itself is important in the context of vas- cular disease. Anti-TNF therapy is commonly accepted as the first line biologic agent for the management of psoriasis; however, some of the patients on this treatment do not have adequate response.20 In psoriasis, TNF inhibitors have also been linked to worsening of cardiometabolic risk factors, including weight gain and a shift in apolipoprotein B,21 and re- cently were shown to not reduce vascular inflammation.22 In that study, however, important inflammatory biomarkers, including TNF-a, interleukin-6, hsCRP, and glycoprotein acetylation, all decreased follow- ing anti-TNF therapy. 3.3 Modulation of coronary plaque characteristic following treatment Table 3 Coronary artery parameters by artery at baseline and one-year follow-up Coronary characterization Biologic treated (n 5 267 arteries) Non-biologic treated (n 5 96 arteries) Baseline One-year Change (%) P-value Baseline One-year Change (%) P-value Total plaque burden (mm2) 1.30 ± 0.60 1.24 ± 0.60 -0.06 (-5) 0.009 1.28 ± 0.53 1.31 ± 0.48 0.03 (2) 0.22 Dense-calciﬁed plaque burden (mm2) 0.064 ± 0.12 0.067 ± 0.14 0.003 (5) 0.36 0.082 ± 0.17 0.084 ± 0.15 0.002 (2) 0.48 Non-calciﬁed plaque burden (mm2) 1.22 ± 0.59 1.15 ± 0.60 -0.07 (-6) 0.005 1.19 ± 0.41 1.25 ± 0.41 0.06 (5) 0.17 Plaque morphology index Fibrous burden (mm2) 0.99 ± 0.45 0.98 ± 0.51 -0.01 (-1) 0.71 0.98 ± 0.32 0.94 ± .31 -0.04 (-4) 0.22 Fibro-fatty burden (mm2) 0.22 ± 0.19 0.10 ± 0.14 -0.12 (-55) 0.004 0.16 ± 0.15 0.22 ± 0.14 0.06 (38) 0.004 Necrotic burden (mm2) 0.07 ± 0.19 0.03 ± 0.09 -0.04 (-57) 0.03 0.06 ± 0.08 0.08 ± 0.22 0.02 (33) 0.27 Values are reported as mean ± SD for continuous data. Two-tailed P-values less than 0.05 deemed signiﬁcant (bold values). Table 3 Coronary artery parameters by artery at baseline and one-year follow-up 725 Biologic therapy and coronary artery plaque in psoriasis light therapy only as our reference. In this referent group, we observed progression of coronary artery disease with conversion of fibrous bur- den to fibro-fatty burden, suggestive of lipid infiltration within the coro- nary plaque. In those treated with biological therapy, we found that inflammatory driven phenotypes including lipid-rich plaque and the ne- crotic core decreased following therapy. Taken together, these data pro- vide preliminary evidence that treatment with biologic modulates coronary artery plaque in psoriasis. Figure 1 Change in coronary plaque burden components over one- year by treatment. (A) Percent change in coronary plaque burden com- ponents over one-year by treatment. (B) Change in non-calcified plaque burden over one-year by treatment. Inflammation is causal in atherosclerosis and still accounts for approxi- mately 20–30% of residual risk for cardiovascular events in the popula- tion.13 Those with inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis have a disproportional rate of cardiovascular events compared with age and gender matched counter- parts. Therefore, these populations serve as a unique vehicle to understand inflammatory atherogenesis. 3.3 Modulation of coronary plaque characteristic following treatment Observational studies have shown that biological therapy, more specifically TNF inhibitors, reduces MI,23 suggesting that longer term observed benefit may relate to coronary plaque modulation over time. The cardiovascular effects of newer, cytokine specific biologic agents have yet to be extensively studied in psoriasis. In a meta-analysis studying the association of major adverse cardiovascular events with the use of anti-IL12/23 agents, the potential of these agents to further in- crease cardiovascular morbidity could not be excluded.24,25 However, previous literature in animal studies has been conflicting regarding whether IL17 is pro- vs. anti-atherogenic.26 While some studies have suggested the pro-atherogenic effects of IL17,27,28 a recent athero- plaque burden on anti-TNF therapy was only significant when compared with non-biologic treated patients (P < 0.01). Finally, we explored the modulation of inflammatory blood markers (IFN-c, TNF-a, IL-6, IL1-b) with treatment of skin disease. PASI score was associated with IFN-c (b = 0.12; P = 0.003), TNF-alpha (b = 0.08; P = 0.05), and IL-6 (b = 0.10; P = 0.02) at baseline. After one-year of bio- logic therapy, there were significant reductions in interferon-c [median (IQR), 10.7 (5.6–28.0) vs. 10.2 (5.2–20.3); P = 0.02], TNF-a [median (IQR), 1.7 (1.0–3.5) vs. 1.3 (0.6–2.5); P = 0.04], and interleukin-6 [median (IQR), 1.4 (0.9–3.0) vs. 1.2 (0.7–2.1); P = 0.01]. These findings were not observed in the non-biologic treated group (Supplementary material on- line, Table S3). .................................. 4. Discussion Herein, this observational study demonstrated favourable modulation in coronary artery plaque disease indices by CCTA in a consecutive sample of severe psoriasis patients treated with commonly used biological clas- ses of drugs: anti-TNF, anti-IL12/23, and anti-IL17, compared with those not treated with biologic therapy. Despite not knowing the specific pro- gression rate of subclinical atherosclerosis on CCTA in psoriasis, we used those with similar disease patterns choosing to receive topical or 726 Y.A. Elnabawi et al. eft anterior descending artery plaque identified before (2A) and after (2B) biologic therapy. (A) (a) Longitudinal planar and (b) curved planar nd d) Representative cross-sectional views with colour overlay for plaque subcomponents. Lumen is encircled in yellow, vessel wall in orange onents in between, including fibrous (dark green), fibro-fatty (light green), necrotic (red), and dense-calcified (white). Non-calcified plaque 3 mm2 and total atheroma volume = 99.2 mm3. (B) (a) Longitudinal planar and (b) curved planar reformat. (c and d) Representative cross- ws with colour overlay for plaque subcomponents. Lumen is encircled in yellow, vessel wall in orange with subcomponents in between, us (dark green), fibro-fatty (light green), necrotic (red), and dense-calcified (white). Non-calcified plaque burden = 0.85 mm2 and total ather- 80.6 mm3. Figure 2 Left anterior descending artery plaque identified before (2A) and after (2B) biologic therapy. (A) (a) Longitudinal planar and (b) curved planar reformat. (c and d) Representative cross-sectional views with colour overlay for plaque subcomponents. Lumen is encircled in yellow, vessel wall in orange with subcomponents in between, including fibrous (dark green), fibro-fatty (light green), necrotic (red), and dense-calcified (white). Non-calcified plaque burden = 1.03 mm2 and total atheroma volume = 99.2 mm3. (B) (a) Longitudinal planar and (b) curved planar reformat. (c and d) Representative cross- sectional views with colour overlay for plaque subcomponents. Lumen is encircled in yellow, vessel wall in orange with subcomponents in between, including fibrous (dark green), fibro-fatty (light green), necrotic (red), and dense-calcified (white). Non-calcified plaque burden = 0.85 mm2 and total ather- oma volume = 80.6 mm3. protective pathway has been proposed through regulation of IFN-c pro- ducing Th1 cells.29 In our present study, we observed the greatest per- cent reduction of non-calcified plaque burden in patients on anti-IL17 therapy with a reduction in necrotic core suggesting a potential role for IL17 in atherosclerotic pathways. Funding This work was supported by the National Heart, Lung and Blood Institute (NHLBI) Intramural Research Program (HL006193-02). This re- search was also made possible through the NIH Medical Research Scholars Program, a public-private partnership supported jointly by the NIH and generous contributions to the Foundation for the NIH from the Doris Duke Charitable Foundation (DDCF Grant #2014194), the American Association for Dental Research, the Colgate-Palmolive Company, Genentech, Elsevier, and other private donors. References 1. Ridker PM, Rifai N, Pfeffer MA, Sacks FM, Moye LA, Goldman S, Flaker GC, Braunwald E. Inflammation, pravastatin, and the risk of coronary events after myocar- dial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events (CARE) Investigators. Circulation 1998;98:839–844. In conclusion, we demonstrate that treatment of psoriasis with biologic therapy is associated with a reduction of non-calcified coronary plaque and improvement in plaque morphology compared with those not treated with biologic therapy. These findings highlight the potential role of quelling residual inflammation in cardiovascular disease and risk reduc- tion. These findings support the conduct of larger randomized trials of bi- ologic therapy on cardiovascular disease in psoriasis and potentially other inflammatory diseases. 2. Ridker PM. Clinical application of C-reactive protein for cardiovascular disease detec- tion and prevention. Circulation 2003;107:363–369. 2. Ridker PM. Clinical application of C-reactive protein for cardiovascular disease detec- tion and prevention. Circulation 2003;107:363–369. 3. Ridker PM, Everett BM, Thuren T, MacFadyen JG, Chang WH, Ballantyne C, Fonseca F, Nicolau J, Koenig W, Anker SD, Kastelein JJP, Cornel JH, Pais P, Pella D, Genest J, Cifkova R, Lorenzatti A, Forster T, Kobalava Z, Vida-Simiti L, Flather M, Shimokawa H, Ogawa H, Dellborg M, Rossi PRF, Troquay RPT, Libby P, Glynn RJ; CANTOS Trial Group. Antiinflammatory therapy with canakinumab for atherosclerotic disease. N Engl J Med 2017;377:1119–1131. 4. Gelfand JM, Dommasch ED, Shin DB, Azfar RS, Kurd SK, Wang X, Troxel AB. The risk of stroke in patients with psoriasis. J Invest Dermatol 2009;129:2411–2418. Authors’ contributions Integrity of the data: Y.A.E. and N.N.M. had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Concept and design: Y.A.E. and N.N.M. conceived the study concept and the study design was by N.N.M. Acquisition, analysis, or interpretation of data: J.R., Y.A.E., M.Y.C. and N.N.M. acquired and analysed the data. Drafting of the manuscript: Y.A.E. drafted the manu- script. Critical revision of the manuscript for important intellectual con- tent: All co-authors provided critical revisions of the manuscript. Statistical analysis: Y.A.E. and A.K.D. performed analyses. Administrative, technical, or material support: N.N.M. provided technical guidance to Y.A.E. and A.K.D. during the study. Study supervision: The study was con- ducted under the supervision of N.N.M. 7. Harrington CL, Dey AK, Yunus R, Joshi AA, Mehta NN. Psoriasis as a human model of disease to study inflammatory atherogenesis. Am J Physiol Heart Circ Physiol 2017; 312:H867–H873. 8. Carlin CS, Feldman SR, Krueger JG, Menter A, Krueger GG. A 50% reduction in the Psoriasis Area and Severity Index (PASI 50) is a clinically significant endpoint in the assessment of psoriasis. J Am Acad Dermatol 2004;50:859–866. 9. von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP. The strengthening the reporting of observational studies in epidemiology (strobe) state- ment: guidelines for reporting observational studies. Ann Intern Med 2007;147:573–577. 10. Kwan AC, May HT, Cater G, Sibley CT, Rosen BD, Lima JAC, Rodriguez K, Lappe DL, Muhlestein JB, Anderson JL, Bluemke DA. Coronary artery plaque volume and obesity in patients with diabetes: the factor-64 study. Radiology 2014;272:690–699. 11. Lerman JB, Joshi AA, Chaturvedi A, Aberra TM, Dey AK, Rodante JA, Salahuddin T, Chung JH, Rana A, Teague HL, Wu JJ, Playford MP, Lockshin BA, Chen MY, Sandfort V, Bluemke DA, Mehta NN. Coronary plaque characterization in psoriasis reveals high-risk features that improve after treatment in a prospective observational study. Circulation 2017;136:263–276. 4. Discussion It has been implicated in literature that IL17 is a central mediator in lipoprotein entrapment, leading to vascular stiffness and promoting early atherosclerosis.30 Mouse studies have also suggested that IL17 increases monocyte adhesion to the vascular walls, promoting inflammatory cytokine production and endothelial dys- function,31 with this phenomenon being normalized by IL17A blockade. Moreover, the potential role of IL17 in linking skin disease and vascular disease in psoriasis was expanded on in another mice study whereby clearance of psoriatic skin manifestations by IL17 blockade was shown to diminish peripheral oxidative stress and vascular dysfunction.32 Taken to- gether, these data provide evidence to support further investigation of IL17 blockade on coronary disease in humans. Our study does have important limitations. This was an observational study, and therefore, is subjected to potential for confounders compared with a randomized clinical trial. Moreover, the use of biologic agents was open-label, non-randomized, in a small sample and with a short duration of follow-up. However, this is the largest consecutive sample of psoriasis patients followed over time using CCTA. Furthermore, the biologic treated groups had variability in baseline coronary parameters due to a small sample size. Finally, we have not studied hard, cardiovascular events, but instead used coronary artery plaque indices by CCTA to un- derstand modulation on cardiovascular disease risk. 727 Biologic therapy and coronary artery plaque in psoriasis ...................................................................................................... Table 4 Change in non-calciﬁed coronary plaque burden over one-year between treatment groups Treatments Change over one-year (mm2) (%) P-value Anti-TNF therapy (n = 48) -0.06 (-5) – vs. Anti-IL12/23 – -0.02 (-2) 0.27 vs. Anti-IL17 – -0.15 (-12) 0.08 vs. NBT – 0.06 (5) 0.009 Anti-IL12/23 therapy (n = 19) -0.02 (-2) – vs. Anti-IL17 – -0.15 (-12) 0.01 vs. NBT – 0.06 (5) 0.09 Anti-IL17 therapy (n = 22) -0.15 (-12) – vs. NBT – 0.06 (5) 0.005 Values are reported as Mean (% change) for continuous data. Two-tailed P-values less than 0.05 signiﬁcant (bold values). IL, interleukin; NBT, non-biologic treated. consultant for Coherus (DSMB), Dermira, Janssen Biologics, Merck (DSMB), Novartis Corp, Regeneron, Dr. Reddy’s labs, Sanofi and Pfizer Inc., receiving honoraria; and receives research grants (to the Trustees of the University of Pennsylvania) from Abbvie, Janssen, Novartis Corp, Regeneron, Sanofi, Celgene, and Pfizer Inc.; and received payment for continuing medical education work related to psoriasis that was sup- ported indirectly by Lilly and Abbvie. J.M.G. Supplementary material 5. Mansouri B, Kivelevitch D, Natarajan B, Joshi AA, Ryan C, Benjegerdes K, Schussler JM, Rader DJ, Reilly MP, Menter A, Mehta NN. Comparison of coronary artery cal- cium scores between patients with psoriasis and type 2 diabetes. JAMA Dermatol 2016;152:1244–1253. Supplementary material is available at Cardiovascular Research online. 6. Dey AK, Joshi AA, Chaturvedi A, Lerman JB, Aberra TM, Rodante JA, Teague HL, Harrington CL, Rivers JP, Chung JH, Kabbany MT, Natarajan B, Silverman JI, Ng Q, Sanda GE, Sorokin AV, Baumer Y, Gerson E, Prussick RB, Ehrlich A, Green LJ, Lockshin BN, Ahlman MA, Playford MP, Gelfand JM, Mehta NN. Association between skin and aortic vascular inflammation in patients with psoriasis: a case-cohort study using positron emission tomography/computed tomography. JAMA Cardiol 2017;2:1013–1018. 4. Discussion is a co-patent holder of resi- quimod for treatment of cutaneous T cell lymphoma. All other authors declared no conflict of interest. Table 4 Change in non-calciﬁed coronary plaque burden over one-year between treatment groups Acknowledgements The IL-17A/IL-17RA axis plays a proa- therogenic role via the regulation of aortic myeloid cell recruitment. Circ Res 2012; 110:675–687. 17. Sandfort V, Lima JA, Bluemke DA. Noninvasive imaging of atherosclerotic plaque progression: status of coronary computed tomography angiography. Circ Cardiovasc Imaging 2015;8:e003316. 27. Erbel C, Chen L, Bea F, Wangler S, Celik S, Lasitschka F, Wang Y, Bockler D, Katus HA, Dengler TJ. Inhibition of IL-17A attenuates atherosclerotic lesion development in apoE-deficient mice. J Immunol 2009;183:8167–8175. 18. Lo J, Lu MT, Ihenachor EJ, Wei J, Looby SE, Fitch KV, Oh J, Zimmerman CO, Hwang J, Abbara S, Plutzky J, Robbins G, Tawakol A, Hoffmann U, Grinspoon SK. Effects of statin therapy on coronary artery plaque volume and high-risk plaque morphology in HIV-infected patients with subclinical atherosclerosis: a randomised, double-blind, placebo-controlled trial. Lancet HIV 2015;2:e52–e63. 28. Madhur MS, Funt SA, Li L, Vinh A, Chen W, Lob HE, Iwakura Y, Blinder Y, Rahman A, Quyyumi AA, Harrison DG. Role of interleukin 17 in inflammation, atherosclero- sis, and vascular function in apolipoprotein e-deficient mice. Arterioscler Thromb Vasc Biol 2011;31:1565–1572. placebo-controlled trial. Lancet HIV 2015;2:e52–e63. 19. Bittencourt MS, Hulten E, Ghoshhajra B, O’Leary D, Christman MP, Montana P, Truong QA, Steigner M, Murthy VL, Rybicki FJ, Nasir K, Gowdak LHW, Hainer J, Brady TJ, Di Carli MF, Hoffmann U, Abbara S, Blankstein R. Prognostic value of non- obstructive and obstructive coronary artery disease detected by coronary computed tomography angiography to identify cardiovascular events. Circ Cardiovasc Imaging 2014;7:282–291. 29. Danzaki K, Matsui Y, Ikesue M, Ohta D, Ito K, Kanayama M, Kurotaki D, Morimoto J, Iwakura Y, Yagita H, Tsutsui H, Uede T. Interleukin-17A deficiency accelerates unsta- ble atherosclerotic plaque formation in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2012;32:273–280. 20. Boehncke WH, Menter A. Burden of disease: psoriasis and psoriatic arthritis. Am J Clin Dermatol 2013;14:377–388. 30. Huang LH, Zinselmeyer BH, Chang CH, Saunders BT, Elvington A, Baba O, Broekelmann TJ, Qi L, Rueve JS, Swartz MA, Kim BS, Mecham RP, Wiig H, Thomas MJ, Sorci-Thomas MG, Randolph GJ. Interleukin-17 drives interstitial entrapment of tissue lipoproteins in experimental psoriasis. Cell Metabol 2018;doi: 10.1016/j.cmet.2018.10.006. 21. Sattar N, Crompton P, Cherry L, Kane D, Lowe G, McInnes IB. Effects of tumor ne- crosis factor blockade on cardiovascular risk factors in psoriatic arthritis: a double- blind, placebo-controlled study. Arthritis Rheum 2007;56:831–839. 31. Acknowledgements 12. Salahuddin T, Natarajan B, Playford MP, Joshi AA, Teague H, Masmoudi Y, Selwaness M, Chen MY, Bluemke DA, Mehta NN. Cholesterol efflux capacity in humans with psoriasis is inversely related to non-calcified burden of coronary atherosclerosis. Eur Heart J 2015;36:2662–2665. We would like to acknowledge and thank the NIH Clinical Center out- patient clinic-7 nurses for their invaluable contribution to the process of patient recruitment. 13. Harrington RA. Targeting inflammation in coronary artery disease. N Engl J Med 2017;377:1197–1198. Conflict of interest: N.N.M. is a full-time US Government Employee and receives research grants to the NHLBI from AbbVie, Janssen, Celgene and Novartis. J.M.G. in the past 12 months has served as a 14. Fischer C, Hulten E, Belur P, Smith R, Voros S, Villines TC. Coronary CT angiography versus intravascular ultrasound for estimation of coronary stenosis and atheroscle- rotic plaque burden: a meta-analysis. J Cardiovasc Comput Tomogr 2013;7:256–266. 728 Y.A. Elnabawi et al. 15. Motoyama S, Ito H, Sarai M, Kondo T, Kawai H, Nagahara Y, Harigaya H, Kan S, Anno H, Takahashi H, Naruse H, Ishii J, Hecht H, Shaw LJ, Ozaki Y, Narula J. Plaque characteriza- tion by coronary computed tomography angiography and the likelihood of acute coro- nary events in mid-term follow-up. J Am Coll Cardiol 2015;66:337–346. 15. Motoyama S, Ito H, Sarai M, Kondo T, Kawai H, Nagahara Y, Harigaya H, Kan S, Anno H, 24. Tzellos T, Kyrgidis A, Trigoni A, Zouboulis CC. Association of ustekinumab and briakinumab with major adverse cardiovascular events: an appraisal of meta- analyses and industry sponsored pooled analyses to date. Dermatoendocrinol 2012;4: 320–323. nary events in mid-term follow-up. J Am Coll Cardiol 2015;66:337–346. 25. Reich K, Langley RG, Lebwohl M, Szapary P, Guzzo C, Yeilding N, Li S, Hsu MC, Griffiths CE. Cardiovascular safety of ustekinumab in patients with moderate to se- vere psoriasis: results of integrated analyses of data from phase II and III clinical stud- ies. Br J Dermatol 2011;164:862–872. 16. Dey D, Achenbach S, Schuhbaeck A, Pflederer T, Nakazato R, Slomka PJ, Berman DS, Marwan M. Comparison of quantitative atherosclerotic plaque burden from cor- onary CT angiography in patients with first acute coronary syndrome and stable cor- onary artery disease. J Cardiovasc Comput Tomogr 2014;8:368–374. onary artery disease. J Cardiovasc Comput Tomogr 2014;8:368–374. 26. Butcher MJ, Gjurich BN, Phillips T, Galkina EV. Acknowledgements Nordlohne J, Helmke A, Ge S, Rong S, Chen R, Waisman A, Haller H, von Vietinghoff S. Aggravated atherosclerosis and vascular inflammation with reduced kid- ney function depend on interleukin-17 receptor A and are normalized by inhibition of interleukin-17A. JACC Basic Transl Sci 2018;3:54–66. 22. Mehta NN, Shin DB, Joshi AA, Dey AK, Armstrong AW, Duffin KC, Fuxench ZC, Harrington CL, Hubbard RA, Kalb RE, Menter A, Rader DJ, Reilly MP, Simpson EL, Takeshita J, Torigian DA, Werner TJ, Troxel AB, Tyring SK, Vanderbeek SB, Van Voorhees AS, Playford MP, Ahlman MA, Alavi A, Jm G. Effect of 2 psoriasis treat- ments on vascular inflammation and novel inflammatory cardiovascular biomarkers: a randomized placebo-controlled trial. Circ Cardiovasc Imaging 2018;11:e007394. J 32. Schuler R, Brand A, Klebow S, Wild J, Protasio Veras F, Ullmann E, Roohani S, Kolbinger F, Kossmann S, Wohn C, Daiber A, Munzel T, Wenzel P, Waisman A, Clausen BE, Karbach S. Antagonization of IL-17A attenuates skin inflammation and vascular dysfunction in mouse models of psoriasis. J Invest Dermatol 2018;doi: 10.1016/j.jid.2018.09.021. omized placebo-controlled trial. Circ Cardiovasc Imaging 2018;11:e0073 23. Wu JJ, Poon KY, Channual JC, Shen AY. Association between tumor necrosis factor inhibitor therapy and myocardial infarction risk in patients with psoriasis. Arch Dermatol 2012;148:1244–1250.
https://openalex.org/W4254040107	https://www.researchsquare.com/article/rs-278592/latest.pdf	English	null	Regeneration Dynamics in Fragmented Landscapes at the Leading Edge of Distribution: Quercus Suber L. as A Study Case	Research Square (Research Square)	2,021	cc-by	11,700	Regeneration Dynamics in Fragmented Landscapes at the Leading Edge of Distribution: Quercus Suber L. as A Study Case Jorge Montero-Muñoz Centro de Investigación y de Estudios Carmen Ureña University of Salamanca Diego Navarro University of Salamanca Valentín Herrera University of Salamanca Pilar Alonso-Rojo University of Salamanca Héctor Hernández-Alonso University of Salamanca María Fernanda Cepeda-González University of Salamanca Luis Carlos Jovellar Junta de Castilla y León Belén Fernández-Santos University of Salamanca Fernando Silla (  fsilla@usal.es ) University of Salamanca https://orc Research Article Keywords: Quercus suber, recruitment dynamics, edge distribution, secondary succession, growth-climate relationships Posted Date: March 11th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-278592/v1 Version of Record: A version of this preprint was published at Plant and Soil on August 5th, 2021. See the published version at https://doi.org/10.1007/s11104-021-05077-7. DOI: https://doi.org/10.21203/rs.3.rs-278592/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/28 Version of Record: A version of this preprint was published at Plant and Soil on August 5th, 2021. See the published version at https://doi.org/10.1007/s11104-021-05077-7. Version of Record: A version of this preprint was published at Plant and Soil on August 5th, 2021. See the published version at https://doi.org/10.1007/s11104-021-05077-7. Page 2/28 Results Succession was arrested in plots without tree remnants after cultivation abandonment. By contrast, remnant trees were accelerators of forest recovery. Tree cover played a fundamental role in Quercus recruitment throughout seed dispersal and facilitation effects that ameliorate summer drought. However, soil variables also significantly explained much of the variance observed and are important for understanding differences in regeneration. Winter and spring precipitation exerted a positive effect on tree growth, as well as temperatures during winter/spring and September. Conclusions Regeneration dynamics are modeled by the density of tree cover in the cold and dry edge of the distribution area of Q. suber where Q. ilex is increasing in abundance. Although temperature has a positive effect on the tree growth of Q. suber, when demographic processes are considered, decreases in water availability likely play a critical role in Q. ilex recruitment. This in turn changes dominance hierarchies, especially in abandoned areas with little or no tree cover. Aims We studied the regeneration dynamics of woodlands and abandoned old fields in a landscape dominated by Quercus suber in its lower limits for rainfall and temperature. Two hypotheses were established: (1) recruitment of Q. suber is restricted more by abiotic variations than other species adapted to more extreme Mediterranean conditions; and (2) decreases in precipitation reduce growth, but temperature positively affects growth in the leading cold edge of this species distribution area. Methods We selected nine sites containing forest stands and old fields with and without tree remnants, and analyzed stand structure, soil parameters and tree growth. Introduction Human activity has influenced the structure and dynamics of forests in the Mediterranean region over centuries (Barbero et al. 1990; Scarascia-Mugnozza et al. 2000; Chauchard et al. 2007; Camisón et al. 2015). This is the case of forests and woodlands dominated by the cork oak, Q. suber L., managed systems that are protected by the European Union (Habitat directive 92/43/EEC). Q. suber is an evergreen tree species present in the western Mediterranean region that extends through the Iberian Peninsula to the western rim of the Italian peninsula, as well as some Mediterranean islands (Corsica, Sardinia, Sicilia and Balearic Islands) and coastal plains and hilly areas in North Africa, ranging from Morocco to Tunisia (Blanco et al. 1997; Magri et al. 2007; Pausas et al. 2009a). Covering about 2.2 million hectares, the current distribution area of this species is rather patchy, suggesting that in the past there was a more Page 3/28 continuous distribution and that much of what we see today is relictual (Blanco et al. 1997, Carrión el al. 2000, Sánchez-Palomares et al. 2007, Pausas et al. 2009a, Jovellar et al. 2010). Additionally, intensification of ongoing climate change is expected to increase temperatures and the length of dry spells in the Mediterranean basin (Kovats et al. 2014). Consequently, areas suitable for cork oak are predicted to become generally reduced over the twenty-first century, owing also to intermediate and high CO2 emission scenarios (Pereira et al. 2009; Vesella et al. 2017). Cork oak can form closed woodlands, but in most parts of its geographic distribution in the Iberian Peninsula and North Africa it usually appears in open woodlands with other dominating or co-dominating Quercus (Q.ilex , Q.faginea) or Pinus (P. pinea, P.pinaster) species (Blanco et al. 1997; Bugalho et al. 2009). Moreover, these open woodlands have been traditionally managed as agro-silvopastoral systems providing goods such as pasture for livestock, acorns for the high-quality pork industry, cereal crops, firewood, cork and game species. However, these woodlands also provide services in the form of recreational tourism, as cork oak systems represent an important cultural heritage in the Mediterranean region (Bugalho et al. 2009; Ovando et al. 2009; Vallejo et al. 2009). In addition, in many parts of Europe, since the second half of the twentieth century, the Iberian Peninsula has been subjected to substantial land abandonment due to socioeconomic factors. Introduction These factors have led to the decline and/or disappearance of traditional management systems, crop abandonment, shrub encroachment and an increased risk of fire (Bugalho et al. 2009; Vallejo et al. 2009). The recruitment of oaks after land abandonment is usually a slow process, especially in shrublands, where succession is strongly delayed or almost halted in a condition known as arrested succession (Pons and Pausas 2006; Acácio et al. 2007; Pausas et al. 2009ab, Acácio and Holmgren 2014). Regeneration of Q. suber from seed shares problems and limitations that are similar to those experienced by other Quercus species in Mediterranean woodlands. These adverse conditions can include: (1) High rates of acorn predation by invertebrates and vertebrates (Branco et al. 2002; Pons and Pausas 2006; Pausas et al. 2009b). (2) Limited dispersion by scatter-hoarding rodents (Pons and Pausas 2007a), since the main long-distance vector the Eurasian jay, Garrulus glandarius (Pons and Pausas 2007b), is a forest species that is very scarce or absent in open woodlands (Pons and Pausas 2006). (3) High seedling mortality due to water stress during the summer in open areas that dramatically restricts its establishment under the plant canopy (Castro et al. 2006, Caldeira et al. 2014; Ibañez et al. 2015), with stronger nurse plant facilitation effects as environmental stress increases (Pugnaire and Luque 2001; Maestre et al. 2009, Costa et al. 2017). And (4), reduced seedling growth and high mortality by herbivore defoliation and trampling (Gómez 2003; Silla and Escudero 2006; Rossetti and Bagella 2014, Costa et al. 2017). In this work, we took advantage of the heterogeneity of the landscape generated by anthropogenic activities in the lower ecological limits of rainfall and temperature supported by Q. suber as a representative case to study the regeneration dynamics and growth at the leading distribution edge. This area is expected to be highly vulnerable to changes in traditional management and can be useful for detecting early warnings signs. As such, we have established two main hypotheses. This first hypothesis states that recruitment of Q. suber is more restricted by the abiotic variations induced by the dominant Page 4/28 Page 4/28 elements of vegetation (nursing effects) than other species more adapted to extreme Mediterranean conditions (e.g.: Q. ilex). Therefore, we expected that Q. suber recruitment would respond more drastically to changes in vegetation structure through the landscape, performing worse than Q. ilex in harsh environments like open old fields. Introduction To test this hypothesis, stand structure, soil parameters and spatial patterns of trees and saplings were analyzed in three types of landscape use. The second hypothesis states that tree growth is driven by local climatic conditions (mainly precipitation and temperature) affected by climate change, and that a decrease in precipitation reduces tree growth in water limited regions. We therefore predicted that temperature would positively affect tree growth in the leading cold edge of its distribution. Study site The study area is located in the north subdivision of the Central plateau of the Iberian Peninsula (Fig. 1a; 41° 07’ N, 5° 47’W; 800-850 m a.s.l.). The mean annual precipitation is around 380 mm, with a typical Mediterranean period of low precipitation during July and August. The mean annual temperature is around 12°C, with mean temperatures between 3-4°C and 20-21°C during the coldest and the warmest months, respectively. This area contains the biggest Q. suber woodland in the northwest part of the Iberian Peninsula, with an extension of almost 2,000 ha (Guerra Velasco 2015). The study area is located in the distributional edge of this species, as Q. suber typically occurs between 0 and 800 m a.s.l., it requires an annual precipitation between 600 and 1000 mm and an average temperature around 15°C (Blanco et al. 1997, Sánchez-Palomares et al. 2007, Houston Durrant et al. 2016). The study area is characterized by a variety of land covers, with open and closed woodlands (dominated by Q. suber and the presence of Quercus ilex subsp. ballota, Q.faginea and Q. pyrenaica), Pinus pinaster plantations, shrublands dominated by Cistus, Halimium and Cytisus species, old fields and abandoned vineyards. Climate data Climate data spanning the 1948-2019 time frame were provided by the Meteorological State Agency (AEMET) from the nearest meteorological station located 30 km southeast and at the same altitude as the study site (Matacán; 40° 94’ N, 5° 50’W; 790 m a.s.l.). Dendrochronological analysis Increment cores from all trees were extracted using Pressler increment borers (Häglof, Sweden) at 0.3-0.4 m above ground level to obtain the most accurate age for each tree (Veblen 1992), and at 0.6 m when the tree centers were rotten. Increment cores were mounted and sanded following the procedure established by Stokes and Smiley (1968), and the annual rings were counted using a stereomicroscope (SMZ800, Nikon, Japan). When the cores did not reach the pith, the number of rings to the center was estimated using the geometric procedure described by Duncan (1989). If the center was rotten, the rings counted in the non-rotten section of the core were considered as the minimum age for that tree. Cores were scanned at 2000 dpi resolution (Perfection V550, Epson, Japan), and tree-ring widths were measured with a 0.01 mm resolution on the scanned JPG images using the software CooRecorder 7.6 (Cybis, Sweden). The visual and statistical cross-dating of the tree-ring width series was done and checked using the software CDendro 7.6 (Cybis, Sweden) and Cofecha (Holmes 1983), respectively. The tree-ring width series were detrended to obtain mean residual chronologies of the tree-ring width indices. First, a spline function was fitted to each tree-ring width series to obtain standardized indices which preserve the high-frequency variability potentially related to climate. Second, an autoregressive model was applied to remove the first- order temporal autocorrelation in the detrended series and generate residual indices. Third, a biweight robust mean was computed to produce residual mean chronologies for each species. Detrending residual chronologies were obtained with the package dplR (Bunn 2008) under R environment (R Development Core Team 2018). Plot establishment and data collection The positions of the trees and saplings were located to the nearest centimeter using measuring tapes that were aligned with the sides of the plots, providing X and Y coordinates in a Cartesian plane. Shrub cover (%) was measured sampling five line transects (5 m in length) per plot. Plot establishment and data collection Nine sites were selected encompassing three forest stands dominated by Q. suber, three old fields with tree remnants and three old fields without tree cover. Site selection was based on changes in landscape use determined by comparing aerial photographs of the Spanish Inter-ministry flight from 1973-1986 (https://fototeca.cnig.es) and aerial photographs taken from Google Earth from 2015 (Fig. 1bc). The sites located in old fields, with and without tree remnants, had been cultivated in the 70s and 80s and abandoned in the late 1990s. The forest stands were non-cultivated areas managed for traditional cork extraction, which is still harvested today. In each site, we established a 40 x 40 m plot (1600 m2) where the origin point of each one was randomized and their sides were oriented in the directions of the cardinal points. All data were collected between October 2015 and June 2017. Page 5/28 Page 5/28 Page 5/28 All trees, both live and dead, and the saplings and seedlings in each plot were recorded. Trees were defined as individuals with a diameter at breast height (dbh) ≥5 cm and saplings as individuals with a dbh < 5 cm and height > 200 cm. Small seedlings were defined as individuals < 50 cm in height and large seedlings were defined as individuals ≥ 50 and ≤ 200 cm in height. Resprouting individuals were not considered, and in some doubtful cases the soil surrounding each plant was excavated to check for independence from the nearest plants. The positions of the trees and saplings were located to the nearest centimeter using measuring tapes that were aligned with the sides of the plots, providing X and Y coordinates in a Cartesian plane. Shrub cover (%) was measured sampling five line transects (5 m in length) per plot. All trees, both live and dead, and the saplings and seedlings in each plot were recorded. Trees were defined as individuals with a diameter at breast height (dbh) ≥5 cm and saplings as individuals with a dbh < 5 cm and height > 200 cm. Small seedlings were defined as individuals < 50 cm in height and large seedlings were defined as individuals ≥ 50 and ≤ 200 cm in height. Resprouting individuals were not considered, and in some doubtful cases the soil surrounding each plant was excavated to check for independence from the nearest plants. Soil analysis Five soil subsamples per plot were taken using a soil core sampler at depths between 0 and 30 cm. One subsample was taken in the center of the plot and four subsamples were taken 20 m apart from the center towards each corner of the plot. The subsamples were pooled in a single sample for structural and Page 6/28 chemical soil analysis. The samples were air-dried and passed through a 2-mm sieve before laboratory analysis. The pH was determined in distilled water (in a ratio 1:2.5) using a CRISON micropH 2001 pH-meter. Soil texture was quantified by the Robinson’s pipette method after previous dispersion with sodium hexametaphosphate (Loveland and Whalley 1991). Organic carbon was determined by wet oxidation with a dichromate-sulphuric acid mixture (Walkley 1947). Residual dichromate was back titrated using ferrous sulphate. The difference in added FeSO4 compared with a blank titration determined the amount of easy oxidizable organic carbon (Walkley 1947). We used a conversion factor of 1.72 to convert organic carbon to organic matter (Nelson and Sommers 1996). Available Ca, Mg and K were extracted using ammonium acetate 1M and determined by plasma ICP-MS. P was determined by the Bray I method, modified from Bray and Kurtz (1945). Statistical analysis To analyze how seedling regeneration (small seedlings, large seedlings and saplings) could be explained by soil and structural stand variables, a Redundant Analysis (RDA) was performed (Borcard et al. 2018). Previously, seedling data was transformed using a hellinger transformation, as suggested by Legendre and Gallagher (2001) for abundance data. To avoid collinearity within sets of soil and structural explanatory variables, we examined the correlation between variables and groups of variables using a hierarchical cluster analysis approach (complete linkage method). When the correlation between variables or groups of variables was greater than 0.6, only one variable was selected, which resulted in variance inflation factors (VIF) always less than five units within the set of variables (Quinn and Keough 2002). For the soil variables we selected organic matter, P (%), pH and sand content (%), and for the structural stand variables we selected basal area and tree density (Table S2, Figs S1 and S2). Then, we performed a variation partitioning analysis (9999 random permutations) to analyze how much of the variance in seedling regeneration was explained by these variables (Borcard et al. 2018). Variation partitioning and RDA were analyzed with the vegan library under the R environment (R Development Core Team 2018), whereas HH library was used to evaluate variance inflation. For each plot, the univariate spatial patterns of all seedlings were analysed using the O-ring statistic derived from the pair correlation function (Wiegand and Moloney 2014). The pair correlation function is the expected number of points between the largest and smallest radii of a ring of fixed width at increasing distances from an arbitrary point, divided by the intensity k of the pattern (Diggle 2003, Wiegand and Moloney 2014). We used the complete spatial randomness model (CSR) implemented as a homogeneous Poisson process to determine whether the distribution of trees or saplings was random, aggregated or regular. For this univariate analysis, O(r) > 1 indicates that the individuals are aggregated at distances r, while O(r) < 1 means they are regularly dispersed. Additionally, the spatial relationships between seedlings and trees (of the same species and all Quercus trees pooled together) were analysed using the bivariate O-ring statistic. We used the antecedent condition as the null model, keeping the tree positions fixed whereas the seedlings were randomized using a CSR model (Wiegand and Moloney Page 7/28 Page 7/28 2014). Statistical analysis Values of O12(r) < 0 indicate repulsion between the two classes of individuals up to distance r. By contrast, values of O12(r) > 0 indicate attraction between the two classes up to distance r. To evaluate the significance of the spatial statistics under the null model considered, 95% of simulated envelopes were generated using 999 Monte Carlo simulations. The twenty-fifth highest and lowest values of the 999 iterated functions were chosen to obtain the upper and lower values of the envelopes. All spatial analyses were performed using the 2014 version of the software Programita (Wiegand and Moloney 2014). Bootstrapped Pearson’s correlation functions were calculated between the mean residual chronologies of each tree species and monthly climatic variables (mean, minimum and maximum temperature, precipitation). Confidence envelopes were obtained by calculating 1000 bootstrap samples and tested for significance using the 95% percentile range method (Dixon 2001). The climatic window for these analyses spanned from the previous September (year t-1), i.e. prior to the year of tree-ring formation, up to the following September (year t). This analysis was performed with the function dcc implemented in the package bootRes (Zang and Biondi 2013) under R environment (R Development Core Team 2018). The trends in monthly or seasonal climatic data were analyzed using the Kendall τ statistic with one-tail tests, as we were interested in decreasing rainfall and increasing temperature patterns. Results Woodlands showed tree densities of 95.8± 29.2 trees ha-1 and basal areas of 11.84±1.34 m2 ha-1 (mean± se), where Q. suber was the dominant tree species. Mean tree dbh was 29.9 cm (confidence interval of 24.7-36.2 cm at 95% level). Quercus species showed a broad and multiage structure, but with most of the trees recruiting between 1930 and 1980 (Fig. 2a). Old fields with tree remnants showed lower tree density and basal area than woodlands (62.5± 12.5 trees ha-1, 5.20±1.95 m2 basal area ha-1, mean± se), and with most of the trees (66.7% of total trees) recruiting after 1970 (Fig. 2b). Mean tree dbh was 19.6 cm (confidence interval of 16.1-23.2 cm at 95% level). Q. suber was also the main species in the old fields with tree remnants, although Q. ilex dominated tree recruitment after 1990 (Fig. 2b). In relation to seedling recruitment, oak recruitment was quite abundant in the woodlands (2410.4± 719.8 seedlings ha-1, mean± se) and dominated by Q. suber, especially in the shortest height classes (Fig. 3a). Significant differences in the mean recruitment of seedlings were observed between species and height classes, with clear differences in the shortest categories but insignificant ones in the highest categories (Table 2). In the old fields with tree remnants, oak recruitment was moderately abundant (1266.7± 445.6 seedlings ha-1, mean± se, Fig. 3b) and seedling recruitment varied between species and height class (Table 2). By contrast, Q. ilex was the only species that recruited in the old fields without tree cover, although with low densities (33.3± 10.4 seedlings ha-1, mean± se, Fig. 3c). Sapling density of Quercus species was low in woodlands (66.7± 29.6 saplings ha-1) and old fields with tree remnants (43.7± 64.9 saplings ha-1) and zero in old fields without tree remnants. Differences in sapling density were found between forest types (df= 2, F= 7.06, p= 0.005) but no differences were found between species (df= 2, F= 0.62, p= 0.56). In relation to seedling recruitment, oak recruitment was quite abundant in the woodlands (2410.4± 719.8 seedlings ha-1, mean± se) and dominated by Q. suber, especially in the shortest height classes (Fig. 3a). Significant differences in the mean recruitment of seedlings were observed between species and height classes, with clear differences in the shortest categories but insignificant ones in the highest categories (Table 2). Results In the old fields with tree remnants, oak recruitment was moderately abundant (1266.7± 445.6 seedlings ha-1, mean± se, Fig. 3b) and seedling recruitment varied between species and height class (Table 2). By contrast, Q. ilex was the only species that recruited in the old fields without tree cover, although with low densities (33.3± 10.4 seedlings ha-1, mean± se, Fig. 3c). Sapling density of Quercus species was low in woodlands (66.7± 29.6 saplings ha-1) and old fields with tree remnants (43.7± 64.9 saplings ha-1) and zero in old fields without tree remnants. Differences in sapling density were found between forest types (df= 2, F= 7.06, p= 0.005) but no differences were found between species (df= 2, F= 0.62, p= 0.56). Page 8/28 Page 8/28 In woodlands and old fields with tree remnants, small seedlings of Q. suber showed a clear clumped spatial pattern at distances up to 8-12 m and were associated to Quercus trees, with more seedlings than expected under a random process up to distances of 5-7 m from each tree (Table 1). Small seedlings of Q. ilex presented a clumped pattern, but at greater distances in old fields with tree remnants than in woodlands. Q. ilex small seedlings were also associated with trees but at lower distances than Q.suber, especially in woodlands (Table 1). By contrast, large seedlings of both Q. suber and Q. ilex mostly showed a random pattern distribution and were independently distributed from trees in woodlands whereas in old fields with tree remnants only large seedlings of Q. suber showed association with trees at distance of 4-7 m (Table 1). In the RDA, the first axis represented a contrast between old fields without tree cover at the right, where only small seedlings of Q. ilex were present, and woodlands at the left, where small and large seedlings of Q. suber dominated tree regeneration (Fig. 4). Basal area, pH and soil organic matter showed a strong correlation with the first axis, with higher values of these three variables associated with woodlands. The second axis was related to the abundance of Q. faginea with regard to tree regeneration, with the sites with the higher scores in the upper panel showing a higher abundance for this species (Fig. 4). Variance partition analyses showed that soil and structural variables explained most (81.0%) of the variance in the abundance of regeneration across sites (Fig. 5). Results Shared explained variation for both sets of variables was 31.6%, with soil variables contributing slightly more to the unshared explained variation in seedling abundances than structural variables (soil variables: 27.1%, F= 3.85, p= 0.014, permutations= 999; structural stand variables: 22.3%, F= 5.67, p= 0.004, permutations= 999). Ring growth was positively correlated with rainfall in January and June (Fig. 6a), and September temperatures (mean, maximum and minimum) and minimum winter-early spring temperatures also positively influenced ring growth (Fig. 6bcd). Monthly rainfall in January and June did not show a decreasing trend over time (Table S1), although annual rainfall significantly decreased (Table S1, Fig. S3). All mean, maximum and minimum temperatures in January, February, March and September did not show temperature increases over time, except for the minimum temperature in September (Table S1). a) Regeneration dynamics of Quercus Page 9/28 Succession was considerably arrested in the plots without tree remnants two to three decades after cultivation abandonment. Similar trends have been observed in other studies, where 45 years after abandonment Cistus-dominated shrublands prevailed with scarce or absent oak recruitment (Acácio and Holmgren 2014). Cistus-dominated shrublands, as well as acorn availability and drought stress, exert competition and inhibition effects over Quercus recruitment and appear as highly resilient systems (Pérez-Devesa et al. 2008, Rolo and Moreno 2011, Acácio and Holmgren 2014). However, shrub cover (mainly characterized by Cistus, Halimium and Lavandula spp.) was quite low in plots in old fields (< 10%, Table S2) and vegetation was dominated by annual herbaceous species, likely due to low rainfall combined with high sand and low organic soil contents that exacerbated hydric stress (Fernández-Ales et al. 1993, Rawls et al. 2003, Nunes et al. 2017). Q. ilex was the only tree species successfully recruiting in the open old fields, although slowly and with low densities, despite Q. suber being the dominant tree species in the landscape with respect to density and basal area. Two processes are likely involved in the success of Q. ilex over other Quercus species in the open plots. First, although Quercus species share the same animal dispersers, most studies have shown that acorns from Q. ilex are preferred over other Quercus species by the main oak disperser the Eurasian jay (Garrulus glandarius) and by small rodents (Pons and Pausas 2007ac, del Arco et al. 2018). Since the Eurasian jay is a forest species that has never been detected in the study area, mice species are the main candidates for acorn dispersal. Pilferage rates are reduced by caching the seeds preferentially outside the canopies of scattered trees, increasing the survival of cached acorns in open areas (Muñoz and Bonal 2011). However, limited facilitative shrub cover reduces successful recruitment causing a low density of Q. ilex seedlings (Pulido and Díaz 2005, Smit et al. 2009, Rolo and Moreno 2011). Second, although both evergreen species, Q. suber and Q. ilex, are well adapted to the summer drought of the Mediterranean climate (Mediavilla and Escudero 2003, González-Rodríguez et al. 2011, San-Eufrasio et al. 2020), previous studies have shown a higher survival rate for Q.ilex seedlings than for Q.suber (Plieninger et al. 2010, González-Rodríguez et al. 2011). In addition, Q. ilex has lower conductance and maximum transpiration rates than Q. a) Regeneration dynamics of Quercus These changes in the spatial pattern with ontogenetic development support previous findings that suggest that the positive effects of shaded microhabitats are reversed for seedling development (Pérez-Ramos et al 2011, Pausas et al. 2009b). In Q. suber and Q. ilex, seedling establishment and survival are improved under shade (Espelta et al. 1995, Pausas et al. 2009b, Smit et al. 2009, Pérez-Ramos et al. 2012). However, it has been also shown that low light suppresses growth, that these species establish ‘seedling banks’ under dense tree cover and that more open canopy conditions are required for saplings and trees to develop (Espelta et al. 1995, Pausas et al. 2009b, Pérez-Ramos et al. 2010, 2012). In addition, we also found significant differences in the regeneration densities of Q. suber,Q. ilex, Q. faginea, with the abundance of Q. suber strongly correlated with the basal area and tree density of stands. In woodlands, Q. suber dominated seedling regeneration in the categories with the shortest height, but the differences in seedling abundance between Q. ilex and Q. suber disappeared in the tallest categories and showed similar densities. In the old fields with tree remnants, Q. ilex and Q. suber showed similar seedling densities, and although the interaction was not significant, the abundance of Q. ilex large seedlings was 5-6 fold higher than that of Q. suber. These results indicate that Q. ilex has a higher seedling survival rate and more likely to reach the sapling stage, probably due to their higher tolerance to shade (Sevilla 2008), and in particular their greater tolerance to hydric stress during the summer (Plieninger et al. 2010, González-Rodríguez et al. 2011, San-Eufrasio et al. 2020). Thus, although Q. suber trees dominate the landscape of our study area, the results indicate that Q. ilex can produce a greater number of young trees as compared to Q. suber (Fig. 2b), owing to the better performance of Q. ilex seedlings. Only in woodlands can Q. suber partially compensate for their lower performance and survival, producing a higher number of seedlings. Conversely, the deciduous Q. faginea present the lowest abundance, especially in the old fields (with or without tree remnants), which is in line with its lower tolerance to drought compared to evergreen species (Silla and Escudero 2004, González-Rodríguez et al. 2011, San-Eufrasio et al. 2020). a) Regeneration dynamics of Quercus suber, which delays leaf desiccation thanks to a more conservative use of water (Mediavilla and Escudero 2003, San-Eufrasio et al. 2020), which decreases growth suppression under high water stress (Caldeira et al. 2014). Our results indicate that remnant oak trees are great accelerators of forest recovery after cultivation abandonment. Also, old fields with tree remnants boost oak regeneration almost 40-fold in comparison to old fields without trees, and woodlands show a 2-fold increase in oak regeneration in comparison to old fields with tree remnants. Tree cover is considered to play a fundamental role in Quercus recruitment through several processes. First, acorns are heavy fruits with limited dispersal, where most of the acorns produced only reach areas situated close to the parent trees (Pulido and Díaz 2005, Acácio et al. 2007, Pausas et al. 2009b). Second, facilitation effects caused by tree cover are key in the regeneration of Quercus species, especially in Mediterranean environments (Caldeira et al. 2014, Costa et al. 2017). Facilitation includes several direct and indirect mechanisms with positive effects on both survival and Quercus seedling growth. Drought stress and summer seedling survival during the first years of establishment is usually considered one of the main bottlenecks in Quercus regeneration (Díaz and Pulido 2005, Silla and Escudero 2004, Smit el al. 2009, Pérez-Ramos et al. 2012). Therefore, tree cover improves microclimate conditions reducing high summer temperatures and alleviating heat stress (Díaz and Pulido 2005, Pausas et al. 2009, Smit el al. 2009) and reduces competition with herbaceous vegetation (Caldeira et al. 2014). Our results are also consistent with these patterns, as tree basal area and density were strongly correlated with the first RDA ordination axis that ordered sites with a strong gradient of seedling density from right to left (Fig. 4). Additionally, the spatial analysis of small seedlings of Q. suber and Q.ilex, which accounted for most of the seedling abundance (71.6%) showed a clustered pattern associated with Quercus trees in both woodlands and old fields with tree remnants, although at shorter distances in Q. ilex than in Q. suber. By contrast, Quercus large seedlings showed a random Page 10/28 Page 10/28 spatial pattern independent of the trees, highlighting that tree cover is only a limiting factor during seedling establishment. a) Regeneration dynamics of Quercus suber distribution limits, as has also been recently observed in Mediterranean forest at the limits of species distribution (Madrigal-González et al. 2018, Marqués et al. 2018). Although no significant decreasing trends were found in the more critical months (Fig. 6a), there is a decreasing trend in mean annual rainfall (Fig. S3), so more detailed studies will be needed to understand the combined effects of temperature and rainfall on tree growth within future climate change scenarios a) Regeneration dynamics of Quercus However, in relation to other studies where temperature has been found to have an insignificant or negative effect over cork or ring growth (Caritat et al. 1996, Costa et al. 2001, Zribi et al. 2016), in our study site, the mean and minimum temperatures during winter and/or spring and the mean and maximum temperatures during September exerted a positive influence on tree growth. This is consistent with the cold temperature conditions occurring in winter and early spring in our study area, located at the leading temperature edge of its distribution limits in the Iberian Peninsula. The large vessels in oak trees are very sensitive to winter embolisms caused by freezing temperatures, and in spring the reactivation of growth is greatly dependent on hydraulic conductivity recovery (Hacke and Sauter 1996, Lebourgeois et al. 2004). This highlights the importance of mild winter-early spring temperatures on Q. suber tree growth in the “cold leading edge” of its distribution. However, although winter temperatures are a limiting factor and warming has room for net positive effects on tree growth (Sánchez-Salguero et al., 2015), climatic data did not reveal a significant temperature increase during winter and early spring in this study site. On the other hand, minimum temperatures during September have significantly increased during the last decades and have had a positive impact on tree growth by most likely extending the favorable weather (Marqués et al. 2018). However, the positive effect of an extended growing season in early autumn can be counteracted by a decrease in rainfall, especially in the study area where annual rainfall is also in the lower edge of Q. suber distribution limits, as has also been recently observed in Mediterranean forest at the limits of species distribution (Madrigal-González et al. 2018, Marqués et al. 2018). Although no significant decreasing trends were found in the more critical months (Fig. 6a), there is a decreasing trend in mean annual rainfall (Fig. S3), so more detailed studies will be needed to understand the combined effects of temperature and rainfall on tree growth within future climate change scenarios. Analysis of the impact of climatic conditions on the tree-ring growth of Q. suber is challenging due to the difficulty in identifying ring boundaries in trees being managed for cork extraction (Costa et al. 2003). Only two previous studies have reported successful tree ring chronologies involving Q. suber in the Mediterranean region (Costa et al. a) Regeneration dynamics of Quercus 2003, Zribi et al. 2016) that have been complemented with short chronologies from cork growth-rings (Caritat et al. 1996, Costa et al 2016, Leite et al. 2019). As shown in these studies, winter and spring precipitation exerted a large positive effect on tree-ring growth due to the replenishment of soil water reserves before the onset of the favorable growing season (Costa et al. 2001, Jovellar et al. 2010, Costa et al 2016, Zribi et al. 2016, Leite et al. 2019). In our study site, Q. suber showed the latest leaf emergence of all coexisting Quercus species, with budbreak and emergence of the new leaf cohort occurring between the end of May and the beginning of June (del Río et al. 2015). However, in relation to other studies where temperature has been found to have an insignificant or negative effect over cork or ring growth (Caritat et al. 1996, Costa et al. 2001, Zribi et al. 2016), in our study site, the mean and minimum temperatures during winter and/or spring and the mean and maximum temperatures during September exerted a positive influence on tree growth. This is consistent with the cold temperature conditions occurring in winter and early spring in our study area, located at the leading temperature edge of its distribution limits in the Iberian Peninsula. The large vessels in oak trees are very sensitive to winter embolisms caused by freezing temperatures, and in spring the reactivation of growth is greatly dependent on hydraulic conductivity recovery (Hacke and Sauter 1996, Lebourgeois et al. 2004). This highlights the importance of mild winter-early spring temperatures on Q. suber tree growth in the “cold leading edge” of its distribution. However, although winter temperatures are a limiting factor and warming has room for net positive effects on tree growth (Sánchez-Salguero et al., 2015), climatic data did not reveal a significant temperature increase during winter and early spring in this study site. On the other hand, minimum temperatures during September have significantly increased during the last decades and have had a positive impact on tree growth by most likely extending the favorable weather (Marqués et al. 2018). However, the positive effect of an extended growing season in early autumn can be counteracted by a decrease in rainfall, especially in the study area where annual rainfall is also in the lower edge of Q. a) Regeneration dynamics of Quercus The presence of tree remnants showed a strong effect over soil parameters with higher concentrations of organic matter, N, exchangeable cations (K+, Ca2+, Mg2+) and slightly less acidic soils than in old fields without tree remnants (Table S2). The deep root system of Q. suber trees uptakes and pumps basic cations, especially Ca2+, from the deep to upper soil layers throughout litterfall production and decomposition, which significantly improves soil nutrient availability (Serrasolses et al. 2009, Rossetti et al. 2015). The results of the variance partitioning showed that soil and stand structural parameters explain a significant amount of the shared variation in the amount of regeneration of Quercus species (Fig. 5), which makes sense since the influence of trees on soil characteristics are spatially correlated. However, soil variables also explain a significant amount of the variance (27.1%) not explained by structural stand variables and are important for understanding the differences found in Quercus regeneration between plots. Among soil variables, organic matter content summarizes most information on nutrient availability with which is strongly correlated (Table S2, Fig. S1), but it is also directly involved in improving soil water retention in abandoned cultivars, especially in soils with high sand content (Rawls et al. 2003, Costa et al. 2017). Page 11/28 b) Climate-growth relationships of suber trees Analysis of the impact of climatic conditions on the tree-ring growth of Q. suber is challenging due to the difficulty in identifying ring boundaries in trees being managed for cork extraction (Costa et al. 2003). Only two previous studies have reported successful tree ring chronologies involving Q. suber in the Mediterranean region (Costa et al. 2003, Zribi et al. 2016) that have been complemented with short chronologies from cork growth-rings (Caritat et al. 1996, Costa et al 2016, Leite et al. 2019). As shown in these studies, winter and spring precipitation exerted a large positive effect on tree-ring growth due to the replenishment of soil water reserves before the onset of the favorable growing season (Costa et al. 2001, Jovellar et al. 2010, Costa et al 2016, Zribi et al. 2016, Leite et al. 2019). In our study site, Q. suber showed the latest leaf emergence of all coexisting Quercus species, with budbreak and emergence of the new leaf cohort occurring between the end of May and the beginning of June (del Río et al. 2015). Acknowledgements This study was funded by project SA013G192 from the Regional Government of Castile and Leon and by research grants awarded for final projects directed in the Master’s degree in Biology and Conservation of Biodiversity at the University of Salamanca, Spain. Emma Keck kindly corrected the English. Funding: This study was funded by project SA013G192 from the Regional Government of Castile and Leon and by research grants awarded for final projects directed in the Master’s degree in Biology and Conservation of Biodiversity at the University of Salamanca, Spain. Conflicts of interest/Competing interests: Authors declare no conflicts of interest cts of interest/Competing interests: Authors declare no conflicts of interest Availability of data and material (data transparency): raw data will be available in the Open Science Framework repository upon acceptance. Code availability: not applicable Conclusions Page 12/28 Regeneration dynamics were strongly modeled by the presence and density of tree cover in the fragmented landscape in the cold and dry edge of Q. suber distribution. We have seen that tree cover affects seedling abundance differently through both direct (acorn availability and shading) and indirect (improving nutrient availability and water retention in soils) mechanisms. Consequently, the stress tolerant species Q. ilex was the only species found to recruit in open old fields decades after being Page 12/28 abandoned. Also, the presence of isolated tree remnants in old fields allowed the recruitment of Q. suber, but Q. ilex had a higher abundance of large seedlings and young trees; only in woodlands did both species show similar recruitment success. These results indicate than Q. ilex is more abundant in this landscape compared to Q. suber, even though the latter species is the dominant tree in our study area. abandoned. Also, the presence of isolated tree remnants in old fields allowed the recruitment of Q. suber, but Q. ilex had a higher abundance of large seedlings and young trees; only in woodlands did both species show similar recruitment success. These results indicate than Q. ilex is more abundant in this landscape compared to Q. suber, even though the latter species is the dominant tree in our study area. In addition, the climate-tree growth relationship of Q. suber presents contrasting results. Higher temperatures were found to have a positive effect on tree ring-growth, especially during winter/early spring and early autumn. On the other hand, winter and spring rainfall recharges water soil reserves promoting tree growth during the favorable season. These opposing effects increase pose uncertainty in predicting Q. suber growth and productivity under climate change scenarios involving higher temperatures and less rainfall. However, when the demographic processes are considered, less water availability is likely to play a critical role in favoring Q. ilex recruitment in contrast to Q. suber, which could lead to changes in dominance hierarchies, especially in abandoned areas with scarce or absent tree cover. In addition, the climate-tree growth relationship of Q. suber presents contrasting results. Higher temperatures were found to have a positive effect on tree ring-growth, especially during winter/early spring and early autumn. On the other hand, winter and spring rainfall recharges water soil reserves promoting tree growth during the favorable season. These opposing effects increase pose uncertainty in predicting Q. Conclusions suber growth and productivity under climate change scenarios involving higher temperatures and less rainfall. However, when the demographic processes are considered, less water availability is likely to play a critical role in favoring Q. ilex recruitment in contrast to Q. suber, which could lead to changes in dominance hierarchies, especially in abandoned areas with scarce or absent tree cover. Authors' contributions Conceptualization: Fernando Silla; Methodology: Carmen Ureña, Diego Navarro, Valentín Herrera, Pilar Alonso-Rojo, Fernando Silla; Formal analysis and investigation: Carmen Ureña, Diego Navarro, Valentín Herrera, Jorge Montero-Muñoz and Fernando Silla; Writing - original draft preparation: Jorge Montero- Muñoz and Fernando Silla; Writing - review and editing: Carmen Ureña, Diego Navarro, Valentín Herrera, Pilar Alonso-Rojo, Héctor Hernández-Alonso, Mª Fernanda Cepeda, Luis Carlos Jovellar, Belén Fernández- Santos Page 13/28 References 1. Acácio V, Holmgren M, Jansen PA, Schrotter O (2007) Multiple recruitment limitation causes arrested auccession in Mediterranean cork oak systems. Ecosystems 10:1220–1230. https://doi.org/10.1007/s10021-007-9089-9 2. Acácio V, Holmgren M (2014) Pathways for resilience in Mediterranean cork oak land use systems. Ann For Sci 71:5–13. https://doi.org/10.1007/s13595-012-0197-0 2. 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Samp.) in a Mediterranean savanna – forest ecosystem. Ann For Sci 66:511. https://doi.org/10.1051/forest/2009028 77. Stokes MA, Smiley TL (1968) An Introduction to Tree-Ring Dating. The University of Chicago Press, Chicago 77. Stokes MA, Smiley TL (1968) An Introduction to Tree-Ring Dating. The University of Chicago Press, Chicago 78. Vallejo VR, Aronson J, Pausas JG, Pereira JS, Fontaine C (2009) The way forward. In: Aronson J, Pereira JS, Pausas JG (eds) Cork Oak Woodlands on the Edge. Island Press, Washington DC, pp 235–245 78. Vallejo VR, Aronson J, Pausas JG, Pereira JS, Fontaine C (2009) The way forward. In: Aronson J, Pereira JS, Pausas JG (eds) Cork Oak Woodlands on the Edge. Island Press, Washington DC, pp 235–245 79. References Vessella F, López-Tirado J, Simeone MC, Schirone B, Hidalgo PJ (2017) A tree species range in the face of climate change: cork oak as a study case for the Mediterranean biome. Eur J Forest Res 136:555–569. https://doi.org/10.1007/s10342-017-1055-2 79. Vessella F, López-Tirado J, Simeone MC, Schirone B, Hidalgo PJ (2017) A tree species range in the face of climate change: cork oak as a study case for the Mediterranean biome. Eur J Forest Res 136:555–569. https://doi.org/10.1007/s10342-017-1055-2 Page 19/28 80. Walkley A (1947) A Critical Examination of a Rapid Method for Determining Organic Carbon in Soils: Effect of Variations in Digestion Conditions and of Inorganic Soil Constituents. Soil Sci 63:251–264 81. Wiegand T, Moloney KA (2014) Handbook of spatial point-pattern analysis in ecology. CRC Press, Boca Raton 81. Wiegand T, Moloney KA (2014) Handbook of spatial point-pattern analysis in ecology. CRC Press, Boca Raton 81. Wiegand T, Moloney KA (2014) Handbook of spatial point-pattern analysis in ecology. CRC Press, Boca Raton 82. Zang C, Biondi F (2013) Dendroclimatic calibration in R: The bootRes package for response and correlation function analysis. Dendrochronologia 31:68–74. http://doi.org/10.1016/j.dendro.2012.08.001 82. Zang C, Biondi F (2013) Dendroclimatic calibration in R: The bootRes package for response and correlation function analysis. Dendrochronologia 31:68–74. http://doi.org/10.1016/j.dendro.2012.08.001 83. Zribi L, Mouillot F, Guibal F, Rejeb S, Rejeb MN, Gharbi F (2016) Deep soil conditions make Mediterranean cork oak stem growth vulnerable to autumnal rainfall decline in Tunisia. Forests 7:245. http://doi.org/10.3390/f7100245 83. Zribi L, Mouillot F, Guibal F, Rejeb S, Rejeb MN, Gharbi F (2016) Deep soil conditions make Mediterranean cork oak stem growth vulnerable to autumnal rainfall decline in Tunisia. Forests 7:245. http://doi.org/10.3390/f7100245 Tables Table 1. Results of the O-ring statistic for the univariate and bivariate analyses. Statistical significance at 95% simulated envelopes for the univariate analyses (grey: random pattern; black: clumped pattern; white: regular pattern) and for the bivariate analyses (grey: independence pattern; black: attraction pattern; white: repulsion pattern). Page 20/28 Table 2 Results of the two-way variance analysis testing for differences in seedling recruitment between species and height classes. Woodlands Oldfields with tree remnants df F p df F p Species 2 5.10 0.003 2 7.58 < 0.001 Table 2 Results of the two-way variance analysis testing for differences in seedling recruitment between species and height classes. Woodlands Oldfields with tree remnants df F p df F p Species 2 5.10 0.003 2 7.58 < 0.001 Height 4 6.09 0.006 4 5.91 0.007 Interaction 8 2.33 0.044 8 0.93 0.500 Table 2 Results of the two-way variance analysis testing for differences in seedling recruitment between species and height classes. A Page 21/28 Figures Page 22/28 Figure 1 (a) Location of the Quercus suber forest (grey area). Valdelosa municipality (black area) is shown. Old field with tree remnants plot in aerial photographs of the Spanish Inter-ministry flight from 1973-1986 ( and aerial photographs taken from Google Earth (© 2015 Google) from 2015 (c). Note: The designatio Figure 1 Page 22/28 (a) Location of the Quercus suber forest (grey area). Valdelosa municipality (black area) is shown. Old field with tree remnants plot in aerial photographs of the Spanish Inter-ministry flight from 1973-1986 (b) and aerial photographs taken from Google Earth (© 2015 Google) from 2015 (c). Note: The designations Page 22/28 employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Figure 2 Age distributions in 10-year establishment classes in woodlands (a) and old fields with tree remnants (b). Solid bars: Q. suber; striped bars: Q. ilex; open bars: Q. faginea. Figure 2 Age distributions in 10-year establishment classes in woodlands (a) and old fields with tree remnants (b). Solid bars: Q. suber; striped bars: Q. ilex; open bars: Q. faginea. Age distributions in 10-year establishment classes in woodlands (a) and old fields with tree remnants (b). Solid bars: Q. suber; striped bars: Q. ilex; open bars: Q. faginea. Page 23/28 Page 23/28 Figure 3 Height distribution classes of Quercus seedlings in woodlands (a), old fields with tree remnants (b) and old fields without tree remnants (c). Solid bars: Q. suber; striped bars: Q. ilex; open bars: Q. faginea. Figure 3 Figure 3 Height distribution classes of Quercus seedlings in woodlands (a), old fields with tree remnants (b) and old fields without tree remnants (c). Solid bars: Q. suber; striped bars: Q. ilex; open bars: Q. faginea. Page 24/28 Page 24/28 Page 24/28 Figure 4 Redundant Analysis (RDA) showing differences in plots in relation to regeneration abundance of seedlings and saplings. The sizes of the circles are proportional to abundance of tree regeneration (seedlings plus saplings). Regeneration codes are Qs: Q. suber; Qi: Q. ilex; Qf: Q. faginea; sa: saplings; ls: large seedlings; ss: small seedlings. Vectors showed structural (BA: basal area; TD: trees density) and soil (OM: organic matter; pH; P: phosphorus; sand: sand content) variables. Redundant Analysis (RDA) showing differences in plots in relation to regeneration abundance of seedlings and saplings. The sizes of the circles are proportional to abundance of tree regeneration (seedlings plus saplings). Regeneration codes are Qs: Q. suber; Qi: Q. ilex; Qf: Q. faginea; sa: saplings; ls: large seedlings; ss: small seedlings. Vectors showed structural (BA: basal area; TD: trees density) and soil (OM: organic matter; pH; P: phosphorus; sand: sand content) variables. Page 25/28 Figure 6 Climate-growth associations calculated for Q. suber trees. The bars indicate the mean Pearson correlation coefficients (error bars show 95% confidence intervals) calculated between ring-width indices and monthly climatic variables. The temporal window of analyses comprises the hydrological year from October of the previous year to September of the current year (x axes). Significant correlations are indicated by bars filled with dark grey color. Climate-growth associations calculated for Q. suber trees. The bars indicate the mean Pearson correlation coefficients (error bars show 95% confidence intervals) calculated between ring-width indices and monthly climatic variables. The temporal window of analyses comprises the hydrological year from October of the previous year to September of the current year (x axes). Significant correlations are indicated by bars filled with dark grey color. Figure 5 Venn diagram of the variation partitioning of regeneration abundance of Quercus saplings and seedlings explained by soil and structural (struct) variables. Numbers indicate percentage of variance explained. Page 26/28 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 27/28 Page 27/28 SUPPLEMENTARYMATERIAL.docx Page 28/28
https://openalex.org/W2901546341	https://pure.tue.nl/ws/files/116901053/materials_11_02253_v2.pdf	English	null	Electrolytic Surface Treatment for Improved Adhesion between Carbon Fibre and Polycarbonate	Materials	2,018	cc-by	13,816	Document status and date: Published: 12/11/2018 Document status and date: Published: 12/11/2018 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. 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If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: Electrolytic surface treatment for improved adhesion between carbon fibre and polycarbonate Citation for published version (APA): Kamps, J. H., Henderson, L. C., Scheffler, C., van der Heijden, R., Simon, F., Bonizzi, T., & Verghese, N. (2018). Electrolytic surface treatment for improved adhesion between carbon fibre and polycarbonate. Materials, 11(11), Article 2253. https://doi.org/10.3390/ma11112253 Electrolytic surface treatment for improved adhesion between carbon fibre and polycarbonate Citation for published version (APA): Kamps, J. H., Henderson, L. C., Scheffler, C., van der Heijden, R., Simon, F., Bonizzi, T., & Verghese, N. (2018). Electrolytic surface treatment for improved adhesion between carbon fibre and polycarbonate. Materials, 11(11), Article 2253. https://doi.org/10.3390/ma11112253 Citation for published version (APA): Kamps, J. H., Henderson, L. C., Scheffler, C., van der Heijden, R., Simon, F., Bonizzi, T., & Verghese, N. (2018). Electrolytic surface treatment for improved adhesion between carbon fibre and polycarbonate. Materials, 11(11), Article 2253. https://doi.org/10.3390/ma11112253 DOI: 10.3390/ma11112253 DOI: 10.3390/ma11112253 Download date: 24. Oct. 2024 Received: 13 October 2018; Accepted: 6 November 2018; Published: 12 November 2018 Abstract: To achieve good mechanical properties of carbon ﬁbre-reinforced polycarbonate composites, the ﬁbre-matrix adhesion must be dialled to an optimum level. The electrolytic surface treatment of carbon ﬁbres during their production is one of the possible means of adapting the surface characteristics of the ﬁbres. The production of a range of tailored ﬁbres with varying surface treatments (adjusting the current, potential, and conductivity) was followed by contact angle, inverse gas chromatography and X-ray photoelectron spectroscopy measurements, which revealed a signiﬁcant increase in polarity and hydroxyl, carboxyl, and nitrile groups on the ﬁbre surface. Accordingly, an increase in the ﬁbre-matrix interaction indicated by a higher interfacial shear strength was observed with the single ﬁbre pull-out force-displacement curves. The statistical analysis identiﬁed the correlation between the process settings, ﬁbre surface characteristics, and the performance of the ﬁbres during single ﬁbre pull-out testing. Keywords: carbon ﬁbre; surface treatment; polycarbonate; composites; interfacial adhesion; single ﬁbre pull out an Henk Kamps 1,, Luke C. Henderson 2, Christina Schefﬂer 3 , Ruud van der Heijden 1, Frank Simon 3 , Teena Bonizzi 4 and Nikhil Verghese 5 1 SABIC, Plasticslaan 1, 4612PX Bergen op Zoom, The Netherlands; ruud.vanderheijden@sabic.com 2 Institute for Frontier Materials, Carbon Nexus, Deakin University, 75 Pigdons Rd, Waurn Ponds, VIC 3216 Australia; luke.henderson@deakin.edu.au 3 Leibniz-Institut für Polymerforschung Dresden e.V. (IPF), Hohe Straße 6, 01069 Dresden, Germany; schefﬂer@ipfdd.de (C.S.); frsimon@ipfdd.de (F.S.) 4 SABIC Technology Center, 6160 AL Geleen, The Netherlands; Teena.Bonizzi@sabic.com 5 SABIC Technology Center, Sugar Land, Houston, TX 77478, USA; nikhil.verghese@sabic.com Correspondence: janhenk.kamps@sabic.com; Tel.: +31-164-293-482 Materials 2018, 11, 2253; doi:10.3390/ma11112253 materials materials www.tue.nl/taverne Take down policy If you believe that this document breaches copyright please contact us at: openaccess@tue.nl providing details and we will investigate your claim. Download date: 24. Oct. 2024 1. Introduction Improving mechanical properties through the addition of reinforcing ﬁbres is a common approach used in a range of thermoplastic materials [1–4]. An important parameter faced in all research in this domain is the role of the interface between the ﬁbre and the resin. To enable and exploit the mechanical property proﬁle of ﬁbre-reinforced thermoplastic composites, ﬁbre-matrix adhesion must be at an optimum level [1–3]. An increase in the adhesion between carbon ﬁbres and the polymer matrix can be achieved using different approaches, which are summarized in detailed review articles [5,6]. In general, wet-chemical (sizing/polymer ﬁnish, acidic modiﬁcation, and electrochemical modiﬁcation), dry-chemical (plasma/high energy irradiation modiﬁcation, nickel surface coating, and thermal modiﬁcation) and multiscale methods by applying nano-particles onto the surface are used to modify the carbon ﬁbre surface. Each combination of ﬁbre and matrix material will have its own ideal approach; for polycarbonate, speciﬁc studies have been conducted, mainly with respect to oxygen plasma-treated carbon ﬁbres [7–9] or electrochemical oxidation [10–13], generally showing a signiﬁcant increase in adhesion to polycarbonate after treatment. Table 1 gives an overview of the studies documented in literature and their results. The interfacial shear strength characterization Materials 2018, 11, 2253; doi:10.3390/ma11112253 www.mdpi.com/journal/materials Materials 2018, 11, 2253 Materials 2018, 11, 2253 2 of 19 can be approached using different micromechanical testing methods, each of which has its own unique procedure [14–18]. In this study, we focused on the single ﬁbre pull-out, which is very suitable for evaluating the interfacial shear strength on a microscopic scale involving viscous polymers like polycarbonate. Combining the modiﬁcation of carbon ﬁbres with surface characterization, followed by single ﬁbre pull-out testing gives a detailed insight into the crucial parameters that control the interface and its impact on composite performance. Linking this approach with statistical studies to demonstrate the value of surface treatment for interface formation and compatibility of speciﬁc process settings with polycarbonate completes the work documented here. 3 of 19 Materials 2018, 11, 2253 Table 1. Overview of the material modiﬁcations, processing conditions, and micromechanical tests applied on carbon ﬁbre—polycarbonate composites to increase and characterize the ﬁbre-matrix adhesion by the interfacial shear strength (IFSS; the results represent the lowest and highest achieved value of the investigated materials for each reference); * Mw = molecular weight, ** SD = standard deviation. for each reference); * Mw = molecular weight, SD = standard deviation. 1. Introduction Fibre Treatment Matrix Testing Method IFSS ± SD Ref. PAN-based unmodiﬁed, unsized CF (Idemitsu Kosan, Tokyo, Japan) Anodic oxidation (electrolyte solution: K2CO3/KOH; KNO3/KOH) PC (Makrofol®, Bayer, Leverkusen, Germany) Microdroplet pull-off test 9.6 ± 1.1 MPa (not oxidized); 14.7 ± 3.1 MPa (2.5 min in KNO3/KOH) [10] PAN-based CF with unknown sizing (12K, HTS40, Toho Inc. Corp., Tokyo, Japan) and self-prepared CF with epoxy sizing Electrochemical oxidation using a 0.1 mol/L NaOH electrolyte PC (Dongguang Plastic Film Corporation, Dongguang, China), focusing on polycarbonate backbone transesteriﬁcation Single ﬁbre fragmentation test 25.04 ± 1.08 MPa (not oxidized); 47.53 ± 1.23 MPa (15 min treatment time) [11] PAN-based unmodiﬁed (UT) and oxidized (ST) CF (Toray Industries Inc., Tokyo, Japan) Electrochemical oxidation Bisphenol-A based PC with varying Mw * PC1 Mw 25,000 g/mol PC2 Mw 32,000–36,000 g/mol (consolidation temperature 230–310 ◦C) Single ﬁbre fragmentation test PC 1: (230/310 ◦C) UT: 30.2/41.0 MPa ST: 43.8/56.5 MPa PC 2: UT: 42.8/48.4 MPa ST: 59.3/67.9 MPa [12] UHM pitch-based CF; HT PAN-based CF; both untreated and unsized Microwave O2-plasma oxidation PC Makrolon® 2805 (Bayer, Leverkusen, Germany) Single ﬁbre fragmentation test HT: 24.0 ± 2 MPa HT-ox.: 27.7 ± 2 MPa UHM: 12.2 ± 1 MPa UHM-ox: 46.7 ± 3 MPa [7] PAN-based CF (Hexcel Magnamite® IM7, Stamford, CT, USA) Commercial oxidative surface treatment at different grades linear amorphous thermoplastic, Bisphenol-A based PC (GE Plastics, Inc., Pittsﬁeld, MA, USA), Mw 31,000 g/mol Microindentation test 100% ox.: 27.0 ± 1.9 MPa 400% ox.: 28.6 ± 3.2 MPa [13] PAN-based CF, Magnamite AS1 and AS4 (Hercules Aerospace, Wilmington, NC, USA) Plasma treatment with ammonia, argon, nitrogen and oxygen Polycarbonate LEXAN™101, (SABIC, Bergen op Zoom, The Netherlands) Single ﬁbre fragmentation test lc/d 102% for ammonia treated lc/d 100% for ammonia treated lc/d 90% for argon treated lc/d 65% for oxygen treated [8] PAN-based CF, C320.00A, Sigri SGL Carbon, Wiesbaden, Germany Low pressure oxygen plasma PC Macrofol® DE 1-1 (Bayer AG, Leverkusen, Gerrmany) Single ﬁbre fragmentation test 11.1 ± 1.2 MPa (no treatment) 9.8 ± 1.4 MPa (20 min treatment) [9] PAN-based CF, unsized Commercial process, undisclosed Functionalized polycarbonate (SABIC, Bergen op Zoom, The Netherlands) Single ﬁbre pull-out 33.9 ± 9.1 MPa (reference) 42.2 ± 9.0 MPa (functionalized PC) [19] Materials 2018, 11, 2253 4 of 19 2. Materials and Methods The carbonization of poly (1-acrylonitrile) ﬁbre, followed by varying surface treatments (adjusting the current, potential, and conductivity), created a range of seven different ﬁbre samples used in this study (Table 2). For the manufacture of carbon ﬁbres in this study, three surface treatment variables were taken into account, which are thought to affect ﬁbre-to-matrix adhesion. These include the current passed through the ﬁbre during surface treatment, the potential applied to the ﬁbre, and the conductivity of the electrolyte used in the bath (in this instance, ammonium hydrogen carbonate, [NH4]+ [HCO3]−). It should be noted that due to the continuous nature of this production methodology, and the fact that the electrolyte is partially consumed during the surface treatment process, maintaining the exact level of each amperage, potential, and conductivity is challenging. Thus, different experiments used values as similar as possible for each of these, and these variables were classed into ‘bands’ of low, medium, and high for current (8, 14, and 26 A, respectively), and potential (5.7–5.8, 8–8.1, and 12.5–13.5 V, respectively). Only medium and high conductivities (17.0–17.5, and 31.2–31.4 mS/cm−1, respectively) were investigated, as low conductivity of the electrochemical bath carries a risk of equipment malfunction or breakage. q p g To minimize the effect of unknown inﬂuences, a control sample (sample number 1), which did not have any surface treatment applied, was included in the experiment. This sample was collected directly after being passed through the high-temperature furnace. Table 2. Surface treatment parameters and physical properties of resultant ﬁbres. Sample Number Current (A) Potential (V) Conductivity (mS/cm) 1 - - - 2 8 5.8 17.5 3 14 8 17.5 4 26 13.5 17 5 26 12.5 31.3 6 14 8.1 31.4 7 8 5.7 31.2 Table 2. Surface treatment parameters and physical properties of resultant ﬁbres. LEXAN™HF1110, a polycarbonate homopolymer (BPA) produced on a commercia LEXAN™HF1110, a polycarbonate homopolymer (BPA) produced on a commercial scale by SABIC (Saudi Basic Industries Corporation, Riyadh, Saudi Arabia) and available as high-ﬂow general-purpose grade, was used for the single ﬁbre pull-out (SFPO) testing, selected for its lower viscosity, enabling efﬁcient ﬁbre embedding. 2.1. Inverse Gas Chromatography (IGC)—Surface Free Energy Analysis (SEA) A series of n-alkanes (n-hexane, n-heptane, n-octane, and n-nonane) and polar probes (chloroform, ethyl acetate, 1,4-dioxane, ethanol, and dichloromethane) were injected into a column, which was ﬁlled with the ﬁbre samples with speciﬁc fractional surface coverages, and their retention times were measured. The retention times (t) were converted into retention volumes. The dispersive free surface energy (γD S ) and speciﬁc free energy of desorption (∆G0 SP) values on the surface of the ﬁbre samples were determined in accordance with the standard method described by Jones [20]. 0 The (∆G0 SP) value obtained from the chloroform and ethyl acetate pair of mono-functional acidic and basic probes was used to determine the acid and base properties of the samples by applying an acid-base theory developed by van Oss [18,21]. The speciﬁc component of the surface free energy (γAB S ) was calculated for this acid (Lewis electron pair acceptor) base (Lewis electron pair donor) pair. The so-called term ‘speciﬁc component of the surface free energy’ is widely used. However, according to the deﬁnition of the surface free energy {(∂f/∂o) = free energy (f), which is necessary to increase the surface (o)} it seems to be wrong to separate the surface free energy into dispersive and speciﬁc 5 of 19 Materials 2018, 11, 2253 components because the surface free energy is an intrinsic value of the solid surface and does not depend on interacting liquids. The energy, which is determined from a solid surface coming into contact with a liquid, must be considered as interaction energy (both two phases contribute to the interaction energy). However, there is no problem in determining the interaction energy using IGC and splitting the interaction energy values into the contributions of dispersive (interaction) energy and speciﬁc (interaction) energy values. The total surface free energy, γT S, was calculated as the sum of the dispersive (γD S ) and speciﬁc (γAB S ) energy contributions. Fitting the data to an exponential decay function, y = y0 + A exp [−x·t−1], allowed for extrapolation across the entire range (0–100%) of the surface coverage (x), where y0 is the value of the function at inﬁnity. 2.3. Tensiometer: Contact Angle and Surface Free Energy The contact angles (CA) of fibre samples 1–7 with deionized water (milli-Q) and 1-bromonaphthalene (97%, Sigma-Aldrich, Taufkirchen, Germany) were measured on a force tensiometer K100SF with LabDesk 3.2.2 software from KRÜSS GmbH (Hamburg, Germany), which was placed on a TS-150 LP dynamic antivibration system supplied by TABLE STABLE (Mettmenstetten, Switzerland). The measurements were performed at ambient conditions. In each test, a force-displacement curve was recorded while immersing a ﬁbre into one of the test liquids at a length of 5 mm with a speed of 3 mm/min and a data acquisition step of 0.02 mm. For the detection of the ﬁbre (a sudden change in force), a detection speed of 6 mm/min was used and the detection sensitivity was set at 2 × 10−5–7.5 × 10−5 g. By regression of the force-displacement curve and extrapolation to 0 mm immersion depth, the wetting force F was determined and the advancing contact angle (θa) was calculated with the Wilhelmy Equation: cosθa = F/(Lσ) (1) (1) where σ is the total surface tension of the liquid (water = 72.8 mN/m, 1-bromonaphthalene = 44.6 mN/m) and L is the perimeter of the ﬁbre based on the average ﬁbre diameter determined for each sample during tensile testing (see Section 2.2). For each sample, 10 ﬁbres were tested per test liquid, resulting in an average θa per test liquid. The average θa with water and 1-bromonaphthalene were used to calculate the surface free energy (SFE) values of all the ﬁbre samples, using the Owens, Wendt, Rabel, and Kaelble (OWRK) method [22]. The total SFE of each sample equals the sum of a polar (σP) and dispersive surface energy component (σD). The surface polarity was determined by taking the ratio—reﬂected as a percentage—of the polar component to the overall SFE. 2.2. Tensile Testing Bare ﬁbre samples were tested using a Favimat+ Robot 2 single ﬁbre tester (Textechno H. Stein, Mönchengladbach, Germany) which automatically records linear density and force extension data for individual ﬁbres loaded into a magazine (25 samples) with a pretension weight of 80 ± 5 mg attached to the bottom of each carbon ﬁbre. Linear density was recorded using a length of 25 mm and a tension of 0.15 mN (as per the supplier speciﬁcations). The tensile load extension curves were collected at 1.0 mm/min using a gauge length of 25 mm and a pretension of 1.0 cN/tex. The load data were normalized by dividing by the linear density to give the speciﬁc stress strain curves from which the tensile strength (ultimate speciﬁc stress or tenacity) and speciﬁc modulus could be determined. 2.4. Single Fibre Pull-Out Test (SFPO) The interfacial adhesion strength between the ﬁbre and matrix was evaluated by means of a SFPO using purpose-built embedding equipment constructed at IPF Dresden (Germany) [15,23]. Samples were prepared by accurately embedding one end of the selected single ﬁbre in the matrix 6 of 19 Materials 2018, 11, 2253 (perpendicularly) with a pre-selected embedding length le (le = 150 µm). For polycarbonate, an embedding temperature of 300 ◦C was required and embedding was carried out at controlled atmosphere and temperature. After embedding, the temperature was held at 300 ◦C for about 30 s, before cooling down to ambient temperature. The pull-out test was carried out with a force accuracy of 1 mN, a displacement accuracy of 0.07 µm, and a loading rate of 0.01 µm/s at ambient conditions (using a self-made pull-out apparatus). The force-displacement curves and the maximum force, Fmax, required for pulling the ﬁbre out of the matrix were measured. After testing, the ﬁbre diameter, df, was measured using optical microscopy; le was determined using the force-displacement curve and cross-checked using scanning electron microscope (SEM) Ultra (Carl Zeiss AG, Oberkochen, Germany). The adhesion bond strength between the ﬁbre and the matrix was characterized by the values of the apparent interfacial shear strength (τapp = Fmax/(π × df × le)). Other interfacial parameters (such as local interfacial shear strength τd and interfacial frictional stress τf) were not considered in this work for analysing the ﬁbre-matrix adhesion. Most of the curves did not follow the characteristic shape of the pull-out curve as described in Reference [18], meaning that the determination of the characteristic points for modelling (debonding force Fd, minimum force after debonding based on friction Fb) were not clearly identiﬁable [24,25]. Instead, the debonding work (from le = 0 to le at Fmax) and pull-out work (from le at Fmax to maximum le achieved at complete ﬁbre pull-out) were used for comparison. Each ﬁbre/matrix combination was evaluated in about 15–20 single tests. The ﬁlament surface before and after the pull-out test was evaluated using (SEM). 2.5. X-ray Photoelectron Spectroscopy (XPS) All the XPS studies were carried out by means of an Axis Ultra photoelectron spectrometer (Kratos Analytical, Manchester, UK), equipped with a monochromatic Al Kα (1486.6 eV) X-ray source of 300 W at 15 kV. A hemispheric analyzer set to pass energy of 160 eV for wide-scan spectra and 20 eV for high-resolution spectra was used to determine the kinetic energy of the photoelectrons. The sample (carbon ﬁbre tow) was mounted on a sample holder using adhesive tape so that the analyzed area was over an opening in the sample holder, enabling exposure to the X-ray source during measurement. Although the carbon ﬁbres were electrically conductive, a low-energy electron source in combination with a magnetic immersion lens was employed to avoid electrostatic charging of the sample that can occur by ﬁxing the ﬁbres on the sample holder with the insolating adhesive tape. All the recorded peaks were shifted by the same value to set the C 1s component peak of the saturated hydrocarbons to 285.00 eV. The quantitative elemental compositions were determined from the peak areas using experimentally determined sensitivity factors and the spectrometer transmission function. Kratos spectra deconvolution software was applied to the high-resolution spectra and the spectrum background was subtracted according to Shirley. The free parameters of the component peaks were their binding energy (BE), height, full width at half maximum, and the Gaussian-Lorentzian ratio. 2.6. Statistical Evaluation The testing results are reported as single values or mean ± standard deviation when multiple repeat evaluations of the ﬁbre sample were conducted. Table 3 lists the average values, standard deviation, and sample size for (Favimat) tensile testing, and the single ﬁbre pull-out measurements. In the results section the average values, standard deviation, and sample size for the contact angle measurements, which are used to calculate the energy and adhesion values listed in Table 3. Inverse gas chromatography was practiced on a bundle of ﬁbres, resulting in responses based on the surfaces of numerous individual ﬁlaments; in all cases the line ﬁt had a R2 > 0.997, showing a good representation of the reported results. XPS was carried out by irradiating an area of approximately 3 mm2 of the analyzed ﬁbre bundles. From this irradiated area, the spectrometer collects nearly all the photoelectrons leaving the sample surface, measures their kinetic energy, and uses them to draw the corresponding spectrum, which reﬂects the average of the analyzed area, representing a large number of ﬁlaments. Materials 2018, 11, 2253 7 of 19 To compare the physical properties of the six surface-treated samples to the control, the Dunnett’s Method test was used, the Pearson correlation coefﬁcient (r) was calculated, and the p-value for statistical signiﬁcance was derived. To evaluate the relationship between the IFSS and the surface treatment parameters (current, potential, and conductivity) a linear regression model was used with both univariate and multivariable results reported as a parameter estimate (95% conﬁdence interval) with a p-value. The ﬁt of the model was assessed visually, and no concerns were noted. y All the analyses were performed on JMP© Pro 13, SAS Institute Inc., Cary, NC, USA, and a p-value of less than 0.05 was considered as statistically signiﬁcant. Table 3. Complete overview of process settings and associated test results. 2.6. Statistical Evaluation Sample Number 1 2 3 4 5 6 7 Testing Method Current (A) 0 8 14 26 26 14 8 - Potential (V) 0 5.8 8 13.5 12.5 8.1 5.7 - Conductivity (mS/cm) 0 17.5 17.5 17 31.3 31.4 31.2 - Elongation at Break (%) 1.58 1.60 1.63 1.64 1.70 1.79 1.63 Favimat Standard deviation (n = 25) 0.24 0.29 0.24 0.28 0.32 0.23 0.24 - Modulus (GPa) 259.85 261.44 266.24 262.06 261.81 263.09 264.22 Favimat Standard deviation (n = 25) 3.59 4.58 11.36 3.26 5.20 4.73 4.26 - Tensile strength (GPa) 3.84 3.88 4.05 4.02 4.13 4.38 4.00 Favimat Standard deviation (n = 25) 0.61 0.72 0.62 0.71 0.80 0.58 0.62 - Diameter (µm) 6.54 6.54 6.5 6.55 6.59 6.52 6.56 Favimat Standard deviation (n = 25) 0.14 0.15 0.13 0.13 0.11 0.19 0.15 - Total surface energy (mJ/m2) 67.0 68.1 72.2 75.7 73.2 72.7 70.5 IGC Dispersive surface energy (mJ/m2) 51.9 47.4 46.1 47.8 46.9 46.4 47.4 IGC Speciﬁc surface energy (mJ/m2) 15.0 20.6 26.0 27.5 26.0 26.1 22.7 IGC Atomic Conc. Hydroxyl (%) 1.50 1.90 2.15 3.68 3.24 3.36 3.10 XPS Atomic Conc. Carboxyl (%) 1.10 1.51 1.62 2.93 3.05 2.15 1.80 XPS Atomic Conc. Nitrile (%) 2.07 4.79 4.48 5.75 7.17 6.52 6.70 XPS Total surface energy (mJ/m2) 41.9 55.9 56.0 64.0 56.5 58.4 56.2 CA Polar surface energy (mJ/m2) 2.7 14.8 17.2 21.8 20.3 18.2 14.4 CA Dispersive surface energy (mJ/m2) 39.2 41.1 38.8 42.2 36.2 40.3 41.8 CA Polarity (%) 6.5 26.4 30.7 34.0 35.9 31.1 25.6 CA Adhesion energy ambient (mJ/m2) 83.7 87.6 85.5 89.5 83.1 87.2 88.3 CA Interfacial tension ambient (mN/m) 1.6 11.6 13.9 17.9 16.8 14.7 11.3 CA Adhesion energy 260 ◦C (mJ/m2) 50.6 71.0 72.6 78.8 74.6 74.4 70.9 CA Interfacial tension 260 ◦C (mN/m) 19.4 13.0 11.4 13.3 9.9 12.2 13.4 CA τapp (N/mm2) 48.8 50.1 55.2 43.2 54.7 49.5 33.3 SFPO Standard deviation (n = 25) 12.4 14.0 11.5 11.1 6.5 18.9 15.1 - Wdebond (mN mm) 1.5 1.2 1.8 0.7 0.9 0.7 0.6 SFPO Standard deviation (n = 25) 0.9 0.6 1.3 0.9 0.6 0.5 0.6 - Wpullout (mN mm) 2.1 1.6 1.5 2.9 2.2 1.3 2.0 SFPO Standard deviation (n = 25) 0.9 0.6 0.7 2.7 2.8 0.7 1.1 - Table 3. Complete overview of process settings and associated test results. Table 3. Complete overview of process settings and associated test results. 3. Results The work documented here spreads across different disciplines and techniques. An overview of the results of the production, ﬁbre characterization, and single ﬁbre pull-out testing are reported in Table 3. 3.1. Fibre Surface Treatment Results and Differences Observed In particular, when the potential setting is low, increasing the current is associated with a higher modulus, but when the potential setting is high, increasing the current is associated with a lower modulus. The application of current appeared to influence the modulus, with lower current settings being associated with a higher modulus. However, this effect can be modified by the potential setting. In particular, when the potential setting is low, increasing the current is associated with a higher modulus, but when the potential setting is high, increasing the current is associated with a lower modulus. The ﬁbre surface was examined to ensure no pitting or surface defects had arisen on the ﬁbre surface due to these oxidative procedures. Given the tensile strength data acquired for these samples, it is unlikely that any defects had been introduced to the surface; nevertheless, imaging the ﬁbre using SEM was undertaken in the interest of thoroughness (Figure 1). modulus. The fibre surface was examined to ensure no pitting or surface defects had arisen on the fibre surface due to these oxidative procedures. Given the tensile strength data acquired for these samples, it is unlikely that any defects had been introduced to the surface; nevertheless, imaging the fibre using SEM was undertaken in the interest of thoroughness (Figure 1). Figure 1. SEM images of all treated fibres from this study; sample 1 is the untreated sample, samples 2–7 show the same surface features and no surface defects have been detected. Figure 1. SEM images of all treated ﬁbres from this study; sample 1 is the untreated sample, samples 2–7 show the same surface features and no surface defects have been detected. Figure 1. SEM images of all treated fibres from this study; sample 1 is the untreated sample, samples 2–7 show the same surface features and no surface defects have been detected. Figure 1. SEM images of all treated ﬁbres from this study; sample 1 is the untreated sample, samples 2–7 show the same surface features and no surface defects have been detected. The visual examination of the fibres displayed no obvious changes compared to sample 1, which had not undergone any surface treatment. The longitudinal striations and fibre diameter (approx. 7 μm) were observed with all samples, suggesting that the surface treatment conditions, while aggressive in some instances, did not result in substantial fibre degradation. 3.1. Fibre Surface Treatment Results and Differences Observed The characterization of the untreated ﬁbres (sample 1) showed a tensile strength and Young’s modulus of 3.84 and 259.85 GPa, respectively. For a comparison with a commercial product, these properties are slightly superior compared to automotive grade carbon ﬁbres (T300, tensile strength and Young’s modulus of 3.53 and 230 GPa, respectively). Samples 3 and 7 had a statistically signiﬁcant increase in Young’s modulus, though elongation at break and tensile strength were unchanged. Further improvements were observed when both the potential and current through the ﬁbre were increased, at the same conductivity, though again, the only statistically signiﬁcant change compared to sample 1 8 of 19 Materials 2018, 11, 2253 was with respect to the Young’s modulus. Interestingly, further increasing the amperage and potential caused the Young’s modulus to decrease slightly (Table 3, sample 4), and increasing the conductivity (Table 3, sample 5) corresponded to no meaningful property changes, suggesting that there is an optimal ratio and interplay between these three variables and that more of each, or even one, does not correspond to improved properties. Reverting to medium amperage and potential, which showed promise in sample 3, but increasing conductivity (Table 3, sample 6), had the most beneﬁcial effects on the performance characteristics. All three measured parameters showed statistically signiﬁcant changes relative to sample 1. Finally, combining low amperage and potential with high conductivity showed excellent improvements in all properties, suggesting that conductivity assists in the inﬂuence of the electrochemical treatments. Materials 2018, 11, x FOR PEER REVIEW 8 of 21 of each, or even one, does not correspond to improved properties. Reverting to medium amperage and potential, which showed promise in sample 3, but increasing conductivity (Table 3, sample 6), had the most beneficial effects on the performance characteristics. All three measured parameters showed statistically significant changes relative to sample 1. Finally, combining low amperage and potential with high conductivity showed excellent improvements in all properties, suggesting that conductivity assists in the influence of the electrochemical treatments. The application of current appeared to influence the modulus, with lower current settings being The application of current appeared to inﬂuence the modulus, with lower current settings being associated with a higher modulus. However, this effect can be modiﬁed by the potential setting. 3.1. Fibre Surface Treatment Results and Differences Observed Given the consistency in surface structure and morphology, we turned our attention next to the examination of the surface chemistry using Inverse Gas Chromatography (IGC). The visual examination of the ﬁbres displayed no obvious changes compared to sample 1, which had not undergone any surface treatment. The longitudinal striations and ﬁbre diameter (approx. 7 µm) were observed with all samples, suggesting that the surface treatment conditions, while aggressive in some instances, did not result in substantial ﬁbre degradation. Given the consistency in surface structure and morphology, we turned our attention next to the examination of the surface chemistry using Inverse Gas Chromatography (IGC). 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC To determine the nature of the acid/base and the dispersive surface energies of the treated carbon fibres, we used IGC. In this technique, a column filled with the carbon fibres is injected with gaseous probes, which interact with various functional groups on the surface of the fibres. Typically, a range of non-polar (n-alkanes) and polar (ethyl acetate, ethanol, etc.) test liquids are used to determine the dispersive and Lewis acid/base properties, respectively (Table 4 and Figure 2). To determine the nature of the acid/base and the dispersive surface energies of the treated carbon ﬁbres, we used IGC. In this technique, a column ﬁlled with the carbon ﬁbres is injected with gaseous probes, which interact with various functional groups on the surface of the ﬁbres. Typically, a range of non-polar (n-alkanes) and polar (ethyl acetate, ethanol, etc.) test liquids are used to determine the dispersive and Lewis acid/base properties, respectively (Table 4 and Figure 2). 9 of 19 9 of 21 Materials 2018, 11, 2253 M t i l 2018 11 FOR Materials 2018, 11, 2253 9 of 19 Materials 2018, 11, x FOR PEER REVIEW 9 of 21 Figure 2. Inverse gas chromatography results (sample 1). Figure 2. Inverse gas chromatography results (sample 1). Figure 2. Inverse gas chromatography results (sample 1). Figure 2. Inverse gas chromatography results (sample 1). Given the nature of this technique, a comparison of the absolute values is not informative, therefore the ratio of dispersive and polar energies is provided to give a more meaningful comparison between the samples. Sample 1, as expected, possessed a very high dispersive energy component, resulting from the highly graphitic nature of this fibre. Given the nature of this technique, a comparison of the absolute values is not informative, therefore the ratio of dispersive and polar energies is provided to give a more meaningful comparison between the samples. Sample 1, as expected, possessed a very high dispersive energy component, resulting from the highly graphitic nature of this ﬁbre. There is some evidence to suggest that increasing the current will decrease the dispersive surface energy and increase the specific surface energy, as can be observed in Table 4. Furthermore, the potential applied is likely to have a modifying effect on the surface properties. 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC The similarity of the specific energy values for samples 3 and 4 is counter-intuitive considering that the oxidative treatment was more aggressive for sample 4 than for sample 3, suggesting that a plateau was reached under these conditions, perhaps dictated by the concentration, and thus conductivity, of the There is some evidence to suggest that increasing the current will decrease the dispersive surface energy and increase the speciﬁc surface energy, as can be observed in Table 4. Furthermore, the potential applied is likely to have a modifying effect on the surface properties. The similarity of the speciﬁc energy values for samples 3 and 4 is counter-intuitive considering that the oxidative treatment was more aggressive for sample 4 than for sample 3, suggesting that a plateau was reached under these conditions, perhaps dictated by the concentration, and thus conductivity, of the electrolyte. electrolyte. Table 4. IGC results of the produced fibres. Sample Number Dispersive Energy (mJ/m2) Specific (Acid-Base) (mJ/m2) Total (mJ/m2) Ratio of Dispersive and Specific Energies a 1 51.94 (77.5%) 15.04 (22.5%) 66.98 3.45:1.0 2 47.41 (69.8%) 20.55 (30.2%) 68.14 2.31:1.0 3 46.06 (63.9%) 26.02 (36.1%) 72.19 1.77:1.0 4 47.83 (63.5%) 27.45 (36.5%) 75.72 1.74:1.0 5 46.88 (64.4%) 25.97 (35.6%) 73.23 1.81:1.0 6 46.40 (64.0%) 26.09 (36.0%) 72.65 1.78:1.0 7 47.38 (67.5%) 22.73 (32.5%) 70.49 2.08:1.0 Table 4. IGC results of the produced ﬁbres. Sample Number Dispersive Energy (mJ/m2) Speciﬁc (Acid-Base) (mJ/m2) Total (mJ/m2) Ratio of Dispersive and Speciﬁc Energies a 1 51.94 (77.5%) 15.04 (22.5%) 66.98 3.45:1.0 2 47.41 (69.8%) 20.55 (30.2%) 68.14 2.31:1.0 3 46.06 (63.9%) 26.02 (36.1%) 72.19 1.77:1.0 4 47.83 (63.5%) 27.45 (36.5%) 75.72 1.74:1.0 5 46.88 (64.4%) 25.97 (35.6%) 73.23 1.81:1.0 6 46.40 (64.0%) 26.09 (36.0%) 72.65 1.78:1.0 7 47.38 (67.5%) 22.73 (32.5%) 70.49 2.08:1.0 a Determined by dispersive/speciﬁc energies. te. Table 4. IGC results of the produced ﬁbres. Table 4. IGC results of the produced ﬁbres. ) ( ) %) 22.73 (32.5%) 70.49 a Determined by dispersive/speciﬁc energies. a Determined by dispersive/specific energies. A similar observation can be made when examining samples 5 and 6, where the polar portion of the surface energy remains at approximately 35–36% of the total surface energy, again suggesting a plateau of oxidative treatment and installation of polar functional groups. 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC Nitrogen, which was found on the carbon ﬁbre surfaces, could be a constituent of functional surface groups but also 10 of 19 Materials 2018, 11, 2253 a residue of the ammonium salt (NH4+), which was used during the electrical oxidation process. Shape-analysis of the high-resolution element spectra is an established method to study the different binding states of the atoms in the surface region of solids. However, due to the so-called ‘shake-up’ phenomena, which were observed in XPS spectra recorded from substances consisting of graphite-like lattices, such as carbon ﬁbres, the deconvolution of the C 1s spectra is generally difﬁcult. Graphite-like lattices consist of sp2-hybridized carbon atoms in which the π-bonded pz-electrons can be extensively delocalized. Each linear combination of two of the pz wave functions gives wave functions of one π-orbital occupied by the two pz-electrons and one unoccupied π-orbital. In the case of graphite-like structures, the high number of possible linear combinations leads to a quasicontinuum of energy levels that can be occupied by electrons. Energy from an external source can be consumed to transfer a pz-electron from its π-orbital (ground state) into a π-orbital (excited states). The C 1s spectra shows the photoelectrons emitted from the ground as well as excited states. The latter contribute to the shake-up peaks mentioned above. Materials 2018 11 x FOR PEER REVIEW 11 of 21 Figure 3. Wide-scan photoelectron spectra (left column), C1s (middle column) and N1s high- resolution photoelectron spectra (right column) recorded from unmodified carbon fibres (sample 1) (a), and electro-chemically modified carbon fibres at low current and low current and low conductivity (sample 2) (b), high current and low conductivity (sample 4) (c), high current and high conductivity (sample 5) (d), and low current and high conductivity (sample 7) (e). B C D shake-up F B C D shake-up F B C D shake-up F B C D shake-up O KLL O 1s 292 288 296 Binding Energy [eV] 1000 800 600 400 200 0 284 300 Binding Energy [eV] Binding Energy [eV] 402 398 406 394 410 Gr A a) b) c) d) e) C 1s N 1s Na KLL Ca 2p Si 2p Si 2s Mg 2p Mg 2s O 2s Ca 2s B C D F shake-up L A F Gr K L A Gr K L C 1s N 1s A Gr K L A Gr K L Figure 3. 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC Wide-scan photoelectron spectra (left column), C 1s (middle column) and N 1s high-resolution photoelectron spectra (right column) recorded from unmodiﬁed carbon ﬁbres (sample 1) (a), and electro-chemically modiﬁed carbon ﬁbres at low current and low current and low conductivity (sample 2) (b), high current and low conductivity (sample 4) (c), high current and high conductivity (sample 5) (d), and low current and high conductivity (sample 7) (e). B C D shake-up F B C D shake-up F B C D shake-up F B C D shake-up 292 288 296 284 300 Binding Energy [eV] Binding Energy [eV] 402 398 406 394 410 Gr A p B C D F shake-up L A F Gr K L A Gr K L C 1s N 1s A Gr K L A Gr K L Figure 3. Wide-scan photoelectron spectra (left column), C1s (middle column) and N1s high- resolution photoelectron spectra (right column) recorded from unmodified carbon fibres (sample 1) (a), and electro-chemically modified carbon fibres at low current and low current and low conductivity (sample 2) (b), high current and low conductivity (sample 4) (c), high current and high conductivity (sample 5) (d), and low current and high conductivity (sample 7) (e). Figure 3. Wide-scan photoelectron spectra (left column), C 1s (middle column) and N 1s high-resolution photoelectron spectra (right column) recorded from unmodiﬁed carbon ﬁbres (sample 1) (a), and electro-chemically modiﬁed carbon ﬁbres at low current and low current and low conductivity (sample 2) (b), high current and low conductivity (sample 4) (c), high current and high conductivity (sample 5) (d), and low current and high conductivity (sample 7) (e). Table 5. Fractions of component peak areas. Sample Number [N]:[C] [O]:[C] [B] [C] [D] [F] 1 0.011 0.022 0.021 0.015 0.008 0.011 2 0.030 0.084 0.048 0.019 0.017 0.015 3 0.028 0.105 0.045 0.022 0.021 0.016 4 0.036 0.163 0.058 0.037 0.034 0.029 5 0 045 0 142 0 072 0 032 0 038 0 031 The C 1s spectra recorded from all the carbon ﬁbre samples are characterized by intense shake-up peaks appearing at binding energy values higher than 286 eV (Figure 3, middle column). In the same region, component peaks identifying different functional groups were expected. 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC Interestingly, sample 7 shows a distinct decrease in polar surface energy (32%), relative to the other oxidized samples, which A similar observation can be made when examining samples 5 and 6, where the polar portion of the surface energy remains at approximately 35–36% of the total surface energy, again suggesting a plateau of oxidative treatment and installation of polar functional groups. Interestingly, sample 7 shows a distinct decrease in polar surface energy (32%), relative to the other oxidized samples, which corresponds to a decrease in both current and applied potential. p gy ( ) p corresponds to a decrease in both current and applied potential. While IGC thermodynamically described the interactions of solid surfaces to the probe molecules in their environment, XPS offered the opportunity to analyze the type and number of functional groups in the surface region of the differently treated carbon fibres. The wide-scan XPS spectra (Figure 3, left column) showed—with the exception of hydrogen—all the elements in the surface region of the carbon fibres. Besides the metal ions, such as sodium, magnesium, silicon, and calcium that occur only as traces (regarding carbon content, their contents were less than 0.5 at-%), While IGC thermodynamically described the interactions of solid surfaces to the probe molecules in their environment, XPS offered the opportunity to analyze the type and number of functional groups in the surface region of the differently treated carbon ﬁbres. The wide-scan XPS spectra (Figure 3, left column) showed—with the exception of hydrogen—all the elements in the surface region of the carbon ﬁbres. Besides the metal ions, such as sodium, magnesium, silicon, and calcium that occur only as traces (regarding carbon content, their contents were less than 0.5 at-%), considerable amounts of nitrogen and oxygen were detected on the carbon ﬁbre surface. y g g considerable amounts of nitrogen and oxygen were detected on the carbon fibre surface. Although oxygen may also be bonded in counter ions of the metal ions, it can be assumed that the majority of the oxygen atoms were covalently bonded to the carbon fibres. Nitrogen, which was Although oxygen may also be bonded in counter ions of the metal ions, it can be assumed that the majority of the oxygen atoms were covalently bonded to the carbon ﬁbres. 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC In order to separate the shake-up peaks overlapping the component peaks, it was assumed that the different surface modiﬁcations had the same effect on the π →π* transition probabilities and thus on the shape and intensities of all the shake-up peaks. The C 1s peak areas remained after subtraction, and 11 of 19 Materials 2018, 11, 2253 the shake-up peaks were deconvoluted into six component peaks, showing different binding states of carbon. The most intense component peaks Gr (at 284.14 eV) resulted from the photoelectrons escaped from the sp2-hybridized carbon atoms, forming the graphite-like lattice of the carbon ﬁbre material. Saturated hydrocarbons in the sp3 hybrid state, which did not have heteroatoms as binding partners, were assigned as component peaks A (at 285.00 eV). The presence of saturated hydrocarbons is frequently observed in surface analysis because non-speciﬁcally adsorbed contaminations mainly consist of alkanes and their derivatives. Component peaks B (at 285.84 eV) show C–N bonds of amines, C=N bonds of imines, and/or C≡N of nitrile groups. Surprisingly, the intensities of all component peaks B ([B]) were signiﬁcantly higher than the [N]:[C] ratios independently determined from the wide-scan spectra ([B] ≈1.6 [N]:[C]). Obviously, considerable amounts of the nitrogen atoms were present as bound to two carbon atoms, which is well-known from the oxidized cyclization of the PAN structure before the carbonization process of the ﬁbres [26]. The introduction of oxygen in the surface region of the carbon ﬁbre samples resulted in the formation of C–O bonds of mainly phenolic groups (component peaks C at 286.69 eV), quinone-like groups (C=O as component peaks D at 287.77 eV), and carboxyl groups (O=C–OH) and their corresponding carboxylates (−O–C=O ↔O=C–O−) both as component peaks F at 288.72 eV. Table 5 summarizes the fractions of the component peak areas and thus gives an overview of the number of different functional groups. Table 5. Fractions of component peak areas. Sample Number [N]:[C] [O]:[C] [B] [C] [D] [F] 1 0.011 0.022 0.021 0.015 0.008 0.011 2 0.030 0.084 0.048 0.019 0.017 0.015 3 0.028 0.105 0.045 0.022 0.021 0.016 4 0.036 0.163 0.058 0.037 0.034 0.029 5 0.045 0.142 0.072 0.032 0.038 0.031 6 0.042 0.107 0.065 0.034 0.032 0.022 7 0.042 0.087 0.067 0.031 0.025 0.018 Table 5. Fractions of component peak areas. Materials 2018, 11, 2253 surface polarity than polar surface energy Wetting envelope for complete wetting (θa = 0◦). Table 6. Tensiometer results of the ﬁbres produced. Sample Number θa [Water] a (◦) θa 1-[Bromonaphthalene] a (◦) Total SFE (mJ/m2) Surface Polarity (%) 1 82.6 ± 3.2 b 28.7 ± 5.1 41.9 6.5 2 54.7 ± 3.9 22.2 ± 6.9 55.9 26.4 3 52.2 ± 3.8 29.1 ± 8.2 56.0 30.7 4 41.4 ± 3.7 17.8 ± 7.1 64.0 34.0 5 48.9 ± 4.5 35.8 ± 8.8 56.5 35.9 6 49.3 ± 4.4 24.4 ± 8.7 58.4 31.1 Figure 4. Wetting envelope for complete wetting (θa = 0°). Figure 4. Wetting envelope for complete wetting (θa = 0◦). Figure 4. Wetting envelope for complete wetting (θa = 0°). Figure 4. Wetting envelope for complete wetting (θa = 0◦). Table 6 Tensiometer results of the fibres produced Table 6. Tensiometer results of the ﬁbres produced. Table 6. Tensiometer results of the fibres produced. Sample Number θa [Water] a (°) θa 1-[Bromonaphthalene] a (°) Total SFE (mJ/m2) Surface Polarity (%) 1 82.6 ± 3.2 b 28.7 ± 5.1 41.9 6.5 2 54.7 ± 3.9 22.2 ± 6.9 55.9 26.4 3 52.2 ± 3.8 29.1 ± 8.2 56.0 30.7 4 41.4 ± 3.7 17.8 ± 7.1 64.0 34.0 5 48.9 ± 4.5 35.8 ± 8.8 56.5 35.9 6 49.3 ± 4.4 24.4 ± 8.7 58.4 31.1 7 54.9 ± 4.5 19.7 ± 6.4 56.2 25.6 Table 6. Tensiometer results of the ﬁbres produced. Sample Number θa [Water] a (◦) θa 1-[Bromonaphthalene] a (◦) Total SFE (mJ/m2) Surface Polarity (%) 1 82.6 ± 3.2 b 28.7 ± 5.1 41.9 6.5 2 54.7 ± 3.9 22.2 ± 6.9 55.9 26.4 3 52.2 ± 3.8 29.1 ± 8.2 56.0 30.7 4 41.4 ± 3.7 17.8 ± 7.1 64.0 34.0 5 48.9 ± 4.5 35.8 ± 8.8 56.5 35.9 6 49.3 ± 4.4 24.4 ± 8.7 58.4 31.1 7 54.9 ± 4.5 19.7 ± 6.4 56.2 25.6 a Based on 10 measurements. b Based on 8 measurements. To further quantify the compatibility with polycarbonate, the adhesion energy (ψ) and interfacial tension (γ) were calculated with the Fowkes/Dupre and Good’s expression, respectively, using the SFE values of the ﬁbres and commercial LEXAN™HF1110 [29]. The adhesion energy describes how energetically favourable the initial formation of an interface is, whereas the interfacial tension describes the tendency of the formed interface to break in the future upon stress. Materials 2018, 11, 2253 surface polarity than polar surface energy Materials 2018, 11, 2253 surface polarity than polar surface energy 12 of 19 easing From the SFE values, a wetting envelope (Figure 4) for complete wetting could be calculated, which describes all the combinations of the polar (y-axis) and dispersive (x-axis) surface tensions of a liquid that would result in a θa of 0◦by solving the OWRK equation. These wetting proﬁles allow for the prediction of the wetting behaviour of the ﬁbres: the combinations inside the envelope will result in complete wetting (θa = 0◦), while the combinations outside the envelope will not (θa > 0◦). Figure 4 shows the wetting envelopes for the untreated ﬁbre (sample 1) and the extremes of the treated ﬁbres (samples 4 and 7). It can be seen that, theoretically, improved wetting can be expected of the surface-treated ﬁbres with commercial LEXAN™HF1110 polycarbonate at both ambient temperature (σP = 0.2 mJ/m2, σD = 43.2 mJ/m2) and 260 ◦C (σP = 19.9 mN/m, σD = 8.2 mN/m) compared to the untreated ﬁbres. which describes all the combinations of the polar (y-axis) and dispersive (x-axis) surface tensions of a liquid that would result in a θa of 0° by solving the OWRK equation. These wetting profiles allow for the prediction of the wetting behaviour of the fibres: the combinations inside the envelope will result in complete wetting (θa = 0°), while the combinations outside the envelope will not (θa > 0°). Figure 4 shows the wetting envelopes for the untreated fibre (sample 1) and the extremes of the treated fibres (samples 4 and 7). It can be seen that, theoretically, improved wetting can be expected of the surface-treated fibres with commercial LEXAN™ HF1110 polycarbonate at both ambient temperature (σP = 0.2 mJ/m2, σD = 43.2 mJ/m2) and 260 °C (σP = 19.9 mN/m, σD = 8.2 mN/m) compared to the untreated fibres. Figure 4. Wetting envelope for complete wetting (θa = 0°). Table 6. Tensiometer results of the fibres produced. Sample Number θa [Water] a (°) θa 1-[Bromonaphthalene] a (°) Total SFE (mJ/m2) Surface Polarity (%) 1 82.6 ± 3.2 b 28.7 ± 5.1 41.9 6.5 2 54.7 ± 3.9 22.2 ± 6.9 55.9 26.4 3 52.2 ± 3.8 29.1 ± 8.2 56.0 30.7 4 41.4 ± 3.7 17.8 ± 7.1 64.0 34.0 5 48.9 ± 4.5 35.8 ± 8.8 56.5 35.9 6 4 3 4 4 4 4 4 31 1 Figure 4. 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC 3.2. Treatment Impact on Surface Energy and Functional Groups, Matching with PC While the N 1s spectrum recorded from the unmodiﬁed carbon ﬁbres showed a unimodal distribution of the photoelectrons around the peak maximum at 400.67 eV, the N 1s spectra of electro-chemically treated were deconvoluted into two component peaks, K and L. According to the binding energy values found for component peaks L (400.22 eV), it was assumed that these component peaks appeared from protonated nitrogen species, such as adsorbed ammonium ions (NH4+) or protonated amino groups (C–N+H). The component peaks K were found at about 399 eV, which is a very small value for organically bonded nitrogen. The chemical shift to small binding energy values indicated increased electron densities at the nitrogen atoms probably caused by C=N bonds of azoles [27] or azabenzenes in the immediate environment of highly conjugated π-electron systems, for example. In contrast, the binding energy for the triply bonded nitrogen in the nitrile groups (C≡N) is expected at 399.5 eV [28]. As H-acidic compounds, phenol and carboxyl groups are Brønsted and Lewis acids. Their deprotonated species, the phenolate and carboxylate ions, act as Brønsted and Lewis bases. Brønsted basic amino groups can be protonated by hydronium ions (H3O+). Amino, azole, and azabenzene groups belong to the group of nitrogen bases. Due to the −I effect of the nitrogen atom and the ability of nitrogen to bind a proton via its free electron pair, the nitrile group has an ambidentate character. Contact angle measurements with a single ﬁbre tensiometer resulted in a total surface free energy (SFE) and a surface polarity, which is the percentage of the total SFE that is due to the polar surface energy component, of the tested ﬁbres (Table 6). All the surface-treated ﬁbres had a numerically higher total SFE compared to the untreated ﬁbre (sample 1, 41.9 mJ/m2), with ﬁbre sample 4 having the highest value, 64.0 mJ/m2. In addition, all the surface-treated ﬁbres had a higher surface polarity than the untreated ﬁbre, and increasing the potential was associated with increasing polar surface energy. Materials 2018, 11, 2253 surface polarity than polar surface energy For good interfacial properties, high adhesion energy and low interfacial tension are targeted. Although it is assumed that the SFE values of the ﬁbres are not dependent on the temperature, the total SFE and surface polarity of the polycarbonate matrix material changes when transitioning from a solid at ambient temperature to a molten polymer at 260 ◦C. Therefore, the interfacial parameters and trends amongst the ﬁbre samples depend on the conditions used to combine the materials. Assuming a melt impregnation process, all 13 of 19 urface mbient Materials 2018, 11, 2253 that the SFE value polarity of the poly surface-treated ﬁbres show improved adhesion energy compared to the untreated ﬁbre (50.6 mJ/m2), with the highest value being for ﬁbre sample 4 (78.8 mJ/m2), which had the highest total SFE and surface polarity (Table 7). The conclusion as to how surface treatment inﬂuenced interfacial tension depends highly on the temperature studied: at ambient temperature, the untreated ﬁbre looks superior, whereas at 260 ◦C all treated ﬁbres are better than the untreated ﬁbre. Single ﬁbre pull-out testing has been attempted next to give more clarity as to which parameters and conditions are most indicative for optimal interfacial adhesion. p p y , p g the fibre samples depend on the conditions used to combine the materials. Assuming a melt impregnation process, all surface-treated fibres show improved adhesion energy compared to the untreated fibre (50.6 mJ/m2), with the highest value being for fibre sample 4 (78.8 mJ/m2), which had the highest total SFE and surface polarity (Table 7). The conclusion as to how surface treatment influenced interfacial tension depends highly on the temperature studied: at ambient temperature, the untreated fibre looks superior, whereas at 260 °C all treated fibres are better than the untreated fibre. Single fibre pull-out testing has been attempted next to give more clarity as to which parameters d di i i di i f i l i f i l dh i Table 7. Adhesion energy and interfacial tension for LEXAN™HF1110 polycarbonate. Sample Number Adhesion Energy (mJ/m2) Interfacial Tension (mN/m) Ambient 260 ◦C Ambient 260 ◦C 1 83.7 50.6 1.6 19.4 2 87.6 71.0 11.6 13.0 3 85.5 72.6 13.8 11.4 4 89.5 78.8 17.9 13.3 5 83.0 74.6 16.8 9.9 6 87.2 74.4 14.7 12.2 7 88.3 70.9 11.3 13.4 onditions are most indicative for optimal interfacial adhesion. Table 7. 3.3. Single Fibre Pull-Out Test (SFPO) 3.3. Single Fibre Pull-out Test (SFPO) Contact angle, IGC, and XPS measurements revealed a signiﬁcant increase in the polarity and functional groups on the ﬁbre surface due to the surface treatment. Accordingly, an increase in the ﬁbre-matrix interaction indicated by higher maximum forces was observed using the SFPO force-displacement curves (Figure 5, showing three selected samples: untreated sample 1, highest (sample 3), and lowest (sample 7) maximum pull-out forces). g Contact angle, IGC, and XPS measurements revealed a significant increase in the polarity and functional groups on the fibre surface due to the surface treatment. Accordingly, an increase in the fibre-matrix interaction indicated by higher maximum forces was observed using the SFPO force- displacement curves (Figure 5, showing three selected samples: untreated sample 1, highest (sample 3), and lowest (sample 7) maximum pull-out forces). Figure 5. Force-displacement curves of sample 1 (no surface treatment), sample 3 (medium current, medium conductivity) as the most extensive, and sample 7 (low current, high conductivity) with the lowest fibre-matrix interaction, respectively. Figure 5. Force-displacement curves of sample 1 (no surface treatment), sample 3 (medium current, medium conductivity) as the most extensive, and sample 7 (low current, high conductivity) with the lowest ﬁbre-matrix interaction, respectively. Figure 5. Force-displacement curves of sample 1 (no surface treatment), sample 3 (medium current, medium conductivity) as the most extensive, and sample 7 (low current, high conductivity) with the lowest fibre-matrix interaction, respectively. Figure 5. Force-displacement curves of sample 1 (no surface treatment), sample 3 (medium current, medium conductivity) as the most extensive, and sample 7 (low current, high conductivity) with the lowest ﬁbre-matrix interaction, respectively. However, it should be noted that even the untreated fibre already reveals a good interaction between the fibre and the PC matrix. To some extent, this might be due to the fact that the PC matrix near the fibre is considerably deformed (stretched) during the pull-out. Figure 6 presents the However, it should be noted that even the untreated ﬁbre already reveals a good interaction between the ﬁbre and the PC matrix. To some extent, this might be due to the fact that the PC matrix near the ﬁbre is considerably deformed (stretched) during the pull-out. Figure 6 presents the deformation of the meniscus on the ﬁbre surface as well as the strong deformation of the matrix material near to the ﬁbre entry point. Materials 2018, 11, 2253 surface polarity than polar surface energy Adhesion energy and interfacial tension for LEXAN™ HF1110 polycarbonate. Sample Number Adhesion Energy (mJ/m2) Interfacial Tension (mN/m) Ambient 260 °C Ambient 260 °C 1 83.7 50.6 1.6 19.4 2 87.6 71.0 11.6 13.0 3 85.5 72.6 13.8 11.4 4 89.5 78.8 17.9 13.3 5 83.0 74.6 16.8 9.9 6 87.2 74.4 14.7 12.2 7 88.3 70.9 11.3 13.4 Table 7. Adhesion energy and interfacial tension for LEXAN™HF1110 polycarbonate. p bl Adh d f l f EXA ™ 111 l b 3.3. Single Fibre Pull-Out Test (SFPO) 3.3. Single Fibre Pull-out Test (SFPO) Besides the contribution of the crack that is growing along the ﬁbre surface during pull-out and the friction between the already debonded surface areas, matrix deformation also contributes to the maximum force achieved. This would explain the high forces in the case of untreated sample 1. 14 of 19 orces in Materials 2018, 11, 2253 deformation also co Figure 6. Stretching/yielding of the meniscus after pull-out testing; SEM image of the remaining part on the pulled-out fibre (left) and the stretched area of the fibre entry point in the PC droplet (right). Figure 6. Stretching/yielding of the meniscus after pull-out testing; SEM image of the remaining part on the pulled-out ﬁbre (left) and the stretched area of the ﬁbre entry point in the PC droplet (right). Figure 6. Stretching/yielding of the meniscus after pull-out testing; SEM image of the remaining part on the pulled-out fibre (left) and the stretched area of the fibre entry point in the PC droplet (right). Figure 6. Stretching/yielding of the meniscus after pull-out testing; SEM image of the remaining part on the pulled-out ﬁbre (left) and the stretched area of the ﬁbre entry point in the PC droplet (right). On the contrary, this kind of meniscus stretching also occurred for sample 7, which revealed not only the lowest values of τapp and Wdebond but also a drop in polar surface; these might be related to each other. The currently known models (stress-controlled model; energy-based model, model of adhesion pressure [25]) used to calculate the interfacial parameters (ultimate interfacial shear strength τult, critical energy release rate Gic) do not involve this kind of meniscus deformation. As mentioned in Section 3.4, the apparent shear strength τapp as well as debonding and pull-out work were used to describe the fibre-matrix interaction for that reason. In general, increased shear strength τapp was found for the treated samples; however, the measurements were accompanied by high scatter due to the non-circular fibre shape. On the contrary, this kind of meniscus stretching also occurred for sample 7, which revealed not only the lowest values of τapp and Wdebond but also a drop in polar surface; these might be related to each other. The currently known models (stress-controlled model; energy-based model, model of adhesion pressure [25]) used to calculate the interfacial parameters (ultimate interfacial shear strength τult, critical energy release rate Gic) do not involve this kind of meniscus deformation. 3.3. Single Fibre Pull-Out Test (SFPO) 3.3. Single Fibre Pull-out Test (SFPO) As mentioned in Section 3.4, the apparent shear strength τapp as well as debonding and pull-out work were used to describe the ﬁbre-matrix interaction for that reason. In general, increased shear strength τapp was found for the treated samples; however, the measurements were accompanied by high scatter due to the non-circular ﬁbre shape. 3.4. Correlations 3.4. Correlations A statistical analysis was carried out to find the correlation between the process parameters, surface characterization techniques, and SFPO results and their significance (Tables 8 and 9). A statistical analysis was carried out to ﬁnd the correlation between the process parameters, surface characterization techniques, and SFPO results and their signiﬁcance (Tables 8 and 9). 15 of 19 Materials 2018, 11, 2253 Table 8. Correlation factors on pair-wise comparisons between results, where 1 is the total positive linear correlation, 0 is no linear correlation, and −1 is the total negative linear correlation. The underlined factors show p-values < 0.05 and are considered as signiﬁcant. Factors Elongation at Break Modulus Tensile Strength Total Surface Energy Dispersive Surface Energy Atomic Conc. Hydroxyl Atomic Conc. Carboxyl Atomic Conc. Nitrile Total Surface Energy Polar Surface Energy Dispersive Surface Energy Polarity Interfacial Tens. (Ambient) Interfacial Tens. (260 ◦C) τapp Wdebond Wpullout Favimat IGC XPS CA SFPO Current (A) 0.46 0.17 0.61 0.92 −0.56 0.77 0.96 0.67 0.77 0.89 −0.21 0.86 −0.73 0.20 −0.40 0.45 0.46 Potential (V) 0.47 0.26 0.57 0.93 −0.66 0.80 0.93 0.73 0.87 0.95 −0.09 0.92 −0.79 0.13 −0.44 0.39 0.47 Conductivity (mS/cm) 0.72 0.45 0.16 0.51 −0.79 0.73 0.56 0.96 0.64 0.70 −0.06 0.75 −0.80 −0.18 −0.66 −0.21 0.72 Elongation at Break - 0.20 −0.09 0.54 −0.56 0.66 0.52 0.67 0.45 0.55 −0.20 0.56 −0.55 0.18 −0.50 −0.32 1.00 Modulus 0.20 - −0.39 0.38 −0.73 0.21 0.01 0.35 0.43 0.44 0.06 0.48 −0.56 −0.04 0.13 −0.39 0.20 Tensile strength 0.98 0.36 - 0.60 −0.63 0.67 0.50 0.66 0.50 0.59 −0.17 0.60 −0.59 0.17 −0.44 −0.34 0.98 Total surface energy (IGC) 0.54 0.38 0.60 - −0.60 0.86 0.87 0.66 0.83 0.88 0.01 0.83 −0.65 0.01 −0.44 0.39 0.54 Dispersive surface energy (IGC) −0.56 −0.73 −0.63 −0.60 - −0.53 −0.45 −0.76 −0.79 −0.85 0.02 −0.90 0.94 −0.14 0.24 0.35 −0.56 Atomic conc. Hydroxyl (XPS) 0.66 0.21 0.67 0.86 −0.53 - 0.86 0.85 0.80 0.79 0.17 0.74 −0.57 −0.33 −0.83 0.37 0.66 Atomic conc. Carboxyl (XPS) 0.52 0.01 0.50 0.87 −0.45 0.86 - 0.74 0.72 0.82 −0.17 0.79 −0.64 0.04 −0.60 0.53 0.52 Atomic conc. 3.4. Correlations 3.4. Correlations Nitrile (XPS) 0.67 0.35 0.66 0.66 −0.76 0.85 0.74 - 0.76 0.81 −0.01 0.83 −0.81 −0.22 −0.76 0.04 0.67 Total surface energy (CA) 0.45 0.43 0.50 0.83 −0.79 0.80 0.72 0.76 - 0.95 0.34 0.91 −0.76 −0.15 −0.56 0.19 0.45 Polar surface energy (CA) 0.55 0.44 0.59 0.88 −0.85 0.79 0.82 0.81 0.95 - 0.03 0.99 −0.89 0.08 −0.46 0.15 0.55 Dispersive surface energy (CA) −0.20 0.06 −0.17 0.01 0.02 0.17 −0.17 −0.01 0.34 0.03 - −0.06 0.25 −0.73 −0.39 0.16 −0.20 Polarity (CA) 0.56 0.48 0.60 0.83 −0.90 0.74 0.79 0.83 0.91 0.99 −0.06 - −0.95 0.14 −0.41 0.05 0.56 Interfacial tension (ambient) (CA) 0.55 0.42 0.59 0.89 −0.84 0.79 0.83 0.81 0.94 1.00 0.01 0.99 - 0.10 −0.46 0.16 0.55 Interfacial tension (260 ◦C) (CA) −0.55 −0.56 −0.59 −0.65 0.94 −0.57 −0.64 −0.81 −0.76 −0.89 0.25 −0.95 −0.89 - 0.26 0.18 −0.55 Table 9. p-values for the correlation factors on pair-wise comparisons; p-values < 0.05 are considered as signiﬁcant and are underlined. Factors Elongation at Break Modulus Tensile Strength Total Surface Energy Dispersive Surface Energy Atomic Conc. Hydroxyl Atomic Conc. Carboxyl Atomic Conc. Nitrile Total Surface Energy Polar Surface Energy Dispersive Surface Energy Polarity Interfacial Tens. (Ambient) Interfacial Tens. (260◦) τapp Wdebond Wpullout Favimat IGC XPS CA SFPO Current (A) 0.30 0.72 0.29 0.00 0.19 0.04 0.00 0.10 0.04 0.01 0.65 0.01 0.01 0.06 0.67 0.38 0.31 Potential (V) 0.29 0.58 0.27 0.00 0.10 0.03 0.00 0.06 0.01 0.00 0.85 0.00 0.00 0.04 0.79 0.32 0.39 Conductivity (mS/cm) 0.07 0.31 0.07 0.24 0.03 0.06 0.19 0.00 0.12 0.08 0.90 0.05 0.08 0.03 0.70 0.11 0.65 Elongation at Break - 0.67 0.00 0.21 0.19 0.11 0.23 0.10 0.31 0.20 0.66 0.19 0.20 0.20 0.70 0.25 0.49 Modulus 0.67 - 0.42 0.41 0.06 0.65 0.99 0.44 0.33 0.32 0.90 0.27 0.34 0.19 0.93 0.79 0.39 Tensile strenght 0.00 0.42 - 0.15 0.13 0.10 0.26 0.11 0.26 0.17 0.71 0.15 0.17 0.17 0.72 0.33 0.45 Total surface energy (IGC) 0.21 0.41 0.15 - 0.16 0.01 0.01 0.11 0.02 0.01 0.98 0.02 0.01 0.11 0.98 0.32 0.39 Dispersive surface energy (IGC) 0.19 0.06 0.13 0.16 - 0.22 0.31 0.05 0.03 0.02 0.96 0.01 0.02 0.00 0.77 0.60 0.44 Atomic conc. Hydroxyl (XPS) 0.11 0.65 0.10 0.01 0.22 - 0.01 0.01 0.03 0.03 0.71 0.06 0.03 0.18 0.47 0.02 0.41 Atomic conc. 3.4. Correlations 3.4. Correlations Carboxyl (XPS) 0.23 0.99 0.26 0.01 0.31 0.01 - 0.06 0.07 0.02 0.71 0.04 0.02 0.12 0.93 0.15 0.22 Atomic conc. Nitrile (XPS) 0.10 0.44 0.11 0.11 0.05 0.01 0.06 - 0.05 0.03 0.99 0.02 0.03 0.03 0.63 0.05 0.93 Total surface energy (CA) 0.31 0.33 0.26 0.02 0.03 0.03 0.07 0.05 - 0.00 0.46 0.00 0.00 0.05 0.74 0.20 0.69 Polar surface energy (CA) 0.20 0.32 0.17 0.01 0.02 0.03 0.02 0.03 0.00 - 0.95 0.00 0.00 0.01 0.87 0.30 0.76 Dispersive surface energy (CA) 0.66 0.90 0.71 0.98 0.96 0.71 0.71 0.99 0.46 0.95 - 0.89 0.99 0.59 0.06 0.39 0.73 Polarity (CA) 0.19 0.27 0.15 0.02 0.01 0.06 0.04 0.02 0.00 0.00 0.89 - 0.00 0.00 0.77 0.36 0.91 Interfacial tension (ambient) (CA) 0.20 0.34 0.17 0.01 0.02 0.03 0.02 0.03 0.00 0.00 0.99 0.00 - 0.01 0.84 0.30 0.73 Interfacial tension (260 ◦C) (CA) 0.20 0.19 0.17 0.11 0.00 0.18 0.12 0.03 0.05 0.01 0.59 0.00 0.01 - 0.57 0.57 0.70 Table 9. p-values for the correlation factors on pair-wise comparisons; p-values < 0.05 are considered as signiﬁcant and are underlined. Factors Elongation at Break Modulus Tensile Strength Total Surface Energy Dispersive Surface Energy Atomic Conc. Hydroxyl Atomic Conc. Carboxyl Atomic Conc. Nitrile Total Surface Energy Polar Surface Energy Dispersive Surface Energy Polarity Interfacial Tens. (Ambient) Interfacial Tens. (260◦) τapp Wdebond Wpullout Favimat IGC XPS CA SFPO Current (A) 0.30 0.72 0.29 0.00 0.19 0.04 0.00 0.10 0.04 0.01 0.65 0.01 0.01 0.06 0.67 0.38 0.31 Potential (V) 0.29 0.58 0.27 0.00 0.10 0.03 0.00 0.06 0.01 0.00 0.85 0.00 0.00 0.04 0.79 0.32 0.39 Conductivity (mS/cm) 0.07 0.31 0.07 0.24 0.03 0.06 0.19 0.00 0.12 0.08 0.90 0.05 0.08 0.03 0.70 0.11 0.65 Elongation at Break - 0.67 0.00 0.21 0.19 0.11 0.23 0.10 0.31 0.20 0.66 0.19 0.20 0.20 0.70 0.25 0.49 Modulus 0.67 - 0.42 0.41 0.06 0.65 0.99 0.44 0.33 0.32 0.90 0.27 0.34 0.19 0.93 0.79 0.39 Tensile strenght 0.00 0.42 - 0.15 0.13 0.10 0.26 0.11 0.26 0.17 0.71 0.15 0.17 0.17 0.72 0.33 0.45 Total surface energy (IGC) 0.21 0.41 0.15 - 0.16 0.01 0.01 0.11 0.02 0.01 0.98 0.02 0.01 0.11 0.98 0.32 0.39 Dispersive surface energy (IGC) 0.19 0.06 0.13 0.16 - 0.22 0.31 0.05 0.03 0.02 0.96 0.01 0.02 0.00 0.77 0.60 0.44 Atomic conc. 4. Discussion Several approaches to modifying carbon ﬁbre surfaces can be followed and their impact on adhesion to polycarbonate is studied, as listed in Table 1. In addition to the differences in testing methods, as described in the introduction, different suppliers and grades of polycarbonates were also used, where the difference in molecular weight will impact viscosity (and therefore wetting/impregnation) and the data comparison. This study focused on the electrolytic surface treatment of carbon ﬁbre during its production process. Improvements in interfacial shear strength of comparable approaches have been documented [7–13], and fragmentation tests or indentation were used to quantify the impact. The authors of this article have also investigated the modiﬁcation of polycarbonate as a means to achieving improved adhesion [19], where an apparent interfacial shear strength of 33.9 MPa was found for the reference polycarbonate (HF1110). By using functional groups, the adhesion was improved to 42.2 MPa, in combination with a commercially available carbon ﬁbre (unsized), without further information on the speciﬁc process parameters of production. By controlling the process parameters of the electrolytic surface treatment, a range of samples were created, which were characterized using chemical and mechanical characterization techniques, to evaluate the impact of treatment as well as the predictability of interfacial shear strength. The mechanical properties of the ﬁbres were in all cases equal or superior to untreated sample 1, showing a slight increase in Young’s modulus for samples 4 and 7, but no detrimental impact of the treatments were found and no obvious changes compared to sample 1 were observed from the SEM analysis. From the inverse gas chromatography data, it would seem that the introduction of polar groups onto the surface of carbon ﬁbres correlates well with the application of potential and current, which is in line with the observations made by contact angle measurements as well as the signiﬁcant increase in the functional groups on the ﬁbre surface observed using XPS. There are challenges in controlling the exact level of amperage, potential, and conductivity applied to the samples, and when this is combined with the complexity of the analytical tests performed and a small sample size, establishing clear relationships between the process settings and the ﬁbre characteristics was always going to be a challenge. However, we were able to demonstrate that current and potential are associated with a number of ﬁbre features. 3.4. Correlations 3.4. Correlations Hydroxyl (XPS) 0.11 0.65 0.10 0.01 0.22 - 0.01 0.01 0.03 0.03 0.71 0.06 0.03 0.18 0.47 0.02 0.41 Atomic conc. Carboxyl (XPS) 0.23 0.99 0.26 0.01 0.31 0.01 - 0.06 0.07 0.02 0.71 0.04 0.02 0.12 0.93 0.15 0.22 Atomic conc. Nitrile (XPS) 0.10 0.44 0.11 0.11 0.05 0.01 0.06 - 0.05 0.03 0.99 0.02 0.03 0.03 0.63 0.05 0.93 Total surface energy (CA) 0.31 0.33 0.26 0.02 0.03 0.03 0.07 0.05 - 0.00 0.46 0.00 0.00 0.05 0.74 0.20 0.69 Polar surface energy (CA) 0.20 0.32 0.17 0.01 0.02 0.03 0.02 0.03 0.00 - 0.95 0.00 0.00 0.01 0.87 0.30 0.76 Dispersive surface energy (CA) 0.66 0.90 0.71 0.98 0.96 0.71 0.71 0.99 0.46 0.95 - 0.89 0.99 0.59 0.06 0.39 0.73 Polarity (CA) 0.19 0.27 0.15 0.02 0.01 0.06 0.04 0.02 0.00 0.00 0.89 - 0.00 0.00 0.77 0.36 0.91 Interfacial tension (ambient) (CA) 0.20 0.34 0.17 0.01 0.02 0.03 0.02 0.03 0.00 0.00 0.99 0.00 - 0.01 0.84 0.30 0.73 Interfacial tension (260 ◦C) (CA) 0.20 0.19 0.17 0.11 0.00 0.18 0.12 0.03 0.05 0.01 0.59 0.00 0.01 - 0.57 0.57 0.70 rs on pair-wise comparisons; p-values < 0.05 are considered as signiﬁcant and are underlined. Materials 2018, 11, 2253 16 of 19 16 of 19 5. Conclusions 5. Conclusions The impact of the electrolytic oxidation of carbon fibre on adhesion to polycarbonate has been studied and the impact of the variation of process parameters discussed. A set of on-purpose fibre samples were produced and characterized with a range of surface characterization techniques (IGC, XPS, CA), and single fibre pull-out testing was used for the quantification of the interfacial shear strength between the fibre and the polycarbonate matrix The impact of the electrolytic oxidation of carbon ﬁbre on adhesion to polycarbonate has been studied and the impact of the variation of process parameters discussed. A set of on-purpose ﬁbre samples were produced and characterized with a range of surface characterization techniques (IGC, XPS, CA), and single ﬁbre pull-out testing was used for the quantiﬁcation of the interfacial shear strength between the ﬁbre and the polycarbonate matrix. strength between the fibre and the polycarbonate matrix. The statistical analysis showed significant correlations between IGC, XPS, and CA, but no predictive model was found in the pair-wise comparison between the surface characterization results and the single fibre pull-out measurements The statistical analysis showed signiﬁcant correlations between IGC, XPS, and CA, but no predictive model was found in the pair-wise comparison between the surface characterization results and the single ﬁbre pull-out measurements. and the single fibre pull out measurements. The dataset produced resulted in a predictive model for interfacial shear strength based on the process parameters used for electrolytic oxidation of the carbon fibre. This model makes it possible to target a certain interfacial shear strength, as desired or specified for the carbon fibre-polycarbonate composite The dataset produced resulted in a predictive model for interfacial shear strength based on the process parameters used for electrolytic oxidation of the carbon ﬁbre. This model makes it possible to target a certain interfacial shear strength, as desired or speciﬁed for the carbon ﬁbre-polycarbonate composite. 4. Discussion Figure 7. Linear correlation between τapp predicted (Equation 2) and τapp measured (samples 1–7). Figure 7. Linear correlation between τapp predicted (Equation 2) and τapp measured (samples 1–7). 4. Discussion In particular, we found that to inﬂuence τapp, there is an important interplay between the current and potential settings which means that tailoring these settings is not straightforward but that it could be possible to use this knowledge to target particular applications. While signiﬁcant correlations were found between the ﬁbre characteristics, we did not ﬁnd a direct correlation between process settings, tensile strength measurement, inverse gas chromatography or contact angle results and the single ﬁbre pull-out parameters. This could be due to the limitations of the correlation test, assuming the relationships are linear. However, at this stage we were unable to ﬁnd a test that correlates well with τapp, leaving the single ﬁbre pull-out test as the most important analytical technique used in this study to predict interfacial shear strength. The XPS characterization results did correlate signiﬁcantly with the SFPO results. All the other techniques showed correlations among each other, but this did not render SFPO results (the most time-consuming and specialized technique) predictable enough for it to be acceptable to depend on it for research screening. Further statistical evaluation of the presented dataset resulted in the predictive model for IFSS based on surface treatment process variables: (2) pp = (−0.32 × V × I) + (0.24 × V × C) + (2.1856 × V) + (2.4512 × I) −(2.4084 × C) + 48.7663 (2 where V is the voltage applied, I is the value of current applied to the electrolytic solution, and C is the value of conductivity of the electrolytic solution. Using this formula to calculate the IFSS makes it possible to select the right process settings targeting a speciﬁc value. 17 of 19 17 of 19 8 f 21 Materials 2018, 11, 2253 Veriﬁcation of this model, using the predicted values based on the process settings used in the preparation of samples 1 to 7 versus the actual measured values, shows a very good correlation (R2 = 0.99, p = 0.0255), as presented in Figure 7. Verification of this model, using the predicted values based on the process settings used in the preparation of samples 1 to 7 versus the actual measured values, shows a very good correlation (R2 = 0.99, p = 0.0255), as presented in Figure 7. Figure 7. Linear correlation between τapp predicted (Equation 2) and τapp measured (samples 1–7). Figure 7. Linear correlation between τapp predicted (Equation 2) and τapp measured (samples 1–7). References 1. Cogswell, F.N. Thermoplastic Aromatic Polymer Composites: A Study of the Structure, Processing and Properties of Carbon Fibre Reinforced Polyetheretherketone and Related Materials; Elsevier: Amsterdam, The Netherlands, 2013; ISBN 9781483164762. 1. Cogswell, F.N. Thermoplastic Aromatic Polymer Composites: A Study of the Structure, Processing and Properties of Carbon Fibre Reinforced Polyetheretherketone and Related Materials; Elsevier: Amsterdam, The Netherlands, 2013; ISBN 9781483164762. 2. Fu, S.Y.; Lauke, B.; Mäder, E.; Yue, C.Y.; Hu, X. Tensile properties of short-glass-ﬁbre-and short-carbon- ﬁbre-reinforced polypropylene composites. Compos. Part A Appl. Sci. Manuf. 2000, 31, 1117–1125. [CrossRef] 2. Fu, S.Y.; Lauke, B.; Mäder, E.; Yue, C.Y.; Hu, X. Tensile properties of short-glass-ﬁbre-and short-carbon- ﬁbre-reinforced polypropylene composites. Compos. Part A Appl. Sci. Manuf. 2000, 31, 1117–1125. [CrossRef] 3 Botelho E C ; Rezende M C ; Lauke B Mechanical behavior of carbon ﬁbre reinforced polyamide composites , ; , ; , ; , ; , p p g ﬁbre-reinforced polypropylene composites. Compos. Part A Appl. Sci. Manuf. 2000, 31, 1117–1125. [CrossRef] 3. Botelho, E.C.; Rezende, M.C.; Lauke, B. Mechanical behavior of carbon ﬁbre reinforced polyamide composites. Compos. Sci. Technol. 2003, 63, 1843–1855. [CrossRef] 3. Botelho, E.C.; Rezende, M.C.; Lauke, B. Mechanical behavior of carbon ﬁbre reinforced polyamide composites. Compos. Sci. Technol. 2003, 63, 1843–1855. [CrossRef] 4. Karger-Kocsis, J.; Mahmood, H.; Pegoretti, A. Recent advances in ﬁbre/matrix interphase engineering for polymer composites. Prog. Mater. Sci. 2015, 73, 1–43. [CrossRef] 4. Karger-Kocsis, J.; Mahmood, H.; Pegoretti, A. Recent advances in ﬁbre/matrix interphase engineering for polymer composites. Prog. Mater. Sci. 2015, 73, 1–43. [CrossRef] 5. Sharma, M.; Gao, S.; Mäder, E.; Sharma, H.; Wei, L.Y.; Bijwe, J. Carbon fibre surfaces and composite interphases. Compos. Sci. Technol. 2014, 102, 35–50. [CrossRef] 5. Sharma, M.; Gao, S.; Mäder, E.; Sharma, H.; Wei, L.Y.; Bijwe, J. Carbon fibre surfaces and composite interphases. Compos. Sci. Technol. 2014, 102, 35–50. [CrossRef] 6. Yao, S.S.; Jin, F.-L.; Rhee, K.Y.; Hui, D.; Park, S.J. Recent advances in carbon-ﬁbre-reinforced thermoplastic composites: A review. Compos. Part B Eng. 2018, 142, 241–250. [CrossRef] 7. Montes-Morán, M.A.; Martınez-Alonso, A.; Tascón, J.M.D.; Paiva, M.C.; Bernardo, C.A. Effects of plasma oxidation on the surface and interfacial properties of carbon ﬁbres/polycarbonate composites. Carbon 2001, 39, 1057–1068. [CrossRef] 8. Bascom, W.D.; Chen, W.-J. Effect of Plasma Treatment on the Adhesion of Carbon Fibres to Thermoplastic Polymers. J. Adhes. 1991, 34, 99–119. [CrossRef] 9. Bismarck, A.; Richter, D.; Wuertz, C.; Kumru, M.E.; Song, B.; Springer, J. 6 Patents 6. Patents 18 of 19 Materials 2018, 11, 2253 Conﬂicts of Interest: The authors declare no conﬂict of interest. 6 Patents 6. Patents The results of this study are documented in patent application “Methods for electrolytic surface treatment of carbon fibers”, USPTO serial number 62539879, published on IP.com with reference number IPCOM000252191D The results of this study are documented in patent application “Methods for electrolytic surface treatment of carbon ﬁbers”, USPTO serial number 62539879, published on IP.com with reference number IPCOM000252191D. Author Contributions: This article is the result of collaboration between the authors, where J.H.K. and N.V. led the efforts and arranged all the contacts and connections. L.H. provided the settings for the fibre surface treatment and produced the samples used, including the IGC and mechanical performance. R.v.d.H. and J.H.K. oversaw the practicalities of the project, characterized the surface by tensiometer and collected all the input for further analysis. C.S., responsible for the coordination at IPF Dresden, provided the SFPO results and F.S. Author Contributions: This article is the result of collaboration between the authors, where J.H.K. and N.V. led the efforts and arranged all the contacts and connections. L.H. provided the settings for the ﬁbre surface treatment and produced the samples used, including the IGC and mechanical performance. R.v.d.H. and J.H.K. oversaw the practicalities of the project, characterized the surface by tensiometer and collected all the input for further analysis. C.S., responsible for the coordination at IPF Dresden, provided the SFPO results and F.S. characterized the samples by XPS. T.B. used her analysis tools for the statistical analysis and for deﬁning the correlations between the SFPO and the process parameters. All the authors contributed to the drafting of this document. characterized the samples by XPS. T.B. used her analysis tools for the statistical analysis and for defining the correlations between the SFPO and the process parameters. All the authors contributed to the drafting of this document. Acknowledgments: The work described in this document is the result of a joint effort from the authors, the SABIC Technology department Carbon Nexus (Geelong Australia) and contributors at the Leibniz Institut für Acknowledgments: The work described in this document is the result of a joint effort from the authors, the SABIC Technology department, Carbon Nexus (Geelong, Australia) and contributors at the Leibniz-Institut für Polymerforschung Dresden e.V. (IPF). Special acknowledgement goes to the contributions of Reema Sinha, Haimanti Datta, Anton Kumanan, Mathilde Delory and Alexander van Goudswaard. Alma Rothe and Stefﬁ Preßler are acknowledged for technical support with micromechanical testing. References Adhesion: Comparison Between Physico-chemical Expected and Measured Adhesion of Oxygen-plasma-treated Carbon Fibres and Polycarbonate. J. Adhes. 2000, 73, 19–42. [CrossRef] 10. Bismarck, A.; Kumru, M.E.; Song, B.; Springer, J.; Moos, E.; Karger-Kocsis, J. Study on surface and mechanical ﬁbre characteristics and their effect on the adhesion properties to a polycarbonate matrix tuned by anodic carbon ﬁbre oxidation. Compos. Part A Appl. Sci. Manuf. 1999, 30, 1351–1366. [CrossRef] 11. Yao, T.T.; Wu, G.P.; Song, C. Interfacial adhesion properties of carbon ﬁbre/polycarbonate composites by using a single-ﬁlament fragmentation test. Compos. Sci. Technol. 2017, 149, 108–115. [CrossRef] 12. Lee, J.; Drzal, L.T. Surface characterization and adhesion of carbon ﬁbres to epoxy and polycarbonate. Int. J. Adhes. 2005, 25, 389–394. [CrossRef] 13. Raghavendran, V.K.; Drzal, L.T.; Askeland, P. Effect of surface oxygen content and roughness on interfacial adhesion in carbon ﬁbre–polycarbonate composites. J. Adhes. Sci. Technol. 2002, 16, 1283–1306. [CrossRef] 14. Lorca, J.L.; Gonzalez, C.; Molina-Aldarequia, J.M.; Segurado, J.; Seltzer, R.; Sket, F.; Rodriguez, M.; Sadaba, S.; Munoz, R.; Canal, L.P. Multiscale Modeling of composite materials: A Roadmap towards virtual testing. Adv. Mater. 2011, 23, 5130–5147. [CrossRef] [PubMed] 15. Pisanova, E.V.; Zhandarov, S.F.; Dovgyalo, V.A. Interfacial adhesion and failure modes in single ﬁlament thermoplastic composites. Polym. Compos. 1994, 15, 147–155. [CrossRef] 16. Mäder, E.; Pisanova, E. Interfacial design in ﬁbre reinforced polymers. Macromol. Symp. 2001, 163, 189–212. [CrossRef] 18. Zhandarov, S.; Mäder, E. Characterization of ﬁbre/matrix interface strength: Applicability of different tests, approaches and parameters. Compos. Sci. Technol. 2005, 65, 149–160. [CrossRef] 19. 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Krüss Application Note #232e; KRÜSS GmbH: Hamburg, Germany, 2003. © 2018 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/W4281614752	https://repository.library.noaa.gov/view/noaa/53903/noaa_53903_DS1.pdf	English	null	Evaluating Twenty‐Year Trends in Earth's Energy Flows From Observations and Reanalyses	Journal of geophysical research. Atmospheres	2,022	cc-by	14,362	© 2022 The Authors. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Loeb, N. G., Mayer, M., Kato, S., Fasullo, J. T., Zuo, H., Senan, R., et al. (2022). Evaluating twenty-year trends in Earth's energy flows from observations and reanalyses. Journal of Geophysical Research: Atmospheres, 127, e2022JD036686. https://doi. org/10.1029/2022JD036686 Special Section: M i i h E Monitoring the Earth radiation budget and its implication to climate simulations: Recent Advances and Discussions 1NASA Langley Research Center, Hampton, VA, USA, 2European Centre for Medium-Range Weather Forecasts, Reading, UK, 3Department of Meteorology and Geophysics, University of Vienna, Vienna, Austria, 4Climate and Global Dynamics Division, National Center for Atmospheric Research, Boulder, CO, USA, 5NOAA/Pacific Marine Environmental Laboratory, Seattle, WA, USA, 6Cooperative Institute for Marine and Atmospheric Research, University of Hawaii at Manoa, Honolulu, HI, USA RESEARCH ARTICLE 10.1029/2022JD036686 Norman G. Loeb1 , Michael Mayer2,3, Seiji Kato1, John T. Fasullo4 , Hao Zuo2 , Retish Senan2 , John M. Lyman5,6, Gregory C. Johnson5 , and Magdalena Balmaseda2 Norman G. Loeb1 , Michael Mayer2,3, Seiji Kato1, John T. Fasullo4 , Hao Zuo2 , Retish Senan2 , John M. Lyman5,6, Gregory C. Johnson5 , and Magdalena Balmaseda2 Norman G. Loeb1 , Michael Mayer2,3, Seiji Kato1, John T. Fasullo4 , Hao Zuo2 , Retish Senan2 , John M. Lyman5,6, Gregory C. Johnson5 , and Magdalena Balmaseda2 Key Points: • Regional and global trends in top-of-atmosphere net radiation from atmospheric reanalyses differ markedly from satellite observations Abstract Satellite, reanalysis, and ocean in situ data are analyzed to evaluate regional, hemispheric and global mean trends in Earth's energy fluxes during the first 20 years of the twenty-first century. Regional trends in net top-of-atmosphere (TOA) radiation from the Clouds and the Earth's Radiant Energy System (CERES), ECMWF Reanalysis 5 (ERA5), and a model similar to ERA5 with prescribed sea surface temperature (SST) and sea ice differ markedly, particularly over the Eastern Pacific Ocean, where CERES observes large positive trends. Hemispheric and global mean net TOA flux trends for the two reanalyses are smaller than CERES, and their climatological means are half those of CERES in the southern hemisphere (SH) and more than nine times larger in the northern hemisphere (NH). The regional trend pattern of the divergence of total atmospheric energy transport (TEDIV) over ocean determined using ERA5 analyzed fields is similar to that inferred from the difference between TOA and surface fluxes from ERA5 short-term forecasts. There is also agreement in the trend pattern over ocean for surface fluxes inferred as a residual between CERES net TOA flux and ERA5 analysis TEDIV and surface fluxes obtained directly from ERA5 forecasts. Robust trends are observed over the Gulf Stream associated with enhanced surface-to-atmosphere transfer of heat. Within the ocean, larger trends in ocean heating rate are found in the NH than the SH after 2005, but the magnitude of the trend varies greatly among datasets. • Observing system changes and model bias in atmospheric and oceanic reanalyses remain a challenge for accurate trend determination • Our results highlight a central role for well-calibrated satellite observations in establishing trend patterns in nature Supporting Information: Supporting Information may be found in the online version of this article. Supporting Information: Supporting Information may be found in the online version of this article. Correspondence to: N. G. Loeb, norman.g.loeb@nasa.gov Correspondence to: N. G. Loeb, norman.g.loeb@nasa.gov Received 23 FEB 2022 Accepted 20 MAY 2022 1. Introduction Earth's energy flows encompass the exchange of energy between Earth and space and between Earth's atmos- phere, ocean, lithosphere, and cryosphere. These exchanges occur over a range of time and space scales and influence weather and climate at any given location and time. A thorough understanding of Earth's energy flows is thus necessary to project how regional and global climate will change in response to radiative forcing. Obser- vations of Earth's energy flows are essential for evaluating and improving the climate models used to make these projections. Ideally, the observations must provide accurate descriptions of the mean state of Earth's energy flows as well as their variations on seasonal, interannual, and decadal time scales. Received 23 FEB 2022 Accepted 20 MAY 2022 The combination of ERB satellite and atmospheric reanalysis has been used not only to study the global mean energy budget and mean meridional transports but also their annual cycles and land-ocean exchanges (Fasullo & Trenberth, 2008a, 2008b), cross-equatorial heat transports (Donohoe et al., 2013; Frierson et al., 2013; Liu et al., 2020; Loeb et al., 2015; Marshall et al., 2013; Mayer et al., 2017; Trenberth & Fasullo, 2008), as well as El Niño–Southern Oscillation (ENSO) and other interannual variability (Loeb et al., 2014; Mayer et al., 2014; Mayer and Haimberger, 2012; Trenberth & Fasullo, 2017). Recently, refinements have been made to the formu- lation of the atmospheric energy budget to include contributions from vertical enthalpy fluxes at the surface associated with precipitation and evaporation (Kato et al., 2021; Mayer et al., 2017; Trenberth & Fasullo, 2018). It has recently been demonstrated that TOA ERB data from the Clouds and the Earth's Energy System (CERES) provide robust trends since 2000 (Loeb et al., 2021). At the same time, there has been tremendous progress made in atmospheric and ocean reanalysis systems, with new versions seeing improvements over their predecessors as a result of updates to the underlying model, assimilation system and input data stream (Buizza et al., 2018; Dee et al., 2014; Gelaro et al., 2017; Hersbach et al., 2020; Storto et al., 2019; Zuo et al., 2019, 2021). A question that has yet to be addressed in detail is to what extent can we trust multi-decadal time-scale trends in different components of Earth's energy budget and energy flows within the climate system. Here “trend” refers to the relatively short 20-year period since 2000, which is likely influenced by both anthropogenic forcing and internal variability (Raghuraman et al., 2021). We do not expect that these trends are necessarily representative of longer-term trends, though aspects have been tied to climate change (e.g., Hartmann & Ceppi, 2014). Rather our goal is to investigate the strengths and weaknesses of using observation-based data to determine trends in TOA radiation, atmospheric energy transport, surface flux, and ocean heating rate. The latter is determined from the tendency in OHCA. While evaluations of atmospheric reanalyses for trends in atmospheric moisture transport (Trenberth et al., 2011) and latent heat flux (Robertson et al., 2020) have been conducted, similar analyses for other components of Earth's energy budget are lacking. Received 23 FEB 2022 Accepted 20 MAY 2022 Received 23 FEB 2022 Accepted 20 MAY 2022 Received 23 FEB 2022 Accepted 20 MAY 2022 Efforts aimed at quantifying Earth's mean energy flows date back to the early twentieth century (Hunt et al., 1986). So-called “radiation budget diagrams” of global mean values of shortwave and longwave radiation within the climate system first appeared in 1908 (Abbot & Fowle, 1908). These diagrams were later extended to include non-radiative contributions (Dines, 1917; London, 1957). Energy budget diagrams were further refined following the launch of the first orbiting satellites, which included instruments designed to observe Earth's radiation budget (ERB; House et al., 1986). A key advance was made by Kiehl and Trenberth (1997), who used adjusted global mean top-of-atmosphere (TOA) radiative fluxes from the Earth Radiation Budget Experiment (ERBE), surface radiative fluxes derived from radiative transfer calculations, surface latent heat flux inferred from estimates of global mean precipitation, and sensible heat flux determined as a residual ensuring a global energy balance at the surface. Subsequent studies by Trenberth et al. (2009), Stephens et al. (2012), Wild et al. (2013) and L’Ecuyer et al. (2015) further refined the global mean energy budget diagram using increasingly more sophisticated data- sets and analysis techniques. Early efforts aimed at quantifying energy transports within the climate system focused primarily on meridi- onal transports (e.g., Oort & Vonder Haar, 1976; Trenberth, 1979; Vonder Haar & Oort, 1973) using satellite 1 of 18 LOEB ET AL. LOEB ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 observations to determine the required total energy transport and radiosonde data to determine the atmospheric transports. The ocean transport was then computed as a residual. Alternately, the ocean heat transport was also determined directly using hydrographic cross sections of temperature and salinity (Bryan, 1982). However, both approaches suffered from large sampling errors due to lack of data coverage over the oceans. The use of reanalysis combined with satellite observations of ERB for determining atmospheric and oceanic transports significantly reduced sampling error (Masuda, 1988; Trenberth & Caron, 2001), leading to more reliable results compared to the earlier studies. Furthermore, Trenberth and Fasullo (2017) show that surface fluxes derived as a residual between satellite TOA net downward radiation and estimates of the divergence of the vertically integrated atmos- pheric energy from reanalysis overcome many of the known issues in determining surface flux directly—such as near-surface meteorological variables and bulk flux parameterizations (Yu, 2019). Received 23 FEB 2022 Accepted 20 MAY 2022 We limit our investigation to satellite observations from CERES, atmospheric and oceanic reanalysis from the European Centre for Medium-Range Weather Forecasts (ECMWF), and ocean heating rate calculations benefit- ing from data collected by the revolutionary Argo array of profiling floats (mapped alone, mapped in combination with sea-surface height data from satellite altimeters, and assimilated into reanalyses). The limited number of datasets used enables a more focused assessment of the impact data assimilation in reanalysis on trends. In addi- tion, to our knowledge, ECMWF data are the only source that have been used to calculate the divergence of lateral atmospheric energy transports using the most recent methodological advances (Mayer et al., 2021). The datasets used in the analysis are described in Section 2. This is followed by a description of the methodology applied to the data in Section 3, and results are presented in Section 4. A summary of our main findings is provided in Section 5. 2.1. CERES TOA and Surface Radiation Data Satellite radiation data are from the CERES Energy Balanced and Filled (EBAF) Ed4.1 product (Loeb et al., 2018a), which provides monthly mean TOA and surface shortwave (SW), longwave (LW), net (NET) radiative fluxes and solar irradiance measurements on a 1° × 1° grid along with imager-derived cloud properties. TOA absorbed solar radiation (ASR) is determined from the difference between spatially and temporally aver- aged monthly solar irradiances and reflected SW fluxes. The solar irradiances are determined from time-varying instantaneous total solar irradiance measurements from various sources (Loeb et al., 2018a). Satellite incoming and outgoing radiative fluxes are presently not at the level of accuracy required to resolve TOA fluxes to a few tenths of a Wm −2 in an absolute sense (Loeb et al., 2018a). However, CERES TOA fluxes are highly precise as the instruments are very stable (Loeb et al., 2016, 2018b, 2021; Shankar et al., 2020). The EBAF product uses an objective constrainment algorithm (Loeb et al., 2009) to adjust SW and LW TOA fluxes within their ranges of uncertainty to remove the inconsistency between average global net TOA flux and heat storage in the earth– atmosphere system, determined primarily from ocean heat content anomaly (OHCA) data (Johnson et al., 2016). Use of this approach to anchor the satellite-derived Earth energy imbalance (EEI) to the in situ EEI does not affect the variability and trends in the data (Loeb et al., 2018a). We also use TOA fluxes from the Terra and Aqua CERES SSF1deg Ed 4.1 products (Doelling et al., 2013; Loeb et al., 2018a) to compare CERES fluxes from different satellite platforms. Unlike CERES EBAF, which combines CERES instruments on different satellites, SSF1deg is determined separately for each satellite CERES instruments fly on. The CERES SSF1deg product is derived directly from the CERES Single Scanner Footprint TOA/Surface Fluxes and Clouds (SSF) Level 2 product, which consists of instantaneous footprint-level fluxes. Two sources of surface radiation are considered. The first is from the CERES EBAF Ed4.1 product (Kato et al., 2018) and the second is Aqua-only SYN1deg-Month. EBAF Ed4.1 surface fluxes are derived by making adjustments to the inputs used to compute all-sky and clear-sky surface fluxes in SYN1deg Ed4.1 (Rutan et al., 2015). The adjustments ensure that computed and EBAF-observed TOA radiative fluxes agree to within observational uncertainty. 2. Data We use TOA and surface radiation fields from CERES and ECMWF reanalyses, total atmospheric energy transport estimates from those same ECMWF reanalyses, as well as ocean heating rate estimates derived from two different ECMWF ocean reanalyses, an observational product combining Argo temperature profiles with sea-surface height maps from satellite altimeters, and an Argo-only observational product. 2 of 18 2 of 18 LOEB ET AL. LOEB ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 2.1. CERES TOA and Surface Radiation Data The modified inputs are then used to derive surface radiative fluxes that are self-consistent with the observed EBAF TOA fluxes. The SYN1deg surface radiative fluxes are determined from radiative transfer model calculations initialized using cloud inputs from the Moderate Resolution Imaging Spectroradiometer (MODIS) instruments aboard the Terra and Aqua satellite platforms and hourly geostationary (GEO) imager data between 60°S and 60°N, atmospheric state inputs from the Goddard Earth Observing System (GEOS), version 5.4.1, reanalysis (Rienecker et al., 2008), surface albedo inputs from Rutan et al. (2009), and aerosol inputs based upon an updated version of the assimilation system described in Collins et al. (2001). Because trends in surface radiative fluxes derived using cloud information from GEO imagers are impacted by changes in the design and quality of the GEO instruments over the CERES period (Doelling et al., 2013; Kato et al., 2018), we also determine surface fluxes using a modified version of SYN1deg, which we refer to as Aqua-only SYN1deg-Month. This version uses the same atmospheric state and surface property inputs as Terra + Aqua + GEO SYN1deg-Month, but replaces the GEO cloud properties with those derived from MODIS-Aqua only (Minnis et al., 2020). Instantaneous MODIS cloud retrievals are averaged into 1° × 1° grid boxes. MODIS-Aqua provides cloud properties twice a day for most of non-polar grid boxes. Hourly daytime cloud properties for a grid box are derived by interpolating daytime cloud properties from MODIS across days within a month for the grid box (Doelling et al., 2013). Hourly nighttime properties for a grid box are derived in a similar manner using nighttime MODIS cloud properties. In addition, daytime or nighttime monthly mean cloud properties are used for all hours before the first MODIS observations in the month and after the last MODIS observations in the month. That is, there is no interpolation of cloud properties across different months. In addition, the MATCH aerosol transport model used in Aqua-only SYN1deg-Month only assimilates aerosol optical thickness derived from MODIS Aqua. Aqua-only surface net shortwave and longwave flux trend plots are determined using anomalies for August 2002-February 2020 since that is the period for which Aqua-only SYN is available. 2.2. ERA5 and Integrated Forecasting System (IFS) AMIP ERA5 is the most recent atmospheric reanalysis effort by ECMWF (Hersbach et al., 2020). 2.3. Ocean Data The Ocean and sea-ice ReAnalyses System 5 (ORAS5) (Zuo et al., 2019) is a reconstruction of ocean and sea-ice states derived from an ocean-sea-ice coupled model driven by atmospheric surface forcing and constrained by ocean observations using data assimilation (Balmaseda et al., 2015). It consists of a behind-real-time component of the OCEAN5 ocean reanalysis-analysis system at ECMWF. The ocean model and data assimilation method are kept frozen during the production of the reanalysis. The Ocean ReAnalysis Pilot system-6 (ORAP6) is a new ocean and sea-ice reanalysis system that has been developed based on the ECMWF operational OCEAN5 system (Zuo et al., 2021). Despite sharing the same model configurations as OCEAN5, ORAP6 uses updated atmos- pheric forcing (based on ERA5) and is produced with the most up-to-date reprocessed observation datasets. The ORAP6 data assimilation system has been updated to include a new flow-dependent SST nudging scheme, and to assimilate L3 sea-ice concentration data, among others. ORAP6 uses 3DVar to assimilate in-situ temperature and salinity profiles from Argo, Moorings, XBTs, shipboard CTDs, gliders, and marine mammals, satellite sea-level anomaly and sea-ice concentration data, as well as SST and sea-surface salinity (SSS) nudging in the surface (Zuo et al., 2021). Two sets of ocean data from ORAP6 system have been used in this study. ORAP6.1 is the first version of ORAP6 reanalysis that includes assimilation of all observations. We also consider a control version of ORAP6.1 called “ORAP6-ctrl,” which uses the same model setup and atmospheric forcing (from ERA5) as ORAP6.1, but only uses SST and SSS nudging at the surface. The difference between ORAP6.1 and ORAP6-ctrl thus indicates the impact of data assimilation on ocean heating rates. In addition to the above ocean reanalyses, we determine ocean heating rates from two Argo-based datasets (Johnson et al., 2022). The first is an Argo-only time series obtained from the combination of a October 2021 update of the Roemmich and Gilson (2009) climatology and the Asia-Pacific Data-Research Center's Argo gridded 3° × 3° monthly product on standard depth levels, documented online (at http://apdrc.soest.hawaii.edu/ projects/Argo/index.php). The second Argo-based dataset is an updated version of the 0–2,000 m ocean heat uptake estimate used in Loeb et al. (2021), which is based upon Argo in-situ and satellite altimetry data. 2.3. Ocean Data It uses local correlations between sea-surface height and ocean heat content anomalies to employ satellite altimetry data as a first guess at ocean heat content where (or when) in situ temperature data are sparse (Willis et al., 2003). Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 atmospheric levels up to a pressure of 0.01 hPa. ERA5 is currently available from 1979 onward and consists of analyses and short-range forecasts. The analyses are a physically consistent blend of observations and a short-range forecast based upon the previous analysis. Short-range forecasts are initialized from the analyzed fields daily at 0600 and 1800 UTC. ERA5 uses forcing files from CMIP5 through 2005 and Representative Concentration Pathways 2.6 (RCP2.6) for 2006–2020 (Hersbach et al., 2015, 2020). Here we use profiles of hourly ERA5 analyses of atmospheric wind, temperature, and humidity to calculate verti- cally integrated divergence of total atmospheric energy transport (TEDIV; Section 3.1). We also consider ERA5 short-term forecasts of TOA and surface radiative fluxes as well as surface turbulent heat fluxes for a check on model fidelity. The IFS AMIP is 10-member ensemble continuous atmospheric model integration with a similar setup as ERA5 but uses a slightly newer model cycle. It is initialized in March 2000 and integrated until the end of February 2020 without data assimilation, but with prescribed SSTs and sea ice. The IFS AMIP data used here are based on the ensemble mean. 2.1. CERES TOA and Surface Radiation Data It provides global data on an N320 Gaussian grid (equivalent to 0.288° spatial resolution) at 1-hourly temporal resolution in 137 3 of 18 LOEB ET AL. LOEB ET AL. LOEB ET AL. Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres e determine “inferred” surface turbulent heat fluxes from: (2) 𝑄𝑄𝑆𝑆= 𝐻𝐻𝐿𝐿+ 𝐻𝐻𝑆𝑆= 𝐹𝐹𝑆𝑆−𝑅𝑅𝑆𝑆 (2) where 𝐴𝐴 𝐴𝐴𝑆𝑆 is the sum of surface latent ( 𝐴𝐴 𝐴𝐴𝐿𝐿 ) and sensible ( 𝐴𝐴 𝐴𝐴𝑆𝑆 ) heat flux and 𝐴𝐴 𝐴𝐴𝑆𝑆 is net downward radiation at the surface. We determine 𝐴𝐴 𝐴𝐴𝑆𝑆 from CERES. where 𝐴𝐴 𝐴𝐴𝑆𝑆 is the sum of surface latent ( 𝐴𝐴 𝐴𝐴𝐿𝐿 ) and sensible ( 𝐴𝐴 𝐴𝐴𝑆𝑆 ) heat flux and 𝐴𝐴 𝐴𝐴𝑆𝑆 is net downward radiation at the surface. We determine 𝐴𝐴 𝐴𝐴𝑆𝑆 from CERES. Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 and humidity profiles using an improved budget formulation that treats lateral and vertical moisture enthalpy fluxes in a consistent manner (Mayer et al., 2017) and ensures mass consistency following J. Mayer et al. (2021). Maps of 𝐴𝐴 ∇⋅𝐹𝐹𝐴𝐴 are smoothed using a tapered filter truncating at T42. The 𝐴𝐴 𝐴𝐴𝐴𝐴𝐴𝐴 term is calculated from differ- ences of analyses of the total atmospheric energy at the first of each month, but AET is small on the time scales considered. and humidity profiles using an improved budget formulation that treats lateral and vertical moisture enthalpy fluxes in a consistent manner (Mayer et al., 2017) and ensures mass consistency following J. Mayer et al. (2021). Maps of 𝐴𝐴 ∇⋅𝐹𝐹𝐴𝐴 are smoothed using a tapered filter truncating at T42. The 𝐴𝐴 𝐴𝐴𝐴𝐴𝐴𝐴 term is calculated from differ- ences of analyses of the total atmospheric energy at the first of each month, but AET is small on the time scales considered. In addition to the estimates described above, availability of ERA5 short-term forecasts and IFS AMIP data provides two additional estimates of 𝐴𝐴 𝐴𝐴TOA and 𝐴𝐴 𝐴𝐴𝑆𝑆 and two alternative estimates of 𝐴𝐴 ∇⋅𝐹𝐹𝐴𝐴 . For ERA5, we use short-term forecasts and subtract 𝐴𝐴 𝐴𝐴TOA and 𝐴𝐴 𝐴𝐴𝑆𝑆 (Equation 1). Neglecting the effect of assimilation increments in this estimate will lead to differences with the divergence estimate based on analyzed state quantities (J. Mayer et al., 2021). The short-term ERA5 forecasts are averaged over the first 12 hours of the forecasts and initialized from analyses that are constrained by observations and in that sense are still influenced by observations. The divergence estimate from short-term forecasts can thus be viewed as falling somewhere between an analysis-based estimate and an estimate from a free-running model. The difference between divergence trends estimated from analyses and short-term forecasts provides insight into the degree to which the model can represent observed changes in the atmosphere. It may also reveal areas where the model damps out potential spurious jumps from changes in the observing system. The third divergence estimate is similar to the one based on short-term forecasts but uses IFS AMIP data. Trends in that estimate reflect changes the model captures due to changes in the bound- ary conditions like SSTs and sea ice. 3.1. Inferred Surface Total and Turbulent Heat Fluxes 3.1. Inferred Surface Total and Turbulent Heat Fluxes The surface energy flux ( 𝐴𝐴 𝐴𝐴𝑆𝑆 ) defined here as positive downwards is inferred using the residual method from the atmospheric energy budget (Liu et al., 2020; Mayer et al., 2017; Trenberth & Fasullo, 2017) as follows: 𝐹𝐹𝑆𝑆= 𝐹𝐹TOA −∇⋅𝐹𝐹𝐴𝐴−𝐴𝐴𝐴𝐴𝐴𝐴 (1) (1) 𝐹𝐹𝑆𝑆= 𝐹𝐹TOA −∇⋅𝐹𝐹𝐴𝐴−𝐴𝐴𝐴𝐴𝐴𝐴 where 𝐴𝐴 𝐴𝐴TOA is the net downward radiation at the TOA, 𝐴𝐴 ∇⋅𝐹𝐹𝐴𝐴 is the divergence of lateral atmospheric energy trans- ports (TEDIV), and 𝐴𝐴 𝐴𝐴𝐴𝐴𝐴𝐴 is the vertically integrated atmospheric energy tendency. We use CERES EBAF Ed4.1 to determine 𝐴𝐴 𝐴𝐴TOA . The 𝐴𝐴 ∇⋅𝐹𝐹𝐴𝐴 term is computed from hourly ERA5 analyses of atmospheric wind, temperature, where 𝐴𝐴 𝐴𝐴TOA is the net downward radiation at the TOA, 𝐴𝐴 ∇⋅𝐹𝐹𝐴𝐴 is the divergence of lateral atmospheric energy trans- ports (TEDIV), and 𝐴𝐴 𝐴𝐴𝐴𝐴𝐴𝐴 is the vertically integrated atmospheric energy tendency. We use CERES EBAF Ed4.1 to determine 𝐴𝐴 𝐴𝐴TOA . The 𝐴𝐴 ∇⋅𝐹𝐹𝐴𝐴 term is computed from hourly ERA5 analyses of atmospheric wind, temperature, 4 of 18 LOEB ET AL. LOEB ET AL. LOEB ET AL. Journal of Geophysical Research: Atmospheres 3.2. Trends Trends are determined from deseasonalized monthly anomalies using least squares linear regression. To deter- mine trend uncertainties, we first calculate residuals of the linear regression fit to a monthly anomaly time series. Next, we calculate the autocorrelation function (ACF) of the residuals and assess whether or not the ACF at any lag is significant by comparing it with confidence intervals given by: 𝐶𝐶𝐶𝐶𝑘𝑘= ±𝑡𝑡𝛼𝛼𝛼𝛼𝛼𝜎𝜎𝑘𝑘 (3) (3) where 𝐴𝐴 𝐴𝐴𝛼𝛼𝛼𝛼𝛼 is the student-t statistic at significance level 𝐴𝐴 𝐴𝐴 with 𝐴𝐴 𝐴𝐴= 𝑁𝑁−𝑘𝑘 degrees of freedom, N is the number of samples, and 𝐴𝐴 𝐴𝐴𝑘𝑘 is the standard deviation at lag 𝐴𝐴 𝐴𝐴 derived using the formulation in Mélard and Roy (1987): 휎2 푘= 1 푁 ( 1 + 2 ∑푘−1 푖=1 휌2 푖 ) (4) (4) where 𝐴𝐴 𝐴𝐴𝑖𝑖 is the ACF at lag 𝐴𝐴 𝐴𝐴 . If 𝐴𝐴 𝐴𝐴𝑖𝑖 at any lag lies outside the confidence intervals in Equation 3, we account for autocorrelation in determining the trend uncertainty by calculating the effective sample size following Gelman et al. (2013): where 𝐴𝐴 𝐴𝐴𝑖𝑖 is the ACF at lag 𝐴𝐴 𝐴𝐴 . If 𝐴𝐴 𝐴𝐴𝑖𝑖 at any lag lies outside the confidence intervals in Equation 3, we account for autocorrelation in determining the trend uncertainty by calculating the effective sample size following Gelman et al. (2013): 𝑁𝑁𝑒𝑒= 𝑁𝑁 1 + 2 ∑𝑚𝑚 𝑖𝑖=1 𝜌𝜌𝑖𝑖 (5) (5) We determine 𝐴𝐴 𝐴𝐴 as the first lag satisfying both 𝐴𝐴 𝐴𝐴𝑚𝑚+1 < 0 and 𝐴𝐴 𝐴𝐴𝑚𝑚+1 + 𝜌𝜌𝑚𝑚+2 < 0. This criterion minimizes uncertainty associated with sampling noise in the ACF. If none of the 𝐴𝐴 𝐴𝐴𝑖𝑖 fall outside the confidence intervals, we assume the effective sample size ( 𝐴𝐴 𝐴𝐴𝑒𝑒 ) is equal to N. Once 𝐴𝐴 𝐴𝐴𝑒𝑒 is known, we calculate the trend uncertainty ( 𝐴𝐴 𝐴𝐴𝑡𝑡 ) following Santer et al. (2000): 𝜖𝜖𝑡𝑡= ±𝑡𝑡𝛼𝛼𝛼𝛼𝛼𝑠𝑠𝑏𝑏 (6) 𝜖𝜖𝑡𝑡= ±𝑡𝑡𝛼𝛼𝛼𝛼𝛼𝑠𝑠𝑏𝑏 (6) 5 of 18 5 of 18 LOEB ET AL. LOEB ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 Figure 1. Trends in TOA Net Radiation for March 2000–February 2020. (a) CERES-EBAF, (b) ERA5 forecasts, and (c) IFS AMIP. Figure 1. Trends in TOA Net Radiation for March 2000–February 2020. (a) CERES-EBAF, (b) ERA5 forecasts, and (c) IFS AMIP. for March 2000–February 2020. (a) CERES-EBAF, (b) ERA5 forecasts, and (c) IFS AMIP. 3.2. Trends where 𝐴𝐴 𝐴𝐴𝛼𝛼𝛼𝛼𝛼 is determined with 𝐴𝐴 𝐴𝐴= 𝑁𝑁𝑒𝑒−2 degrees of freedom, and sb is given by: 𝑠𝑠𝑏𝑏= 𝑠𝑠𝑒𝑒 [∑𝑁𝑁 𝑖𝑖=1 (𝑥𝑥𝑖𝑖−𝑥𝑥)2]1∕2 (7) (7) where 𝐴𝐴 𝐴𝐴𝑖𝑖 is time and 𝐴𝐴 𝐴𝐴2 𝑒𝑒 is the variance of the residuals about the regression line ( 𝐴𝐴 𝐴𝐴𝑖𝑖 ), given by: 푠2 푒= 1 푁푒−2 ∑푁 푖=1 푒2 푖 (8) 푠2 푒= 1 푁푒−2 ∑푁 푖=1 푒2 푖 (8) While trend uncertainties in Equation 6 are evaluated using 2.5–97.5% confidence intervals, we use 10–90% confidence intervals in Equation 3 in order to use a less stringent test for autocorrelation in the data. In practice, we generally find statistically significant autocorrelation for monthly data but that is not always the case when using annual mean data with a short 20-year record. 4. Results 4. Results 4.1. Top-of-Atmosphere Regional trends in TOA net radiation for March 2000–February 2020 show marked differences between CERES, ERA5, and IFS AMIP (Figures 1a–1c). As noted in prior studies (Loeb et al., 2018b, 2020; Myers et al., 2018), CERES shows pronounced positive trends in net TOA flux over the Eastern Pacific Ocean off of North America. This increase is driven mainly by an ASR increase associated with a reduction in low cloud cover, which in turn is due to increasing SSTs (Loeb et al., 2018b; Mayer et al., 2018; Myers et al., 2018). In contrast, ERA5 shows negative net TOA flux trends throughout most of the Eastern Pacific Ocean region (Figure 1b), while IFS AMIP shows weaker positive trends and stronger positive trends along the equator (Figure 1c). That neither ERA5 nor IFS AMIP capture the large positive trend off the west coast of North America observed by CERES may suggest that the low cloud response to SST change is too weak in ERA5 and IFS AMIP. In a similar comparison between CERES and seven CMIP6 models run in AMIP mode (Loeb et al., 2020), most of the models show increases in net TOA flux in this region but the magnitude of the change varies amongst the models. As expected, daytime cloud fraction changes from MODIS (Trepte et al., 2019) over the Eastern Pacific (EP) region considered in Loeb et al. (2020) are very similar to those for CERES SW TOA flux (Figure S1 in Supporting Information S1). Over the Arctic, CERES shows weak trends in net TOA flux (Figure 1a)—the result of a cancellation between larger trends in ASR and outgoing longwave radiation (not shown). Net TOA flux trends over the Arctic from IFS AMIP are closer to CERES than ERA5, which shows strong negative trends. All three products show positive net TOA flux trends along the climatological Arctic sea ice edge, where the net radiative effect of the retreating sea ice is visible as noted in Hartmann and Ceppi (2014). ERA5 and IFS AMIP show better agreement with CERES over the Atlantic off the coast of North America, to the southwest of Spain and in the west-east trend dipole in the Indian Ocean around 20°–30°S. There is also very good agreement over the sea ice regions off the coast of Antarctica. 6 of 18 6 of 18 LOEB ET AL. LOEB ET AL. LOEB ET AL. 4.1. Top-of-Atmosphere However, ERA5 mean net fluxes are about half as large as CERES in the SH and more than nine times larger in the NH, while the global mean difference is only 0.05 Wm −2. The ERA5 hemispheric asymmetry in mean net TOA flux implies a −0.006 PW SH-to-NH cross-equatorial heat transport by the ocean-atmosphere system, which is in marked contrast to CERES. For IFS AMIP, the discrepancy with CERES is even greater as the hemispheric contrast in net TOA flux implies a −0.22 PW SH-to-NH cross-equatorial heat transport, and the global mean net flux is negative, both of which are unrealistic. The latter is related to model inconsistencies in the version of the IFS used (see Roberts et al., 2018). A possible reason for the inconsistent hemispheric values could be due to an unrealistic representation of how clouds are distributed between the hemispheres in ERA5 and IFS AMIP. Stephens et al. (2015) showed that increased reflection by clouds in the SH offsets greater reflection by the larger land mass in the NH, resulting in hemispheric symmetry in albedo. Average southern hemisphere (SH) and northern hemisphere (NH) TOA fluxes from CERES for March 2000– February 2020 show hemispheric symmetry in ASR, stronger LW cooling in the NH, and a larger net heat uptake in the SH (Table 1). The hemispheric asymmetry in net TOA flux requires a 0.17 PW SH-to-NH cross-equatorial heat transport by the ocean-atmosphere system for energy budget closure (Donohoe et al., 2013; Frierson et al., 2013; Liu et al., 2020; Loeb et al., 2016; Marshall et al., 2013). With the exception of NH ASR, the ERA5 ASR and −OLR values in Table 1 fall within the 95% uncertainty of CERES (Loeb et al., 2018a). However, ERA5 mean net fluxes are about half as large as CERES in the SH and more than nine times larger in the NH, while the global mean difference is only 0.05 Wm −2. The ERA5 hemispheric asymmetry in mean net TOA flux implies a −0.006 PW SH-to-NH cross-equatorial heat transport by the ocean-atmosphere system, which is in marked contrast to CERES. For IFS AMIP, the discrepancy with CERES is even greater as the hemispheric contrast in net TOA flux implies a −0.22 PW SH-to-NH cross-equatorial heat transport, and the global mean net flux is negative, both of which are unrealistic. 4.1. Top-of-Atmosphere Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 ASR −OLR Net SH NH Global SH NH Global SH NH Global CERES Mean (Wm −2) 241.0 240.9 241.0 −239.6 −240.9 −240.2 1.39 0.076 0.73 Stdev (Wm −2) 0.98 0.94 0.67 0.74 0.83 0.51 0.89 0.88 0.69 Trend (Wm −2 dec −1) 0.65 (0.29) 0.72 (0.28) 0.68 (0.24) −0.27 (0.33) −0.26 (0.26) −0.26 (0.24) 0.38 (0.32) 0.46 (0.27) 0.42 (0.23) ERA5 Forecasts Mean (Wm −2) 242.2 243.4 242.8 −241.6 −242.7 −242.1 0.66 0.71 0.68 Stdev (Wm −2) 0.89 0.76 0.52 0.69 0.72 0.41 0.82 0.79 0.61 Trend (Wm −2 dec −1) 0.10 (0.29) 0.19 (0.24) 0.15 (0.24) −0.11 (0.28) −0.13 (0.22) −0.12 (0.21) −0.01 (0.29) 0.06 (0.26) 0.026 (0.25) IFS AMIP Mean (Wm −2) 239.6 242.0 240.1 −241.0 −241.7 −241.2 −1.4 0.34 −1.1 Stdev (Wm −2) 0.62 0.64 0.39 0.49 0.56 0.25 0.60 0.68 0.49 Trend (Wm −2 dec −1) 0.24 (0.25) 0.28 (0.26) 0.26 (0.19) −0.019 (0.27) −0.046 (0.24) −0.034 (0.17) 0.22 (0.24) 0.24 (0.29) 0.23 (0.23) Note. Numbers in parentheses correspond to uncertainty at 95% confidence level. Bold indicates trend is above the 95% confidence level. Table 1 CERES and ERA5 Southern Hemisphere (SH), Northern Hemisphere (NH) and Global ASR, −OLR and NET TOA Flux Average, Monthly Anomaly Standard Deviation (Stdev) and Trend for March 2000-February 2020 phere (SH), Northern Hemisphere (NH) and Global ASR, −OLR and NET TOA Flux Average, Monthly Anomaly Standard rch 2000-February 2020 Note. Numbers in parentheses correspond to uncertainty at 95% confidence level. Bold indicates trend is above the 95% confidence level. in parentheses correspond to uncertainty at 95% confidence level. Bold indicates trend is above the 95% confidence level. Average southern hemisphere (SH) and northern hemisphere (NH) TOA fluxes from CERES for March 2000– February 2020 show hemispheric symmetry in ASR, stronger LW cooling in the NH, and a larger net heat uptake in the SH (Table 1). The hemispheric asymmetry in net TOA flux requires a 0.17 PW SH-to-NH cross-equatorial heat transport by the ocean-atmosphere system for energy budget closure (Donohoe et al., 2013; Frierson et al., 2013; Liu et al., 2020; Loeb et al., 2016; Marshall et al., 2013). With the exception of NH ASR, the ERA5 ASR and −OLR values in Table 1 fall within the 95% uncertainty of CERES (Loeb et al., 2018a). 4.1. Top-of-Atmosphere The latter is related to model inconsistencies in the version of the IFS used (see Roberts et al., 2018). A possible reason for the inconsistent hemispheric values could be due to an unrealistic representation of how clouds are distributed between the hemispheres in ERA5 and IFS AMIP. Stephens et al. (2015) showed that increased reflection by clouds in the SH offsets greater reflection by the larger land mass in the NH, resulting in hemispheric symmetry in albedo. Anomaly standard deviations and trends in ASR, −OLR and NET are fairly symmetric between the hemispheres for CERES (Table 1). The hemispheric trends in CERES between SH and NH differ by only 0.07 W m −2 per decade for ASR, 0.01 W m −2 per decade for −OLR, and 0.08 W m −2 per decade for NET, implying an insig- nificant 0.02 ± 0.1 PW change to the combined ocean-atmosphere cross-equatorial heat transport over the past 20 years. We note that this does not preclude the possibility of strong but opposing trends in atmospheric and 7 of 18 LOEB ET AL. LOEB ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 Figure 2. CERES net TOA flux trends against record length for CERES SSF1deg Terra (top) and Terra – Aqua (bottom) for (a), (d) SH, (b), (e) NH, (c), (f) Global. tart date is March 2000 for Terra and July 2002 for Terra–Aqua. Gray shading corresponds to 95% confidence interval. Figure 2. CERES net TOA flux trends against record length for CERES SSF1deg Terra (top) and Terra – Aqua (bottom) for (a), (d) SH, (b), (e) NH, (c), (f) Global. Start date is March 2000 for Terra and July 2002 for Terra–Aqua. Gray shading corresponds to 95% confidence interval. oceanic transport. In contrast to CERES, none of the ERA5 hemispheric and global mean trends in TOA radiation are significant at the 95% confidence level (Table 1). Monthly anomaly standard deviations from ERA5 differ by −22% to −7% relative to CERES. Accordingly, ERA5 monthly anomalies track CERES (Figure S2 in Supporting Information S1), but systematic differences are apparent in ASR and −OLR prior to 2003, and in ASR and NET after 2012. Systematic differences between ERA5 and IFS AMIP for ASR and NET are also apparent after 2012 (Figure S3 in Supporting Information S1). This is likely due to changes in the input data stream in ERA5. 4.1. Top-of-Atmosphere (a) ERA5 Analysis (directly from wind, T, q, etc.), (b) ERA5 forecasts (net TOA – FS), and (c) IFS AMIP (net TOA – FS). in Supporting Information S1). Similarly, −OLR trends from Terra and Aqua differ by 0.06 W m −2 per decade of one another, which is also within the 95% confidence level (Figures S5d–S5f in Supporting Information S1). Further validation of the CERES record is provided in Loeb et al. (2021), who compared CERES EBAF variations in global mean net TOA flux with estimates of planetary heat uptake from in situ data for mid-2005 to mid-2019. The in situ data used is derived by an inventory of the rates of changes of energy stored in all components of the climate system, with the primary contribution from differences of overlapping annual 0–2,000 m ocean heat content anomalies from Argo float profiles. As shown in Loeb et al. (2021), the trend in the difference between the CERES and in situ data is 0.068 ± 0.29 W m −2 decade −1, which is similar in magnitude to the comparison between CERES Terra and Aqua. An independent analysis of the CERES data by Datseris and Stevens (2021) confirm our findings. Additionally, Hakuba et al. (2021) use a combination of altimetric and gravimetric observa- tions from GRACE to find a similar trend in EEI. These results stand in marked contrast with Matthews (2021), who claim that there are “spurious calibration drifts” in the CERES record based upon an analysis of lunar reflectance measured by CERES. A direct comparison between the adjusted CERES Terra reflected SW values proposed by Matthews (2021) and the official CERES SSF1deg Ed4.1 product reveals that Matthews (2021) made the largest “corrections” to the CERES record (reaching −0.8 Wm −2) prior to when CERES Terra even started making lunar observations in October 2002 (Figure S6a in Supporting Information S1). If we restrict the comparison only to dates when CERES scans of the moon exist (Figure S6b in Supporting Information S1), there is virtually no trend difference between the two records (trend difference of −0.012 Wm −2 per decade). The CERES lunar data thus confirms that CERES onboard calibration sources are performing nominally. 4.1. Top-of-Atmosphere After removing the trends in the hemispheric and global monthly mean anomaly time series, the correlation coefficient between ERA5 and CERES is 0.80 for ASR and 0.9 for −OLR and NET. Anomaly standard deviations for IFS AMIP are weaker than ERA5 Forecasts and CERES, but trends are in better agreement with CERES, albeit much smaller in magnitude. To examine the robustness of the CERES trends, we compare SH, NH and global trends between CERES instru- ments flying aboard the Terra and Aqua satellites (Figure 2). The CERES data products in this comparison are the SSF1deg-Terra and SSF1deg-Aqua products, which are used as input to CERES EBAF (Loeb et al., 2018a). Importantly, in-orbit calibration adjustments with time for CERES instruments on Terra and Aqua are entirely independent of one another. The CERES Terra net TOA flux trends as a function of record length from March 2000 onwards for SH, NH and global (Figures 2a–2c) show large fluctuations for record lengths shorter than 10 years due to internal variability, but patterns of change remain stable for longer record lengths. Global and NH trends exceed the 95% confidence level for record lengths greater than 12 years, while it takes 17 years in the SH. Trend differences between Terra and Aqua are smaller than 0.05 Wm −2 per decade and fall within the 95% confidence level for July 2002–February 2020 (Figures 2d–2f). The Terra ASR trends (Figures S4a–S4c in Supporting Information S1) decrease rapidly with record length early in the record but begin to increase after the record length reaches 14 years, which corresponds to when the Pacific Decadal Oscillation shifted from negative to positive (Loeb et al., 2021). ASR trends from Terra and Aqua are within 0.04 Wm −2 per decade of one another for the full period and remain below the 95% confidence level for most shorter record lengths (Figures S4d–S4f 8 of 18 8 of 18 LOEB ET AL. LOEB ET AL. LOEB ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 Figure 3. Trends in TEDIV for March 2000–February 2020. (a) ERA5 Analysis (directly from wind, T, q, etc.), (b) ERA5 forecasts (net TOA – FS), and (c) IFS AMIP (net TOA – FS). 000–February 2020. (a) ERA5 Analysis (directly from wind, T, q, etc.), (b) ERA5 forecasts (net TOA – FS), and (c) IFS AM Figure 3. Trends in TEDIV for March 2000–February 2020. 4.2. Within-Atmosphere Transport Trends in TEDIV for March 2000–February 2020from ERA5 Analysis, ERA5 Forecasts and IFS AMIP are provided in Figures 3a–3c. Regions with positive TEDIV trends correspond to increasingly divergent lateral energy fluxes, and negative trends correspond to convergent fluxes. The trends based on ERA5 forecasts and IFS-AMIP are similar to those from ERA5 Analysis over ocean, suggesting that the ERA5 Analysis patterns are not a spurious signal from changes in the observing system. All three results show that the magnitudes of TEDIV trends generally exceed those for net TOA flux (Figures 1a–1c). Large positive trends in TEDIV are observed over the eastern Pacific Ocean to the north and south of the Intertropical Convergence Zone (ITCZ), where trends are weakly negative but intensify toward the west over the Maritime Continent. Trends over the Indian Ocean and North Atlantic are generally negative, except over the Gulf Stream, where a strong positive trend is apparent in all three results. Except for the area of positive TEDIV trends stretching from the Barents and Kara Seas, trends over the Arctic Ocean are generally negative, but the magnitude of the trends differs between these three results, with ERA5 forecasts showing the strongest negative trends. Over the Barents and Kara Seas, the increase in divergence is likely due to sea ice loss, which leads to enhanced surface-to-atmosphere heat flux and divergence of energy. Over land, trends for ERA5 analysis are notably greater in magnitude compared to both ERA5 forecast and IFS-AMIP. This points to a greater uncertainty associated with TEDIV derived directly from atmospheric profiles over land. Before computing TEDIV, we perform a vertically uniform correction to the winds to achieve mass conservation. As such, there is no correction for errors in the vertical structure of the wind divergence (as the vertical error structure is hard to estimate), which over topography are likely larger (also arising from numerical LOEB ET AL. 9 of 18 LOEB ET AL. LOEB ET AL. 4.2. Within-Atmosphere Transport Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 TEDIV ERA5 analysis ERA5 forecasts IFS AMIP SH NH Global SH NH Global SH NH Global Mean (Wm −2) −1.6 1.6 0.0 −5.8 −1.1 −3.4 −3.9 −0.13 −2.1 Stdev (Wm −2) 0.69 0.69 0.0 1.5 1.2 0.93 0.79 0.75 0.47 Trend (Wm −2 dec −1) 0.092 (0.15) −0.092 (0.15) 0.0 0.32 (1.7) −0.87 (0.70) −0.27 (1.4) 0.003 (0.26) 0.039 (0.17) 0.022 (0.11) 𝐴𝐴 𝑭𝑭𝑺𝑺 Inferred ERA5 forecasts IFS AMIP SH NH Global SH NH Global SH NH Global Mean (Wm −2) 3.0 −1.5 0.71 6.4 1.8 4.1 2.5 0.47 1.0 Stdev (Wm −2) 1.6 1.5 1.2 1.7 1.4 1.2 0.84 1.0 0.64 Trend (Wm −2 dec −1) 0.24 (0.35) 0.55 (0.31) 0.40 (0.25) −0.38 (1.5) 0.92 (0.69) 0.27 (1.1) 0.22 (0.26) 0.20 (0.36) 0.21 (0.21) Note. Numbers in parentheses correspond to uncertainty at 95% confidence level. Bold indicates trend is above the 95% confidence level. Table 2 Average, Standard Deviation and Trend for March 2000-February 2020 in TEDIV and 𝐴𝐴 𝐴𝐴𝑆𝑆 for the SH, NH and Global TEDIV Table 2 Average, Standard Deviation and Trend for March 2000-February 2020 in TEDIV and 𝐴𝐴 𝐴𝐴𝑆𝑆 for the SH, NH and Global noise). TEDIV also contains vertical covariances between atmospheric energy and wind divergence (i.e., the vertical error structure of the wind divergence will project on TEDIV). Since the wind divergence errors likely have trends as well (e.g., from changes in the observing system), we see noisy trend patterns in TEDIV over land. While a substantial fraction of the noisy patterns seen from the ERA5 fields is related to numerical noise over topography, some of the non-zero trends over land are similar in ERA5 analysis and ERA5 forecasts (e.g., over central Africa), which suggests spurious jumps in the observing system in that area affecting both analyses and short-term forecasts. Some of the features of TEDIV trends over land may also be realistic and balance observed trends in net TOA flux, such as the negative trends over northern China (compare Figures 3b and 1a). Despite large regional trends in TEDIV, hemispheric average trends are near zero for ERA5 Analysis and IFS AMIP, whereas the NH trend for ERA5 exceeds the 95% confidence interval (Table 2). Time series of hemi- spheric and global average anomalies in TEDIV (Figures S7a–S7c in Supporting Information S1) clearly show spurious variations in ERA5 forecast results. 4.2. Within-Atmosphere Transport The drifts are likely due to changes in the observing system, which are pronounced because assimilation increments are not considered here. Global TEDIV trends based ERA5 Analysis are zero by construction, while they are close to zero for IFS-AMIP TEDIV since the model conserves energy (to a relatively high degree). For the EP region, ERA5 Forecasts is in better agreement with ERA5 Anal- ysis (Figure S7d in Supporting Information S1) since there is a much greater signal-to-noise ratio compared to hemispheric averages, while IFS AMIP shows weaker variability since it is determined from an average of 10 realizations. 4.3. Surface The trends are generally more pronounced for Inferred than for ERA5 Forecasts. In contrast, this trend pattern is less evident for IFS AMIP (Figure 4c), which entirely misses the positive trends along the equator. off of North and South America and a line of positive trends along the equatorial Pacific stretching from the Mari- time Continent to Central America. All three products show significant positive trends in the Atlantic between the equator and ∼40°N. The trends are generally more pronounced for Inferred than for ERA5 Forecasts. In contrast, this trend pattern is less evident for IFS AMIP (Figure 4c), which entirely misses the positive trends along the equator. In order to further decompose trends in 𝐴𝐴 𝐴𝐴𝑆𝑆 in terms of radiative and non-radiative components, we compute trends in net total radiative flux at the surface ( 𝐴𝐴 𝐴𝐴𝑆𝑆 ) from CERES (Figure 5a) and determine the “inferred” surface turbulent flux ( 𝐴𝐴 𝐴𝐴𝑆𝑆 ) trends based upon Equation 2 in Figure 6a. These are compared with regional trends in 𝐴𝐴 𝐴𝐴𝑆𝑆 and 𝐴𝐴𝑆𝑆 for ERA5 Forecasts (Figures 5b and 6b). In Figure 6b, 𝐴𝐴 𝐴𝐴𝑆𝑆 is obtained directly from the sum of 𝐴𝐴 𝐴𝐴𝐿𝐿 and 𝐴𝐴𝑆𝑆 . While the trend patterns in 𝐴𝐴 𝐴𝐴𝑆𝑆 are similar between CERES and ERA5 Forecasts, their magnitudes are quite different. Large differences are evident over the west tropical Pacific Ocean, where ERA5 Forecasts shows large positive trends that are absent in CERES. Regional trend patterns in 𝐴𝐴 𝐴𝐴𝑆𝑆 are similar over ocean, but the inferred method produces generally larger values. There is good agreement over the eastern Pacific off the west coast of the Americas, where trends are predominantly negative. Trends in 𝐴𝐴 𝐴𝐴𝑆𝑆 are generally much larger than those in 𝐴𝐴 𝐴𝐴𝑆𝑆 , suggesting a dominant role for surface turbulent heat flux over net surface radiation at regional scales. Trends in surface turbulent flux from the SeaFlux V3 (Roberts et al., 2020) and OAFlux V3 (Yu & Weller, 2007) products for August 2002–July 2018 (Figures S8a and S8b in Supporting Information S1) are generally in poor agreement everywhere. The lack of agreement is surprising since SeaFlux and OAFlux are dedicated surface turbulent heat products. According to Robertson et al. 4.3. Surface Trend maps of 𝐴𝐴 𝐴𝐴𝑆𝑆 using the Inferred method (Equation 1), ERA5 forecasts and IFS AMIP are provided in Figures 4a–4c. In the latter two cases, 𝐴𝐴 𝐴𝐴𝑆𝑆 is determined from the sum of 𝐴𝐴 𝐴𝐴𝑆𝑆 , 𝐴𝐴 𝐴𝐴𝐿𝐿 , and 𝐴𝐴 𝐴𝐴𝑆𝑆 . Comparing Figures 3a and 4a, it is evident that regional trend patterns and magnitudes in 𝐴𝐴 𝐴𝐴𝑆𝑆 are mainly determined by those in TEDIV. This is consistent with what previous studies have shown for spatial patterns in climatological mean 𝐴𝐴 𝐴𝐴𝑆𝑆 (Liu et al., 2020; Mayer et al., 2017; Trenberth & Fasullo, 2017). Consequently, trends for the Inferred method over land are largely spurious (see Section 4.2). Over ocean, large-scale patterns of surface flux trends from the three methods are similar over the Southern Ocean, Southern Indian Ocean, Barents Sea, and the Kuroshio Current and Gulf Stream. Trends over the Gulf Stream are particularly noteworthy, as all three results show large negative trends, implying increased surface-to-atmosphere heat flux. In this region, the climatological mean 𝐴𝐴 𝐴𝐴𝑆𝑆 is also strongly negative since the atmosphere is supplied energy from warm water masses transporting energy poleward (Mayer et al., 2021; Trenberth & Fasullo, 2017). We also find large negative trends for Inferred (Figure 4a) and ERA5 forecasts (Figure 4b) over the East Pacific 10 of 18 LOEB ET AL. LOEB ET AL. LOEB ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 Figure 4. Trends in surface flux (positive downward) for March 2000–February 2020. (a) Inferred (CERES TOA Net − ERA5 TEDIV), (b) ERA5 forecasts, and (c) IFS AMIP. Figure 4. Trends in surface flux (positive downward) for March 2000–February 2020. (a) Inferred (CERES TOA Net − ERA5 TEDIV), (b) ERA5 forecasts, and (c) IFS AMIP e downward) for March 2000–February 2020. (a) Inferred (CERES TOA Net − ERA5 TEDIV), (b) ERA5 forecasts, and (c) Figure 4. Trends in surface flux (positive downward) for March 2000–February 2020. (a) Inferred (CERES TOA Net − ERA5 TEDIV), (b) ERA5 forecasts, and (c) IFS AMIP. Figure 4. Trends in surface flux (positive downward) for March 2000–February 2020. (a) Inferred (CERES TOA Net − IFS AMIP. off of North and South America and a line of positive trends along the equatorial Pacific stretching from the Mari- time Continent to Central America. All three products show significant positive trends in the Atlantic between the equator and ∼40°N. 4.3. Surface (2020), trends from these products are less reliable due to problems with wind speed retrievals from Special Sensor Microwave Imager/Sounder satellite sensors and exces- sive upward trends in Optimal Interpolation Sea Surface Temperature (OISST) data. Figure 5. Trend for August 2002–February 2020 in net total radiative flux at the surface (positive down) from (a) CERES and (b) ERA5 forecasts. Figure 5. Trend for August 2002–February 2020 in net total radiative flux at the surface (positive down) from (a) CERES and (b) ERA5 forecasts. 11 of 18 LOEB ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 Figure 6. Trends in surface turbulent heat flux (positive downward) for August 2002–February 2020. (a) Inferred (CERES TOA Net − ERA5 TEDIV − CERES Surface Net) and (b) ERA5 forecasts. Figure 6. Trends in surface turbulent heat flux (positive downward) for August 2002–February 2020. (a) Inferred (CERES TOA Net − ERA5 TEDIV − CERES Surface Net) and (b) ERA5 forecasts. 4.4. Within Ocean A benefit of ocean reanalysis is that it provides continuous coverage of the global oceans and therefore can resolve higher frequency variability of ocean heating rate than methods that rely primarily on in situ data like Argo. We compare ORAS5, ORAP6.1, and ORAP6-ctrl global monthly anomalies in full-depth global ocean heating rate for March 2000-February 2020 (Figures 7a–7c) and the hemispheric and global averages, anomaly standard deviations and trends (Table 3). Anomalies for ORAS5 and ORAP6.1 are similar and show a correlation of 0.5. In contrast, ORAP6-ctrl shows much weaker variability than the other two reanalyses, with a monthly standard deviation that is 38% smaller than ORAP6.1, and a correlation with ORAP6.1 of only 0.31. This implies that assimilating more data significantly increases higher-frequency variability. ORAP6.1 shows a sudden decrease around 2005 (Figure 7b) that is not apparent in ORAS5 or ORAP6.1-ctrl. This dip causes the trend in ORAP6.1 Figure 7. Monthly global anomalies in ocean heating rate for (a) ORAS5, (b) ORAP6.1, and (c) ORAP6-ctrl. Figure 7. Monthly global anomalies in ocean heating rate for (a) ORAS5, (b) ORAP6.1, and (c) ORAP6-ctrl. 12 of 18 LOEB ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 0–700 m Full depth SH NH Global SH NH Global ORAS5 Mean (Wm −2) 0.65 0.50 0.57 1.2 0.87 1.0 Stdev (Wm −2) 2.4 2.5 1.0 2.6 2.8 1.1 Trend (Wm −2 dec −1) −0.11 (1.0) −0.062 (0.98) −0.086 (0.31) −0.24 (1.1) −0.13 (1.1) −0.19 (0.47) ORAP6.1 Mean (Wm −2) 0.43 0.39 0.41 0.38 0.64 0.51 Stdev (Wm −2) 2.6 2.5 1.2 2.9 2.8 1.3 Trend (Wm −2 dec −1) −0.11 (1.0) 0.13 (0.98) 0.009 (0.36) 0.013 (1.2) 0.31 (1.1) 0.16 (0.60) ORAP6-ctrl Mean (Wm −2) 0.48 0.29 0.38 0.77 0.30 0.54 Stdev (Wm −2) 2.2 2.0 0.82 2.5 2.2 0.81 Trend (Wm −2 dec −1) −0.10 (1.0) 0.022 (0.87) −0.038 (0.38) 0.037 (1.1) −0.026 (0.95) 0.006 (0.38) Note. Numbers in parentheses correspond to uncertainty at 95% confidence level. Bold indicates trend is above the 95% confidence level. Table 3 Mean, Anomaly Standard Deviation and Trend in Monthly Ocean Heating Rate During March 2000–February 2020 for the SH, NH and Global (Total Area) for July 2005–December 2019 (Table 4) to be much larger than for March 2000–February 2020 (Table 3). The dip in ORAP6.1 is likely caused by the model bias correction method. 4.4. Within Ocean Prior to 2005, ORAP6.1 heating rates are similar to ORAS5, but ocean temperatures are much warmer than ORAS5 in the Southern Ocean. When Argo data are assimilated, the ORAP6.1 data assimilation increment cools the ocean, causing a sudden decrease in ocean heating rate around 2005. This problem illustrates one of the greatest challenges in ocean reanalyses: how to balance the temporal consistency of the model simulation with the increased accuracy of the state estimation in the data rich periods. This underscores the need for improved treatments of model error in reanalyses. Agreement among global annual variations in CERES net TOA flux and ocean heating rate for the three ocean reanalyses, Argo-only, and combined Argo and satellite altimetry data (Argo + SA) is mixed (Figures 8a–8e). Of the three reanalyses (Figures 8a–8c), ORAP6-ctrl provides the best agreement with CERES prior to 2013. After 2013, ORAP6-ctrl ocean heating rates are smaller than those for ORAS5 and ORAP6.1, which show better agree- ment with CERES for that period. This suggests that surface forcing and SST information alone are sufficient to estimate ocean heating rate variability during some periods, but in other periods subsurface information may be necessary. When only Argo data are considered, annual variations are very noisy (Figure 8d). The variability is much greater for 0–2,000 m than 0–700 m, a finding also noted in Trenberth et al. (2016). The noise is signif- icantly reduced when Argo and satellite altimetry data are combined (Figure 8e). Nevertheless, the Argo-only and Argo + SA global trends are similar to CERES while ORAP5 and ORAP6-ctrl show weaker trends (Tables 1 and 4). As noted earlier, ORAP6.1 trends are much larger due to the discontinuity around 2005. Except for ORAP6.1, all the datasets show larger hemispheric mean ocean heating rates for the SH than the NH for July 2005–December 2019 (Table 4). Overall, ORAP6-ctrl shows the best agreement with Argo and Argo + SA. For ORAP6.1, the SH heating rate is a factor of 2.5 smaller than the NH value, and a factor of three smaller compared to the SH values from the other datasets in Table 4. A general consensus amongst all of the ocean datasets is a tendency for larger trends in ocean heating rate in the NH than the SH after 2005 (Table 4), but there is poor agreement on the magnitude of the trends. 4.4. Within Ocean This makes determination of trends in ocean heat transport derived as a residual between net surface flux and ocean heating rate highly uncertain. 13 of 18 LOEB ET AL. LOEB ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 0–700 m Full depth SH NH Global SH NH Global ORAS5 Mean (Wm −2) 0.54 0.38 0.46 1.1 0.65 0.88 Stdev (Wm −2) 1.3 1.3 0.40 1.4 1.4 0.45 Trend (Wm −2 dec −1) 0.15 (1.2) 0.56 (1.2) 0.36 (0.55) −0.37 (1.3) 0.84 (1.3) 0.24 (0.70) ORAP6.1 Mean (Wm −2) 0.30 0.34 0.32 0.26 0.67 0.46 Stdev (Wm −2) 1.2 1.3 0.45 1.4 1.4 0.59 Trend (Wm −2 dec −1) 0.42 (1.2) 0.85 (1.2) 0.63 (0.53) 0.87 (1.4) 1.1 (1.3) 0.98 (0.67) ORAP6-ctrl Mean (Wm −2) 0.43 0.33 0.38 0.78 0.33 0.55 Stdev (Wm −2) 1.3 1.1 0.45 1.4 1.2 0.44 Trend (Wm −2 dec −1) 0.31 (1.3) 0.35 (1.1) 0.33 (0.64) 0.23 (1.4) 0.34 (1.2) 0.28 (0.67) Argo Mean (Wm −2) 0.45 0.23 0.34 0.77 0.40 0.60 Stdev (Wm −2) 2.2 2.1 0.76 3.0 2.3 1.2 Trend (Wm −2 dec −1) −0.049 (2.1) 1.2 (2.0) 0.49 (0.72) −0.22 (3.0) 1.4 (2.2) 0.46 (1.2) Argo + SA Mean (Wm −2) – – – 0.76 0.41 0.59 Stdev (Wm −2) – – – 1.0 1.0 0.37 Trend (Wm −2 dec −1) – – – 0.11 (1.6) 0.74 (1.6) 0.42 (0.44) Note. Numbers in parentheses correspond to uncertainty at 95% confidence level. Bold indicates trend is above the 95% confidence level. Table 4 Mean, Standard Deviation and Trend in Annual Ocean Heating Rate During July 2005–December 2019 for the SH, NH, and Global (Total Area) Note. Numbers in parentheses correspond to uncertainty at 95% confidence level. Bold indicates trend is above the 95% confidence level. 5. Summary Global annual mean variation in CERES net TOA flux and ocean heating rate for 0–700 m and full-depth or 0–2,000 m for: (a) ORAS5; (b) ORAP6.1; (c) ORAP6-ctrl; (d) Argo; and (e) Argo + SLA. in CERES net TOA flux and ocean heating rate for 0–700 m and full-depth or 0–2,000 m for: (a) ORAS5; (b) ORAP6.1; (c) LA. of the 21st century. ERA5 and IFS AMIP also show insignificant hemispheric trend differences, but their SH, NH and global mean trends are smaller than CERES. Surprisingly, ERA5 climatological average net TOA fluxes are approximately half as large as CERES in the SH and more than nine times larger in the NH, while the global mean difference is only 0.05 W m −2. The ERA5 and IFS AMIP hemispheric asymmetries in mean net TOA flux imply a NH-to-SH cross-equatorial heat transport by the ocean-atmosphere system. That is in marked contrast to CERES, which shows a 0.17 PW SH-to-NH cross-equatorial heat transport, consistent with expectation (Frierson et al., 2013). A possible reason for the inconsistent hemispheric values in ERA5 and IFS AMIP could be due to an unrealistic representation of how clouds are distributed between the hemispheres. We compare TEDIV calculated directly from ERA5 analyzed profiles of temperature, humidity and winds (ERA5 Analysis) with TEDIV obtained as a residual between TOA and surface fluxes from ERA5 short-term forecasts and IFS AMIP. Trends based on ERA5 forecasts and IFS AMIP are similar to those from ERA5 Analysis over ocean, suggesting that the ERA5 Analysis patterns are not a spurious signal from changes in the observing system. Regional trends in 𝐴𝐴 𝐴𝐴𝑆𝑆 are determined mainly by those in TEDIV, and therefore exhibit similar features. We find consistent negative trends over the Gulf Stream, implying increased surface-to-atmosphere heat flux. Increases surface-to-atmosphere heat flux are also observed over large portions of the eastern Pacific Ocean off the coasts of North and South America. While trend patterns in net surface radiation are similar between CERES and ERA5 Forecasts, large differences are evident over the west tropical Pacific Ocean, where ERA5 Forecasts show large positive trends not observed by CERES. Regional trends in surface turbulent heat flux from an inferred method that uses an energy budget constraint involving CERES and ERA5 analysis data show a similar pattern over ocean to that obtained from the direct sum of sensible and latent heat from ERA5 Forecasts. 5. Summary This study uses satellite and atmospheric and oceanic reanalysis datasets to address the following question: To what extent can we trust observed 20-year trends in different components of Earth's energy budget and energy flows within the climate system. We focus on trends after 2000 in TOA radiation, TEDIV, surface flux, and within-ocean heating rate using satellite observations from CERES and different versions of atmospheric and oceanic reanalysis datasets from ECMWF. As the trends are likely influenced by both anthropogenic forcing and internal variability, there is no expectation that these are solely representative of longer-term trends. Regional trends in TOA net downward radiation from CERES, ERA5, and IFS AMIP are markedly different over the Eastern Pacific Ocean off North America, where large increases in SST have been observed during the CERES period. Whereas CERES observes large positive trends associated with a reduction in low cloud cover, ERA5 shows negative net TOA flux trends throughout most of the Eastern Pacific Ocean region and IFS AMIP shows weaker positive trends. These results suggest that the low cloud response to SST change may be too weak in ERA5 and IFS AMIP. ERA5 and IFS AMIP show better agreement with CERES over the Atlantic off of North America and Europe, the Indian Ocean between 20° and 30°S, and over sea ice regions off the coast of Antarctica. Trends are generally inconsistent over the Arctic Ocean, except in areas near the sea ice edge that are associated with steep declines in sea ice concentration. We find that CERES global mean trends appear to be robust based upon multiple lines of evidence, including direct comparisons between CERES instruments on Terra and Aqua (consistent to < 0.1 W m −2 decade −1), comparisons with in situ measurements from Argo and results that use a combination of altimetric and gravimetric observations from GRACE. CERES trends in net TOA flux between the SH and NH are very close to one another, implying an insignificant 0.02 ± 0.1 PW change to the combined ocean-atmosphere cross-equatorial heat transport over the first 20 years 14 of 18 LOEB ET AL. LOEB ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 Figure 8. Global annual mean variation in CERES net TOA flux and ocean heating rate for 0–700 m and full-depth or 0–2,000 m for: (a) ORAS5; (b) ORAP6.1; (c) ORAP6-ctrl; (d) Argo; and (e) Argo + SLA. Figure 8. Data Availability Statement Data Availability Statement CERES_EBAF Ed4.1 was obtained from the CERES ordering page at http://ceres.larc.nasa.gov/order_data.php. ERA5 data are publicly available via the Copernicus Climate Change Service climate (https://confluence.ecmwf. int/display/CKB/). CERES_EBAF Ed4.1 was obtained from the CERES ordering page at http://ceres.larc.nasa.gov/order_data.php. ERA5 data are publicly available via the Copernicus Climate Change Service climate (https://confluence.ecmwf. int/display/CKB/). References Acknowledgments We thank the CERES science, algorithm, and data management teams and the NASA Science Mission Directorate for supporting this research. MM's contri- bution was funded by EU H2020 Grant agreement No. 862626 (EUROSEA) as well as Austrian Science Fund project P33177. This is PMEL Contribution Number 5351. The efforts of Dr. Fasullo in this work were supported by NASA Awards 80NSSC17K0565 and 80NSSC22K0046, and by the Regional and Global Model Analysis (RGMA) component of the Earth and Environ- mental System Modeling Program of the U.S. Department of Energy's Office of Biological & Environmental Research (BER) via National Science Foundation IA 1947282. Abbot, C. G., & Fowle, F. E. (1908). Radiation and Terrestrial Temperatures. Annals of the Astrophysical Observatory of the Smithsonian Insti- tution (Vol. 2). Smithsonian Institution. Balmaseda, M., Hernandez, F., Storto, A., Palmer, M. D., Alves, O., & Shi, L. (2015). The ocean reanalyses intercomparison project (ORA-IP). Journal of Operational Oceanography, 8, s80–s97. https://doi.org/10.1080/1755876X.2015.1022329 Balmaseda, M., Hernandez, F., Storto, A., Palmer, M. D., Alves, O., & Shi, L. (2015). 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Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD036686 While anomalies for ORAS5 and ORAP6.1 are similar, variability for ORAP6-ctrl is 38% weaker than ORAP6.1. This implies that assimilating more data significantly increases higher-frequency variability. ORAP6.1 also shows a sudden decrease around 2005 that is not apparent in ORAS5 or ORAP6.1-ctrl, which causes a spurious trend in ORAP6.1. This dip is likely associated with a warm bias in the model that gets corrected after the introduction of Argo data in 2005 by the data assimilation increment, leading to a steep decline in ocean heating rate. Balancing the temporal consistency between the model simulation and introduction of new data in a time series remains a challenge in both ocean and atmosphere reanalysis systems. The impact on trends can be especially large, depend- ing on the magnitude of the model bias and the location within the time series new data are introduced/removed. Global annual variations in CERES net TOA flux and ocean heating rate for the three ocean reanalyses, Argo-only, and combined Argo and satellite altimetry data (Argo + SA) are also compared. Of the three reanaly- ses, ORAP6-ctrl provides the best agreement with CERES up to 2013, while ORAS5 and ORAP6.1 are in better agreement with CERES after 2013. From this we conclude that surface forcing and SST information may be sufficient to estimate ocean heating rate variability for some periods, but other periods may also require subsur- face information. All the ocean datasets except ORAP6.1 show larger hemispheric mean ocean heating rates for the SH than the NH. ORAP6-ctrl shows the best overall agreement with Argo and Argo + SA. For ORAP6.1, the SH heating rate is a factor of 2.5 smaller than the NH value, and a factor of 3 smaller compared to the SH values from the other datasets. All the ocean datasets show larger trends in ocean heating rate in the NH than the SH after 2005, but there is poor agreement on the magnitude of the trends. Consequently, determination of trends in ocean heat transport derived as a residual between net surface flux and ocean heating rate is highly uncertain. Journal of Geophysical Research: Atmospheres R., Wang, H., Su, W., Nguyen, C., Corbett, J. G., et al. (2018). Clouds and the Earth's Radiant Energy System (CERES) energy balanced and filled (EBAF) top-of-atmosphere (TOA) Edition 4.0 data product. Journal of Climate, 31, 895–918. https://doi. org/10.1175/JCLI-D-17-0208.1 eb, N. G., Johnson, G. C., Thorsen, T. J., Lyman, J. M., Rose, F. G., & Kato, S. (2021). Satellite and ocean data reveal marked increase in Earth’s heating rate. 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https://openalex.org/W2884073353	https://europepmc.org/articles/pmc6335064?pdf=render	English	null	Transgenic micro<scp>RNA</scp>‐14 rice shows high resistance to rice stem borer	Plant biotechnology journal	2,018	cc-by	10,008	Introduction Rice (Oryza sativa) is one of the most widely consumed foods in the world (FAOSTAT, 2018). Unfortunately, rice production suffers severe damage from more than 200 insect pests at different life stages (Cheng, 1996). Among them, rice stem borer (RSB, Chilo suppressalis Walker) is one of the most serious insect pests (Bottrell and Schoenly, 2012; Chen et al., 2011). Although many methods have been developed for controlling these insect pests, application of chemical insecticides is still the most widely used (Huang et al., 2017). Unfortunately, overuse of insecticides has caused several serious problems, such as insecticide resis- tance, environment pollution, and food safety issues (Chagnon et al., 2015). Therefore, developing alternative pest control methods is highly necessary. A breakthrough in pest control involving RNA interference (RNAi) occurred in 2007. Transgenic crops expressing double- stranded RNA (dsRNA) against a suitable target gene were shown to exhibit resistance to insect pests (Baum et al., 2007; Mao et al., 2007). Since then, an increasing number of reports demonstrated that repressing insect genes by expressing a small RNA normally expressed in plants conferred protection against insect pests (Auer and Frederick, 2009; Ossowski et al., 2008). The success of transgenic crops using RNAi signiﬁcantly broad- ened the scope of target genes that can be used for pest control (Huvenne and Smagghe, 2010; Price and Gatehouse, 2008). Much effort has been devoted to ﬁnding more suitable insecti- cidal target genes. Most of the newly identiﬁed targets for RNAi pest control are protein-encoding genes (Zhang et al., 2013). However, non-coding RNA genes have seldom been used to control insect pests. Insect-resistant genetically modiﬁed (GM) crops are an increasingly utilized method of pest control (Chen et al., 2011). In the mid-1980s, the ﬁrst GM cotton expressing insecticidal Crystal (Cry) proteins, derived from the bacterium Bacillus thuringiensis (Bt), was generated, which provided a new advantage for crops against cotton bollworm (Vaeck et al., 1987). In the past two decades, transgenic Bt cotton has been widely adopted as a GM insect-resistant crop (Li et al., 2016). In China, dozens of Bt rice and Bt maize lines have been developed, some of which showed high efﬁciency in controlling target lepidopteran pests on rice (Liu et al., 2016). ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Summary Summary Rice stem borer (RSB, Chilo suppressalis) is an insect pest that causes huge economic losses every year. Control efforts rely heavily on chemical insecticides, which leads to serious problems such as insecticide resistance, environment pollution, and food safety issues. Therefore, developing alternative pest control methods is an important task. Here, we identiﬁed an insect-speciﬁc microRNA, miR-14, in RSB, which was predicted to target Spook (Spo) and Ecdysone receptor (EcR) in the ecdysone signalling network. In-vitro dual luciferase assays using HEK293T cells conﬁrmed the interactions of Csu-miR-14 with CsSpo and with CsEcR. Csu-miR-14 exhibited high levels of expression at the end of each larval instar stage, and its expression was negatively correlated with the expression of its two target genes. Overexpression of Csu-miR-14 at the third day of the ﬁfth instar stage led to high mortality and developmental defects in RSB individuals. We produced 35 rice transformants to express miR-14 and found that three lines had a single copy with highly abundant miR-14 mature transcripts. Feeding bioassays using both T0 and T1 generations of transgenic miR-14 rice indicated that at least one line (C#24) showed high resistance to RSB. These results indicated that the approach of miRNAs as targets has potential for improving pest control methods. Moreover, using insect-speciﬁc miRNAs rather than protein- encoding genes for pest control may prove benign to non-insect species, and thus is worthy of further exploration. Keywords: miR-14, rice stem borer, ecdysone biosynthesis, insect-resistant, insect pest control. CpTi, SKTI and BTI-CMe) were also used to produce various GM crops resistant against to herbivory planthoppers, stem borers and rice water weevils (Alfonso-Rubi et al., 2003; Duan et al., 1996; Lee et al., 1999; Xu et al., 1996). Transgenic microRNA-14 rice shows high resistance to rice stem borer Kang He1, Huamei Xiao2,3,, Yang Sun3,4, Simin Ding1, Gongming Situ3 and Fei Li1, g g g g g 1Institute of Insect Sciences/Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, College of Agriculture Zhejiang University, Hangzhou, China 2College of Life Sciences and Resource Environment, Yichun University, Yichun, China 3Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China 4Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences, Nanchang, China nces/Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, College of Agriculture and Biotechnolo ngzhou China 1Institute of Insect Sciences/Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, College of 2College of Life Sciences and Resource Environment, Yichun University, Yichun, China 3Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China 4Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences, Nanchang, China Received 12 April 2018; revised 10 July 2018; accepted 19 July 2018. Correspondence (Tel +86-571-88982679; fax +86-571-88982679; emails xiaohuamei625@163.com (XH); lifei18@zju.edu.cn (LF)) Keywords: miR-14, rice stem borer, ecdysone biosynthesis, insect-resistant, insect pest control. Plant Biotechnology Journal (2019) 17, pp. 461–471 doi: 10.1111/pbi.12990 doi: 10.1111/pbi.12990 doi: 10.1111/pbi.12990 Introduction Botanically derived insecticidal genes, such as Galanthus nivalis agglutinin (GNA; Rao et al., 1998), and protein inhibitor genes (e.g., pin II, MicroRNAs (miRNA) are a class of small, non-coding RNAs that down-regulate the targets by degrading messenger RNA (mRNA) transcript or repressing translation, mainly by targeting the 30-ends of the transcript (Huntzinger and Izaurralde, 2011). In insects, miRNAs have been reported to regulate metamor- phosis in development (Bushati and Cohen, 2007; Ylla et al., 2016). As a result of the evidence indicating the important roles of miRNAs in regulating insect development, researchers 461 Kang He et al. 462 began to explore the application of miRNA in pest control. The larvae of cotton bollworm, Helicoverpa armigera, that fed on bacteria expressing an artiﬁcial miRNA (amiRNA) sequence speciﬁcally targeting the ecdysone receptor (EcR) gene showed greater mortality, developmental defects, and a signiﬁcant decline in reproductive ability (Yogindran and Rajam, 2016). Feeding on transgenic tobacco plants producing an amiRNA based on endogenous miR-24 against a chitinase gene signif- icantly reduced the level of chitinase transcripts in H. armigera larvae, causing cessation of molt (Agrawal et al., 2015). Although RSB larvae that were continuously fed with trans- genic Csu-novel-miR-15 rice showed a 4-day delay of pupation, no signiﬁcant lethal effect was observed (Jiang et al., 2016), the target genes and biological function of this novel miRNA remain unclear. began to explore the application of miRNA in pest control. The larvae of cotton bollworm, Helicoverpa armigera, that fed on bacteria expressing an artiﬁcial miRNA (amiRNA) sequence speciﬁcally targeting the ecdysone receptor (EcR) gene showed greater mortality, developmental defects, and a signiﬁcant decline in reproductive ability (Yogindran and Rajam, 2016). Feeding on transgenic tobacco plants producing an amiRNA based on endogenous miR-24 against a chitinase gene signif- icantly reduced the level of chitinase transcripts in H. armigera larvae, causing cessation of molt (Agrawal et al., 2015). Although RSB larvae that were continuously fed with trans- genic Csu-novel-miR-15 rice showed a 4-day delay of pupation, no signiﬁcant lethal effect was observed (Jiang et al., 2016), the target genes and biological function of this novel miRNA remain unclear. CsSpo and CsEcR are putative targets of Csu-miR-14 CsSpo and CsEcR are putative targets of Csu-miR-14 We downloaded 2,474 insect miRNA sequences from miRbase 22.0 and 517 miRNAs from publicly reported references (Chang et al., 2016; Yang et al., 2017), covering 14 species across ﬁve insect orders. To amass more insect miRNA information, we sequenced small RNA libraries of three notorious insect pests, Scirpophaga incertulas (yellow stem borer, YSB), Nilaparvata lugens (brown planthopper, BPH) and Laodelphax striatellus (small brown planthopper, SBPH), yielding 76, 110 and 118 miRNAs, respectively (Tables S1 and S2). Conservation analysis of all miRNAs from 17 insect species identiﬁed an insect-speciﬁc miRNA, miR-14, which was conserved among stem borers, and planthoppers (Figure 1a). Then, we focused on RSB to study the function of miR-14 and its potential application in pest control. To predict the targets of Csu-miR-14, we identiﬁed the 30 UTRs of all genes with a customized Perl script from the transcriptomes and genome of RSB. Then, we predicted the targets of Csu-miR-14 by using miRanda (John et al., 2004) and RNAHybrid (Kruger and Rehmsmeier, 2006), which indicated that two genes in the ecdysone signalling network, CsSpo and CsEcR, were putative targets of Csu-miR-14 (Figure 1b). Target prediction in other insect pests suggested that miR-14 also targeted genes in the ecdysone signalling network (Table S3), indicating that miR-14 regulation of the ecdysone signalling network is a conserved Insect-speciﬁc miRNAs present a promising target for pest control that may have less negative impacts on the environ- ment and non-insect species than chemical pesticides. Here, we identiﬁed an insect-speciﬁc miRNA, Csu-miR-14, that targets two genes, CsSpo and CsEcR, in the ecdysone signalling pathway of C. suppressalis. In vitro and in vivo experiments conﬁrmed the interactions between Csu-miR-14 and its molec- ular targets. We produced transgenic rice expressing artiﬁcial miRNA (amiR-14). Feeding on transgenic miR-14 rice led to a signiﬁcantly higher mortality and developmental defects in RSB. Figure 1 Insect-speciﬁc miRNA miR-14 and its two predicted targets in rice stem borer (RSB). Spook (Spo) and ecdysone receptor (EcR) are two genes in the ecdysone signaling network. (a) Mature sequence alignment of miR-14 among rice pests. Csu, Chilo suppressalis; Sin, Scirpophaga incertulas; Nlu, Nilaparvata lugens; Lst, Laodelphax striatellus; Sfu, Sogatella furcifera; Lor, Lissorhoptrus oryzophilus. (b) Sequence alignment of Csu-miR-14 with the predicted target sites in the 30UTR of Spo and EcR. 18 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Son CsSpo and CsEcR are putative targets of Csu-miR-14 Mutants (mut) were generated by deletion (red dash) or mutating (red letters) of complementary bases in the binding region of Csu-miR-14. Seed sequences were indicated in purple. Wild-type (WT) sequences of those mutations are indicated in green. Mfe, minimum free energy of the binding between miRNA and UTRs predicted by RNAhybrid. (c) Dual-luciferase activities compared between miR-14 with negative control (NC) in HEK293T cells (n = 6). The data are shown as means SE, P < 0.05, P < 0.01, P < 0.001 ª 2018 The Authors Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd 17 461–471 Figure 1 Insect-speciﬁc miRNA miR-14 and its two predicted targets in rice stem borer (RSB). Spook (Spo) and ecdysone receptor (EcR) are two genes in the ecdysone signaling network. (a) Mature sequence alignment of miR-14 among rice pests. Csu, Chilo suppressalis; Sin, Scirpophaga incertulas; Nlu, Nilaparvata lugens; Lst, Laodelphax striatellus; Sfu, Sogatella furcifera; Lor, Lissorhoptrus oryzophilus. (b) Sequence alignment of Csu-miR-14 with the predicted target sites in the 30UTR of Spo and EcR. Mutants (mut) were generated by deletion (red dash) or mutating (red letters) of complementary bases in the binding region of Csu-miR-14. Seed sequences were indicated in purple. Wild-type (WT) sequences of those mutations are indicated in green. Mfe, minimum free energy of the binding between miRNA and UTRs predicted by RNAhybrid. (c) Dual-luciferase activities compared between miR-14 with negative control (NC) in HEK293T cells (n = 6). The data are shown as means SE, P < 0.05, P < 0.01, P < 0.001 Transgenic miR-14 rice shows pest resistance 4 463 implying that the role of Csu-miR-14 is to eliminate the functions of ecdysone. process among insects. We used an in vitro dual-luciferase reporter assay to validate the interactions between Csu-miR-14 and its target genes. The 30-UTRs containing the predicted target sites of CsSpo and CsEcR were cloned into the downstream of luciferase genes in the pMIR-REPORT vector (Obio, China) and then transfected into HEK293T cells. The results indicated that luciferase activities were signiﬁcantly reduced in the presence of Csu-miR-14 mimics (P < 0.05, Student’s t-test; Figure 1c), con- ﬁrming the interactions of Csu-miR-14 with each of its two targets, CsSpo and CsEcR. The expression of Csu-miR-14 negatively correlated with the expression of CsSpo and CsEcR The expression of Csu-miR-14 negatively correlated with the expression of CsSpo and CsEcR Expression analysis indicated that the level of transcripts of Csu- miR-14 were high at the end of each instar and then declined sharply in the initial point of the following stage (Figure 2a). In our previous report (He et al., 2017; Sun et al., 2017), cus- tomized microarrays were designed to study the expression of small RNA and mRNA from the same batch of RSB samples including the stages of ageing larval, prepupal, early pupal, compound eye formation, pretarsal formation, pupal elongation and adult. This enabled us to calculate the co-expression patterns between Csu-miR-14 and its target genes. Poisson correlation analysis showed that the abundance of Csu-miR-14 was nega- tively correlated with the abundances of CsSpo (r = 0.614, P = 0.143) and CsEcR (r = 0.714, P = 0.072; Figure 2b–d), Csu-miR-14 controls metamorphosis development of RSB To study the in vivo function of Csu-miR-14, we overexpressed it by injecting the miRNA mimic (agomir-14) into third-day larvae at ﬁfth-instar stage (5L3D) of RSB. Chemically synthesized agomir- 14 (100 pmol) was injected into each 5L3D individual. The abundance of Csu-miR-14 was signiﬁcantly greater, by 6.86 3.36-fold at 24 h post-injection than in the control (Figure 3a, Mann–Whitney U-test, P < 0.05), whereas the expres- sion of the two targets, CsEcR and CsSpo, was lower than their corresponding controls by 46.6% (Student’s t-test, P < 0.05) and 67.7% (Student’s t-test, P < 0.05), respectively (Figure 3b). Moreover, a signiﬁcantly higher mortality was observed in the agomir-14-treated group than in the control group from 24 to 96 h post-injection (Figure 3c). The individuals treated with agomir-14 showed an abnormal phenotype with developmental defects such as an unusually dark body colour (Figure 3d). GM rice expressing mature Csu-miR-14 The mature sequence of Csu-miR-14 was used to design the miRNA expression cassette ubi::amiR-14-nos-hpt (Figure 4a), with the expression of artiﬁcial miR-14 driven by a ubiquitin promoter. The amiRNA vector was introduced into embryonic callus of japonica rice variety Zhonghua (ZH11) via Agrobacterium- Figure 2 Expression proﬁles of Csu-miR-14 and its two target genes, CsSpo and CsEcR at different developmental stages. (a) qRT-PCR analysis of Csu-miR- 14 from the ﬁfth day of the fourth-instar larvae (4L5D) to the third-day pupa (P3) stage, indicating that Csu-miR-14 is highly expressed at the end of each larval instar stage. (b) The changes in expression levels of Csu-miR-14, (c) CsSpo, and (d) CsEcR estimated by a previous microarray analysis at seven developmental stages of RSB (He et al., 2017; Sun et al., 2017) including aging larvae (aL), prepupae (pP), early pupae (eP), compound eye formation stage (cE), pupae elongation stage (Pe), and adult (Ad). The data are shown as means SE (n = 3). Signal values were compared by one-way ANOVA among various stages and signiﬁcant difference was indicated with different letters after Turkey’s multiple comparison test. ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 461–471 Figure 2 Expression proﬁles of Csu-miR-14 and its two target genes, CsSpo and CsEcR at different developmental stages. (a) qRT-PCR analysis of Csu-miR- 14 from the ﬁfth day of the fourth-instar larvae (4L5D) to the third-day pupa (P3) stage, indicating that Csu-miR-14 is highly expressed at the end of each larval instar stage. (b) The changes in expression levels of Csu-miR-14, (c) CsSpo, and (d) CsEcR estimated by a previous microarray analysis at seven developmental stages of RSB (He et al., 2017; Sun et al., 2017) including aging larvae (aL), prepupae (pP), early pupae (eP), compound eye formation stage (cE), pupae elongation stage (Pe), and adult (Ad). The data are shown as means SE (n = 3). Signal values were compared by one-way ANOVA among various stages and signiﬁcant difference was indicated with different letters after Turkey’s multiple comparison test. ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 461–471 Kang He et al. 464 Figure 3 Overexpression of Csu-miR-14 led to high mortality and developmental defects in RSB. GM rice expressing mature Csu-miR-14 (a) The transcript abundance of Csu-miR-14 signiﬁcantly increased at 24 h after injection of agomir-14 (Student t-test, P < 0.05, *P < 0.01). (b) The transcript abundance of target genes CsSpo and CsEcR were signiﬁcantly reduced at 24 h post-injection. (c) Overexpression of Csu-miR-14 led to higher mortality from 24 h to 96 h post-injection. (d) The abnormal phenotype of the agomir-14 treated group compared to the control. Kang He et al. 4 Kang He et al. 64 Figure 3 Overexpression of Csu-miR-14 led to high mortality and developmental defects in RSB. (a) The transcript abundance of Csu-miR Figure 3 Overexpression of Csu-miR-14 led to high mortality and developmental defects in RSB. (a) The transcript abundance of Csu-miR-14 signiﬁcantly increased at 24 h after injection of agomir-14 (Student t-test, P < 0.05, *P < 0.01). (b) The transcript abundance of target genes CsSpo and CsEcR were signiﬁcantly reduced at 24 h post-injection. (c) Overexpression of Csu-miR-14 led to higher mortality from 24 h to 96 h post-injection. (d) The abnormal phenotype of the agomir-14 treated group compared to the control. Figure 3 Overexpression of Csu-miR-14 led to high mortality and developmental defects in RSB. (a) The transcript abundance of Csu-miR-14 signiﬁcantly increased at 24 h after injection of agomir-14 (Student t-test, P < 0.05, *P < 0.01). (b) The transcript abundance of target genes CsSpo and CsEcR were signiﬁcantly reduced at 24 h post-injection. (c) Overexpression of Csu-miR-14 led to higher mortality from 24 h to 96 h post-injection. (d) The abnormal phenotype of the agomir-14 treated group compared to the control. expression of amiR-14 can potentially be effective in pest control applications (Figure 5b), we chose three GM rice lines, C#24, C#15 and C#18, for further analysis. mediated transformation (Figure 4b), yielding 35 rice transfor- mants after kanamycin screening. The seedlings then were transplanted into pots of soil and grown in the greenhouse (Figure 4c–g). To conﬁrm the insertion of the amiR-14 expression cassette in the genome of GM rice, a pair of primers was designed to amplify a 557-bp fragment of the selective marker gene hygromycin B phosphotransferase (Hpt). Positive PCR products were ampliﬁed from 33 transformants, resulting in a transformation efﬁciency of 94.29% (Figure 4h). ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 461–471 Feeding on transgenic amiR-14 rice led to high mortality and developmental defects in RSB To measure insect resistance in transgenic amiR-14 rice, the stems of three positive transformants, C#24, C#15 and C#18, were used to feed ﬁrst-instar larvae of RSB. The japonica rice variety ZH11 was used as the negative control. The results indicated that the survival rates of groups fed with transgenic amiR-14 were signiﬁcantly lower than that of the control group. The average mortality of the control group was 20% at 7-day after treatment. However, more than 60% of the tested larvae were dead at 3 days after feeding on C#24, while mortality of the C#24 group reached >80% at 12 days after treatment with GM rice (Figure 6a), showing a relatively high resistance to RSB. The mortalities of the groups fed with C#15 and C#18 were about 60% at 9 days after feeding on GM-rice, suggesting that they have moderate resistance to RSB (Figure 6a). A southern blot was used to determine the copy number of the amiR-14 transgene expression cassette in the GM rice genome. A 557-bp DNA probe was designed based on the complementary sequence of Hpt. The results showed that 12 of the 33 positive transformants were detected with a single copy of amiR-14, 11 were detected with two copies, four were detected with three copies, ﬁve were detected with four copies, and one was detected with eight copies (Figure 5a). The expression of amiR-14 was measured by qRT-PCR in T0 GM rice (Table S4). The highest levels of expression were observed in three transformants, C#22 (205.17 9.40), C#25 (149.02 12.46) and C#24 (65.79 1.47). However, Spearman’s correlation analysis showed that there was no positive relation between the expression of amiR-14 and its copy number in the GM rice genome (r = 0.172). Observing that transgenic rice with a single copy and high In addition, the survivors in the groups fed on GM rice exhibited apparent developmental defects. The larvae feeding on GM rice of the C#15 and C#18 lines had a slightly longer lifespan (36.0 2.8 days for C#15; 37.2 4.2 days for C#18) than the Transgenic miR-14 rice shows pest resistance 465 465 (a) (b) (c) (d) (e) (f) (g) (a) (b) (h) (c) (d) (e) (f) (g) Figure 4 Genetically modiﬁed rice expressing miR-14 and PCR validation of rice transformants. (a) Schematic diagram of amiR-14 expression cassette pUbi-amiR-14 introduced into rice variety ZH11 via Agrobacterium-mediated transformation. Feeding on transgenic amiR-14 rice led to high mortality and developmental defects in RSB Pink box indicates amiRNA-14 precursor. Hpt, hygromycin B phosphotransferase; NOS, Nos terminator; RB, right border; LB, left border. (b) Embryonic callus induction of the seeds processed by shelling and disinfection. (c) Subculture of the callus grown in the dark for 20 days. (d) Screening for positive rice transformants by kanamycin. (e) Seedlings from resistant callus at 40 days. (f) Callus rooting for 20 days in the climate incubator. (g) Seedlings were transferred into a greenhouse and grown to obtain the T0 generation. WT, wild type plants; Trans, transgenic plants. (h) Selection of the positive rice transformants based on PCR ampliﬁcation of the hpt gene. M, DL2000 k B bl k t l (a) (b) (c) (g) (f) (h) (h) (h) (h) Figure 4 Genetically modiﬁed rice expressing miR-14 and PCR validation of rice transformants. (a) Schematic diagram of amiR-14 expression cassette pUbi-amiR-14 introduced into rice variety ZH11 via Agrobacterium-mediated transformation. Pink box indicates amiRNA-14 precursor. Hpt, hygromycin B phosphotransferase; NOS, Nos terminator; RB, right border; LB, left border. (b) Embryonic callus induction of the seeds processed by shelling and disinfection. (c) Subculture of the callus grown in the dark for 20 days. (d) Screening for positive rice transformants by kanamycin. (e) Seedlings from resistant callus at 40 days. (f) Callus rooting for 20 days in the climate incubator. (g) Seedlings were transferred into a greenhouse and grown to obtain the T0 generation. WT, wild type plants; Trans, transgenic plants. (h) Selection of the positive rice transformants based on PCR ampliﬁcation of the hpt gene. M, DL2000 marker; B, blank control. Figure 4 Genetically modiﬁed rice expressing miR-14 and PCR validation of rice transformants. (a) Schematic diagram of amiR-14 expression cassette pUbi-amiR-14 introduced into rice variety ZH11 via Agrobacterium-mediated transformation. Pink box indicates amiRNA-14 precursor. Hpt, hygromycin B phosphotransferase; NOS, Nos terminator; RB, right border; LB, left border. (b) Embryonic callus induction of the seeds processed by shelling and disinfection. (c) Subculture of the callus grown in the dark for 20 days. (d) Screening for positive rice transformants by kanamycin. (e) Seedlings from resistant callus at 40 days. (f) Callus rooting for 20 days in the climate incubator. (g) Seedlings were transferred into a greenhouse and grown to obtain the T0 generation. WT, wild type plants; Trans, transgenic plants. (h) Selection of the positive rice transformants based on PCR ampliﬁcation of the hpt gene. M, DL2000 marker; B, blank control. Feeding on transgenic amiR-14 rice led to high mortality and developmental defects in RSB resistance to RSB. The japonica rice variety ZH11 was used as the negative control. Thirty ﬁrst-instar larvae were transferred to each plant. 50 days later, all ZH11 rice plants were dead with 100% dead hearts because of the extensive damage caused by RSB, whereas all six transgenic miR-14 rice plants in two replicates were alive and lacked any indication of damage by RSB herbivory (Figure 7). other two groups, but without signiﬁcant difference. The average lifespan of the C#24 group was 34.5 5.1 days, which was similar to that of the control (34.1 1.4 days; Figure 6b). Compared to the control group, 30% of the survivors that fed on GM rice showed early pupation, while the remaining 70% was delayed in pupation (Figure 6c). The control population showed a peak in the frequency of pupating individuals within a similar time period, which corresponds to the natural feature of developmen- tal synchrony that ensures concurrent sexual maturation of adult females and males. However, this peak did not appear in GM-rice treated groups (Figure 6d), suggesting that the deaths of the majority of RSB individuals caused by ingestion of the transgenic rice would likely hamper population growth. Discussion Application of transgenic insect-resistant crops has been shown to be an efﬁcient method for pest control (Vaeck et al., 1987). The widely used transgenic Bt cotton is a successful example of controlling insecticide-resistant insect pests (Wu et al., 2008). Many insect-resistant Bt rice lines have been developed, although they are not commercially planted so far (Liu et al., 2016). Transgenic Bt rice showed high resistance to early-instar larvae of RSB (90%–100% mortality; Liu et al., 2016), however, the mortalities signiﬁcantly decreased when feeding it to higher instar larvae (Hu et al., 2005). At present, only a limited number of insecticidal genes have been identiﬁed at present (Tabashnik ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 461–471 Transgenic amiR-14 rice showed high resistance to RSB Given the observation that line C#24 had the highest resistance to RSB in feeding assays, we focused on this GM rice line for further analysis. The transgenic line C#24 was grown in the greenhouse and was harvested for seeds of the T1 generation. The germination rate of the seeds of line C#24 was 87.5%. Six T1-generation plants of C#24 were selected for estimating their Figure 5 The copy number and mRNA expression for miR-14 detected in the T0 generation of rice. (a) Southern-blot detection with a 557-bp probe against the hpt gene. M, k DNA marker. (b) Relative mRNA abundance of miR-14 detected with qRT-PCR. Signiﬁcant differences (P < 0.05) are indicated with different letters detected by one-way ANOVA analysis after Turkey’s multiple comparison test. Kang He et al. 6 Kang He et al. 466 Kang He et al. 466 Figure 5 The copy number and mRNA expression for miR-14 detected in the T0 generation of rice. (a) Southern-blot detection with a 557-bp probe against the hpt gene. M, k DNA marker. (b) Relative mRNA abundance of miR-14 detected with qRT-PCR. Signiﬁcant differences (P < 0.05) are indicated with different letters detected by one-way ANOVA analysis after Turkey’s multiple comparison test. Figure 6 The bioassay of RSB response to ingestion of three different lines of T0 transgenic rice plants or the wild-type. (a) Survival rates, (b) The days of survival for each larva. (c) Cumulative pupation rates. (d) The pupation frequency, where the naturally occurring peak indicating synchrony of pupation among individuals is notably lacking in the transgenic rice groups. Figure 5 The copy number and mRNA expression for miR-14 detected in the T0 generation of rice. (a) Southern-blot detection with a 557-bp probe against the hpt gene. M, k DNA marker. (b) Relative mRNA abundance of miR-14 detected with qRT-PCR. Signiﬁcant differences (P < 0.05) are indicated with different letters detected by one-way ANOVA analysis after Turkey’s multiple comparison test. Figure 5 The copy number and mRNA expression for miR-14 detected in the T0 generation of rice. (a) Southern-blot detection with a 557-bp probe against the hpt gene. M, k DNA marker. (b) Relative mRNA abundance of miR-14 detected with qRT-PCR. Signiﬁcant differences (P < 0.05) are indicated with different letters detected by one-way ANOVA analysis after Turkey’s multiple comparison test. Figure 5 The copy number and mRNA expression for miR-14 detected in the T0 generation of rice. Small RNAs sequencing and identiﬁcation Total RNA was isolated from samples consisting of a mix of the eggs, larvae (nymphs), pupae and adults for each species (S. incertulas, N. lugens and L. striatellus) using TRIzol (Invitro- gen, Carlsbad, CA) reagent following the instructions of the manufacturer. Small RNA sequencing was performed as described in He et al. (2017). Puriﬁed RNA fragments were ﬁrst separated by polyacrylamide gel electrophoresis (PAGE) and then ligated to two adaptors (50/30) for PCR primers binding. Then these small RNAs were ampliﬁed with RT-PCR, followed by sequencing with an Illumina HiSeq 2000 to construct RNA libraries. After quality control, the cleaned data were used for further analysis. Data statistics from library sequencing are provided in Table S1. Insect metamorphosis is a successful life-history strategy for fully exploring various environmental conditions (Truman and Riddiford, 1999). The steroid hormone 20-hydroxyecdysone (20E) coordinates with juvenile hormone (JH) to regulate moulting (Dubrovsky, 2005; Jindra et al., 2013; Liu et al., 2018; Yamanaka et al., 2013). Interfering with ecdysone biogenesis can result in morphological defects (Neubueser et al., 2005), and thus can be used for developing pest control methods (Luan et al., 2013). The genes in the ecdysone pathway and the miRNAs targeting these genes are promising targets of RNAi pest control, as evidenced by results of this study. To identify small RNAs in these three pests, cleaned reads were annotated by Blastn against the nr database (https://www.ncbi. nlm.nih.gov), Rfam (Kalvari et al., 2018) and RepBase (Jurka et al., 2005), and then two algorithms were used to identify the miRNAs. One algorithm involved homology-searching against arthropod miRNAs in the miRbase (Kozomara and Grifﬁths-Jones, 2014) using a cut-off of 0-2 nt mismatches or deletions. The other algorithm identiﬁed both conserved and novel miRNAs with miRDeep (An et al., 2013) against the respective genome sequence of N. lugens (Xue et al., 2014) and L. striatellus (Zhu et al., 2017) with the default parameter of ignoring reads of length <18 nt and mapping to no more than 5 genomic loci. For S. incertulas, the C. suppressalis genomic sequence (Yin et al., 2014) was used as the reference. MicroRNA sequences identiﬁed We also investigated the control effect of transgenic miR-14 on BPH. Experimental procedures Insect rearing Rice stem borer larvae were maintained on newly germinated rice seeds in glass bottles at 28 1°C with a relative humidity of 70%–80% as described in He et al. (2017). Nymphs of N. lugens and L. striatellus also were maintained on rice seedlings. All of these rice pests were cultured under the same environmental conditions, including a photoperiod of 16 h light/8 h dark. RNAi is a promising genetic method for pest control (Huvenne and Smagghe, 2010; Price and Gatehouse, 2008). Suppressing the expression of genes participating in important physiological processes may prove lethal to insect pests (Baum et al., 2007; Mao et al., 2007). RNAi has an advantage over previous transgenic methods in that it can signiﬁcantly enlarge the scope of target genes that can potentially be used for pest control. At present, most target genes used in RNAi pest control are protein- encoding genes (Zhang et al., 2013). Here, we showed that noncoding RNA genes also can be used for RNAi pest control. Moreover, the ability to select from a wider variety of target genes implies that we are more likely to develop safer alternatives to chemical pesticides by using insect-speciﬁc miRNAs that cannot affect non-target species because its homologs may not be found in non-insect species. ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 461–471 Transgenic amiR-14 rice showed high resistance to RSB (a) Southern-blot detection with a 557-bp probe against the hpt gene. M, k DNA marker. (b) Relative mRNA abundance of miR-14 detected with qRT-PCR. Signiﬁcant differences (P < 0.05) are indicated with different letters detected by one-way ANOVA analysis after Turkey’s multiple comparison test. Figure 5 The copy number and mRNA expression for miR-14 detected in the T0 generation of rice. (a) Southern-blot detection with a 557-bp probe against the hpt gene. M, k DNA marker. (b) Relative mRNA abundance of miR-14 detected with qRT-PCR. Signiﬁcant differences (P < 0.05) are indicated with different letters detected by one-way ANOVA analysis after Turkey’s multiple comparison test. Figure 6 The bioassay of RSB response to ingestion of three different lines of T0 transgenic rice plants or the wild-type. (a) Survival rates, (b) The days of survival for each larva. (c) Cumulative pupation rates. (d) The pupation frequency, where the naturally occurring peak indicating synchrony of pupation among individuals is notably lacking in the transgenic rice groups. Figure 6 The bioassay of RSB response to ingestion of three different lines of T0 transgenic rice plants or the wild-type. (a) Survival rates, (b) The days of survival for each larva. (c) Cumulative pupation rates. (d) The pupation frequency, where the naturally occurring peak indicating synchrony of pupation among individuals is notably lacking in the transgenic rice groups. C#24, showed high resistance to RSB. miR-14 has been reported to regulate metamorphosis in a variety of insects (Jayachandran et al., 2013; Liu et al., 2013; Varghese and Cohen, 2007). and Carriere, 2017). Here, we demonstrated that an insect- speciﬁc miRNA, miR-14, can be used for producing transgenic insect-resistant rice. At least one line of transgenic miR-14 rice, and Carriere, 2017). Here, we demonstrated that an insect- speciﬁc miRNA, miR-14, can be used for producing transgenic insect-resistant rice. At least one line of transgenic miR-14 rice, ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 461–471 ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 461–471 Transgenic miR-14 rice shows pest resistance 467 (a) (b) (b) (a) (b) (b) (a) Figure 7 The bioassay of insect resistance in C#24 T1 transgenic rice plants exposed to 30 RSB individuals. Transgenic amiR-14 rice showed high resistance to RSB (a) GM rice C#24 showed high resistance to RSB. (b) There was no obvious tissue damage observed in C#24 GM stems caused by RSB. Red arrow indicates damage of worm holes. recent study showed that less caterpillar damage in Bt rice attracts fewer planthoppers, implying possible ecological resis- tance against BPH (Wang et al., 2018). Whether transgenic miR- 14 rice also has this kind of resistance to nontarget planthoppers requires further investigation. Overexpression of Csu-miR-14 led to the death of RSB individuals on such plants which indicates that Csu-miR-14 can interfere with normal metamorphosis development in RSB. The role of Csu-miR- 14 is to eliminate the functions of ecdysone after molt. Its ﬁrst effect is to clean unwanted transcripts of CsSpo to prevent the biogenesis of ecdysone; the second effect is to repress CsEcR, the ecdysone receptor. Using miRNA in developing transgenic insect- resistant rice lines signiﬁcantly broadens the scope of target genes that can potentially be used for pest control. Cell culture and luciferase assay The HEK293T cell line has been commonly used in the luciferase assay for the largely conserved mechanisms underlying the processing of pre-miRNAs between mammalian and insect cells (Bogerd et al., 2014; Liu et al., 2017). Cells were maintained at 37 °C with 5% CO2 in DMEM high-glucose medium (Gibco, Grand Island, NY) containing 10% foetal bovine serum (Gibco). 30UTR fragments of the CsSpo and CsEcR genes were cloned into the pMIR-REPORT vector (Obio, China) between the ﬁreﬂy luciferase open reading frames (ORF) and the SV40 polyA signal. Cells were transfected with the mixture of 200 ng of the reporter plasmid, and 10 ng of the pRL-CMV control plasmid in 0.25 mL Lipofectamine 2000 Transfection Reagent (Invitrogen) in 5-mL Opti-MEM I Reduced Serum Medium (Gibco) in each well of a 96-well plate. The miRNA mimics were synthesized by RiboBio Small RNAs sequencing and identiﬁcation Feeding on transgenic miR-14 rice only caused a moderate degree of mortality in BPH, not signiﬁcantly different from the control (Figure 8), suggesting that the transgenic miR-14 rice did not have resistance to BPH, which has piercing-sucking mouth- parts. We suggested that this might be due to only a limited number of miR-14 transcripts expressed in the phloem sap of the rice plant. Actually, improving the efﬁciency of controlling pests with piercing-sucking mouthparts is still an important task. A Kang He et al. 468 Figure 8 The bioassay of BPH in response to being fed on T0 transgenic or wild type rice. Transgenic miR-14 rice did not exhibit strong resistance to BPH herbivory. (a) Survival rates of BPH that fed on transgenic rice stems. (b) The days of survival for each nymph. (c) Cumulative eclosion rates. (d) Eclosion frequency. Kang He et al. 8 Figure 8 The bioassay of BPH in response to being fed on T0 transgenic or wild type rice. Transgenic miR-14 rice did not exhibit strong resistance to BPH herbivory. (a) Survival rates of BPH that fed on transgenic rice stems. (b) The days of survival for each nymph. (c) Cumulative eclosion rates. (d) Eclosion frequency. by the two methods were pooled, and the redundancies were removed to generate a ﬁnal set of miRNAs for the three rice pests. (Guangzhou, China) and diluted to a concentration of 100 nM. The Dual-Luciferase Reporter (DLR TM) Assay System (Promega, Madison City, WI) was performed 24 h after transfection according to the manufacturer’s protocol. The experiment was performed in six independent replicates. The mean of the relative luciferase expression ratio (renilla luciferase/ﬁreﬂy luciferase) of the control was set to 1. MicroRNA agomir and antagomir treatment in vivo Mimics of miR-14 (agomir-14) and a negative control (agomir-NC) were synthesized by RiboBio. For each individual insect, 100 nmol of miRNA mimic (agomir-14) was injected at the intersegmental membrane in the abdomen of larvae. A total of 30 insects was injected for each treatment. Then, 24 and 48 h after the injection, larvae were ﬂash-frozen in liquid nitrogen and stored at 80 °C before measurement of mRNA levels. Target prediction of miR-14 To predict targets of miR-14 in rice pests, the 30 UTR sequences of 19 genes in the ecdysone signalling pathway network were obtained by searching the genome and transcriptome of all four rice pests using a customized Perl script. Two miRNA target prediction methods, miRanda (John et al., 2004) and RNAHybrid (Kruger and Rehmsmeier, 2006), were used to predict the targets of miR-14 with the default parameters. The cut-off for free energy was <18 kJ/mol. Targets predicted by either algorithm were used for further analysis. ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 461–471 Detection of amiR-14 copies by southern blot hybridization To determine the copy number of amiR-14, 10 lg genomic DNA isolated from GM and WT rice leaves of 40-day old seedlings was digested with 15 U of the restriction enzyme HindIII (NEB) for 16 h at 37 °C, and the digested DNA was resolved on a 0.8% agarose gel for 16 h at 30 V. Then, the electrophoresis gel was blotted onto a Hybond N+ nylon membrane (GE healthcare, Amersham, UK) by overnight capillary transfer. The blot was hybridized with the complementary strand of the hpt gene-based DNA probe at 48 °C. The blot was subjected to photography using X-ray ﬁlm by exposure at 80 °C for 4 h in a darkroom. Statistical analysis All data sets are shown as means SE (n = 3). The dual- luciferase reporter assay and quantitative real-time PCR results were analysed by a two-tailed unpaired Student’s t-test. Signif- icance was set at P < 0.05. Laboratory resistance assay of T1 transgenic miR-14 rice to RSB Laboratory resistance assay of T1 transgenic miR-14 rice to RSB For the ﬁeld trial assay, the percentage of rice plants with dead- heart was taken as the main indicator of damage caused by RSB (Liu et al., 2016; Shu et al., 2000). To examine the resistance of amiR-14 transgenic rice plants to RSB in laboratory, 30 newly hatched RSB larvae were transferred to limited numbers of whole rice plants at the booting stage (60-days old) using a small brush and kept in nylon mesh cages (80-cm in length and 18-cm in diameter). Each cage was sealed with glue to prevent the larvae from escaping. The rice plants were grown under the same conditions as mentioned above. Six replications for each amiRNA rice line were examined. After infestation, rice plants were checked for white heads or dead hearts caused by RSB every 2 days, and the sheaths were dissected to verify any damage made by RSB. qRT-PCR for mRNA and miRNA Fifth day of fourth-instar larvae (4L5D) to third-day of the pupa (P3) stage RSB were collected for the measurement of expression of Csu-miR-14. Total RNA was isolated from the whole-larval insect body of 40-day old or rice leaves using TRIzol Reagent (Invitrogen) following the instructions. A ﬁrst-strand cDNA synthesis kit (Vazyme, Nanjing, China) was used to prepare the oligo (dT)-primed cDNA. A stem-loop cDNA synthesis kit (RiboBio) was used to prepare stem-loop cDNA. The mRNAs and miRNAs were subjected to qRT-PCR for the respective gene expression assays using AceQ SYBR Green Master Mix (Vazyme) and Bulge- Loop TM miRNA qRT-PCR Starter Kit (RiboBio) according to man- ufacturers’ directions. Expression of the RSB Actin, 18s rRNA, and rice U6 snRNA genes were used as the internal controls for data analysis. qPCR was performed on ABI7500 instrument (Applied Biosystems, Foster City, CA, USA). The program for mRNA qPCR was as follows: 95 °C for 30 s at the initial denaturation step; and 40 cycles at 95 °C for 5 s, and 60 °C for 34 s. The program for miRNA qPCRs was 95 °C for 10 min at the initial denaturation step; and 40 cycles at 95 °C for 15 s, 55 °C for 30 s and 70 °C for 34 s. Data were analysed using the 2DDCt method of relative quantiﬁcation (Livak and Schmittgen, 2001). Differences of amiR- Selection of amiR-14 T0 GM rice plants Selection of amiR-14 T0 GM rice plants Genomic DNA was isolated from the leaves of T0 GM and wild- type ZH11 (WT) rice plants according to the CTAB-based protocol (Doyle and Doyle, 1987; Porebski et al., 1997). To identify positive T0 transformants, the hygromycin B phosphotransferase (hpt) gene was detected with PCR using the genomic DNA as a template. The PCR reaction included a mixture of dNTPs, 0.2 U Takara rTaq DNA polymerase, and the primers which are listed in Table S5 (Hpt557-F, Hpt557-R). The PCR conditions were as follow: initial denaturation at 94 °C for 3 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s; and a ﬁnal extension at 72 °C for 10 min. Genomic DNA from WT plants and double-distilled water were both used as negative controls. The PCR products were analysed by agarose gel electrophoresis. BPH feeding test Rice stems were individually placed in a glass tube (20-cm in length and 3-cm in diameter) for the consecutive BPH feeding test. A fresh rice stem from each amiRNA rice line was infested with 20 newly molted, third-instar BPH nymphs. The tubes were sealed with nylon mesh to prevent the larvae from escaping and then incubated under the same conditions as described above. The BPH nymphs were inspected twice each day until they emerged as adults. The time-periods for achievement of devel- opmental stages of the nymphs were recorded. Detection of amiR-14 copies by southern blot hybridization RSB feeding test After determination of the amiRNA-14 expression levels in rice plants using stem-loop qRT-PCR, fresh stems of the rice lines with high levels of expression of amiRNA were harvested at the booting stage and cut into 8-cm segments. Six fresh stem cuttings from each amiRNA rice line with 20 newly hatched ﬁrst-instar RSB larvae were placed in a Petri dish (9-cm in diameter). The dish was covered with a piece of moist ﬁlter paper and sealed with breathable tape, then incubated in darkness at 28 °C with 80% relative humidity. The rice stems were dissected to ﬁnd the RSB individuals and fresh rice stems were used to replace old ones every 3 days until all the RSB larvae had developed to pupae, and the mortality of RSB larvae was recorded. Three replications for each amiRNA rice line were examined. The data were analysed using Student’s t-test to compare RSB mortalities between the groups feeding on ZH11 or transgenic rice. Transgenic miR-14 rice shows pest resistance Transgenic miR-14 rice shows pest resistance 14 expression between treatments were compared by one-way ANOVA, followed by Turkey’s multiple comparison test. All primers used in the present study are listed in Table S5. Ubi-nos (Chen et al., 2013), between the maize ubiquitin promoter and the Nos terminator, to form the ﬁnal amiRNA expression vectors. The expression vectors were introduced into the japonica rice variety ZH11 via Agrobacterium-mediated transformation following the protocol of Lin et al. (2002). After transformation and 3 days in culture, calluses were washed three times with distilled water and dried on absorbent paper. Then, calluses were pre-selected with kanamycin and transferred for differentiation and rooting. Regenerated plantlets were cultivated in a greenhouse for selection. 14 expression between treatments were compared by one-way ANOVA, followed by Turkey’s multiple comparison test. All primers used in the present study are listed in Table S5. Vector construction, rice transformation and screening for expression of amiRNA Genetic transformation of rice was entrusted to Towin Biotech- nology (Wuhan, China). Expression vectors for amiRNA-14 were designed and constructed according to the sequence of Csu-miR- 14. The amiRNA precursors were generated based on the precursor of rice miRNA Osa-miR-528 (Warthmann et al., 2008). Subsequently, the 554-bp amiRNA precursors were cloned using the pEASY-T3 Cloning Kit (TransGen, Beijing, China). After validation by sequencing, the amiRNA precursors were released from the T-vector by digestion with BamHI and KpnI restriction enzymes and cloned into the plant expression vector pC1300- 469 RSB and BPH feeding assay RSB and BPH feeding assay ª 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 17, 461–471 References Jiang, S., Wu, H., Liu, H., Zheng, J., Lin, Y. and Chen, H. (2016) The overexpression of insect endogenous small RNAs in transgenic rice inhibits growth and delays pupation of striped stem borer (Chilo suppressalis). Pest Manag. Sci. 73, 1453–1461. Agrawal, A., Rajamani, V., Reddy, V.S., Mukherjee, S.K. and Bhatnagar, R.K. (2015) Transgenic plants over-expressing insect-speciﬁc microRNA acquire insecticidal activity against Helicoverpa armigera: an alternative to Bt-toxin technology. Transgenic Res. 24, 791–801. Jindra, M., Palli, S.R. and Riddiford, L.M. 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(1987) Transgenic plants protected from insect attack. Nature, 328, 33–37. Additional supporting information may be found online in the Supporting Information section at the end of the article. Additional supporting information may be found online in the Supporting Information section at the end of the article. Varghese, J. and Cohen, S.M. (2007) microRNA miR-14 acts to modulate a positive autoregulatory loop controlling steroid hormone signaling in Drosophila. Genes Dev. 21, 2277–2282. Table S1 Raw reads of three rice pests small RNA libraries. Table S2 Gene numbers of miRNAs predicted in three rice pests. Wang, X., Liu, Q., Meissle, M., Peng, Y., Wu, K., Romeis, J. and Li, Y. (2018) Bt rice could provide ecological resistance against non-target planthoppers. Plant Biotechnol. J. https://doi.org/10.1111/pbi.12911. Table S3 Potential targets of miR-14 predicted in ﬁve rice pests. Table S4 Copy number and mRNA expression level of miR-14 detected in T0 generation transgenic rice plants. 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https://openalex.org/W4200182658	https://ora.ox.ac.uk/objects/uuid:999d8c45-6591-4f53-833a-7f837eec04ab/files/smc87pr18p	English	null	Impact of metabolic disorders on the structural, functional, and immunological integrity of the blood‐brain barrier: Therapeutic avenues	The FASEB journal	2,021	cc-by	17,533	eived: 13 August 2021 \| Revised: 4 November 2021 \| Accepted: 3 December 2021 eived: 13 August 2021 \| Revised: 4 November 2021 \| Accepted: 3 December 2021 Received: 13 August 2021 \| Revised: 4 November 2021 \| Accepted: 3 December 2021 DOI: 10.1096/fj.202101297R DOI: 10.1096/fj.202101297R R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Impact of metabolic disorders on the structural, functional, and immunological integrity of the blood-brain barrier: Therapeutic avenues Correspondence Egle Solito, Centre for Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Email: e.solito@qmul.ac.uk Funding information British Heart Foundation, Grant/ Award Number: FS 16/60/32739; Abstract Mounting evidence has linked the metabolic disease to neurovascular disorders and cognitive decline. Using a murine model of a high-fat high-sugar diet mim- icking obesity-induced type 2 diabetes mellitus (T2DM) in humans, we show that pro-inflammatory mediators and altered immune responses damage the blood- brain barrier (BBB) structure, triggering a proinflammatory metabolic phenotype. We find that disruption to tight junctions and basal lamina due to loss of con- trol in the production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) causes BBB impairment. Together the disruption to the structural and Abstract Egle Solito, Centre for Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Mounting evidence has linked the metabolic disease to neurovascular disorders and cognitive decline. Using a murine model of a high-fat high-sugar diet mim- icking obesity-induced type 2 diabetes mellitus (T2DM) in humans, we show that pro-inflammatory mediators and altered immune responses damage the blood- brain barrier (BBB) structure, triggering a proinflammatory metabolic phenotype. Mounting evidence has linked the metabolic disease to neurovascular disorders and cognitive decline. Using a murine model of a high-fat high-sugar diet mim- icking obesity-induced type 2 diabetes mellitus (T2DM) in humans, we show that pro-inflammatory mediators and altered immune responses damage the blood- brain barrier (BBB) structure, triggering a proinflammatory metabolic phenotype. We find that disruption to tight junctions and basal lamina due to loss of con- trol in the production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) causes BBB impairment. Together the disruption to the structural and We find that disruption to tight junctions and basal lamina due to loss of con- trol in the production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) causes BBB impairment. Together the disruption to the structural and \| 1 of 27 wileyonlinelibrary.com/journal/fsb2 Impact of metabolic disorders on the structural, functional, and immunological integrity of the blood-brain barrier: Therapeutic avenues Madeeha H. Sheikh1 \| Mariella Errede2 \| Antonio d'Amati2,3 \| Noorafza Q. Khan1 \| Silvia Fanti1 \| Rodrigo A. Loiola1,4 \| Simon McArthur5 \| Gareth S. D. Purvis1,6 \| Caroline E. O'Riordan1 \| Davide Ferorelli7 \| Alessandro Dell'Erba7 \| Julius Kieswich1 \| Chis Reutelingsperger8 \| Eugenio Maiorano3 \| Magdi Yaqoob1 \| Christoph Thiemermann1 \| Andrea Baragetti9,10 \| Alberico Luigi Catapano9,10 \| Giuseppe Danilo Norata9,10,11 \| Federica Marelli-Berg1 \| Daniela Virgintino2 \| Egle Solito1,12 Madeeha H. Sheikh1 \| Mariella Errede2 \| Antonio d'Amati2,3 \| Noorafza Q. Khan1 \| Silvia Fanti1 \| Rodrigo A. Loiola1,4 \| Simon McArthur5 \| Gareth S. D. Purvis1,6 \| Caroline E. O'Riordan1 \| Davide Ferorelli7 \| Alessandro Dell'Erba7 \| Julius Kieswich1 \| Chis Reutelingsperger8 \| Eugenio Maiorano3 \| Magdi Yaqoob1 \| Christoph Thiemermann1 \| Andrea Baragetti9,10 \| Alberico Luigi Catapano9,10 \| Giuseppe Danilo Norata9,10,11 \| Federica Marelli-Berg1 \| Daniela Virgintino2 \| Egle Solito1,12 1William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London 2Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari School of Medicine, Bari, Italy 3Department of Emergency and Organ Transplantation, Section of Anatomic Pathology, University of Bari, Bari, Italy 4Laboratoire de la Barrière Hémato-Encéphalique, Faculty Jean Perrin, EA 2465, Université d'Artois, Arras, France 5Institute of Dentistry, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK 6Sir William Dunn School of Pathology, University of Oxford, Oxford, UK 7Department of Interdisciplinary Medicine, Section of Legal Medicine, University of Bari, Bari, Italy 8Cardiovascular Research Institute, Maastricht University, Maastricht, The Netherlands 9Department of Pharmacological and Biomolecular Sciences, Milan University, Milan, Italy 10IRCCS Multimedica, Sesto San Giovanni, Italy 11S.I.S.A. Centre for the Study of Atherosclerosis-Bassini Hospital, Cinisello Balsamo, Italy 12Department of Medicina Molecolare e Biotecnologie Mediche, University of Naples “Federico II”, Naples, Italy FASEB J. 2022;36:e22107. \| 1 of 27 https://doi.org/10.1096/fj.202101297R wileyonlinelibrary.com/journal/fsb2 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology. Abbreviations: ANXA1, annexin A1; FPR, formyl peptide receptor. 1 \| BACKGROUND 1 shown reduced grey and white matter, a hallmark of de- mentia.10–12 In fact, rodents fed an unhealthy “western diet” have impaired memory retention and faster cogni- tive decline compared to chow-fed rodents.13,14 Metabolic Syndrome (MetS)1—a cluster of at least three of the following traits: hyperglycemia, insulin resistance, ab- dominal obesity, high blood pressure, and dyslipidemia— have been linked with the development of neurovascular disease, including vascular dementia and neurodegener- ative conditions such as Alzheimer's disease,2 together termed the “metabolic-cognitive syndrome”.3 Low-grade chronic inflammation in MetS and subsequent develop- ment of Type 2 diabetes mellitus (T2DM) results in mac- rovascular and microvascular complications in peripheral vessels, but the impact on the brain microvasculature and blood-brain barrier (BBB) function remains elusive. In this study, we have used a mouse model of high-fat high-sugar (HFHS) diet-induced T2DM to examine the structural, metabolic, and immunological changes at the BBB. We report that HFHF-feeding causes the dis- mantling of endothelial tight junctions and loss of ves- sel basal lamina (VBL) molecular components to impair BBB integrity. Moreover, metabolic overload is accompa- nied by a marked release of pro-inflammatory mediators and brain endothelial cell activation, which promote leu- kocyte migration into the brain parenchyma resulting in activation of microglia. We further report that therapeu- tic treatment with the pro-resolving protein hrANXA1 or via dietary changes can attenuate T2DM development with downstream effects on restoration of BBB struc- tural, functional, and immunology integrity. In addition, we show the translatable applicability of our results using a humanized in vitro BBB model with T2DM pa- tient serum and T2DM patient post-mortem brain stud- ies. Our results find that T2DM patients have increased BBB leakage attributed to structural alterations at endo- thelial TJs and laminins that result in a marked increase in leukocyte presence within the brain parenchyma. These pathophysiological changes could be key in the development of the metabolic-cognitive syndrome. The BBB forms a specialized barrier between the in- terface of the peripheral and central systems, to maintain a homeostatic brain microenvironment. Ministry of Education-CAPES, Brazil, Grant/Award Number: 7326/2014- 09; Vini di Batasiolo S.p.A, Grant/ Award Number: AL-RIC19ABARA_01; Fondazione Umberto Veronesi, Grant/Award Number: 2020-3318; Fondazione Cariplo, Grant/Award Number: 2016-0852, 2015-0524 and 2015-0564; Fondazione Telethon, Grant/Award Number: GGP19146; PRIN, Grant/Award Number: 2017K55HLC and 2017H5F943; H2020 REPROGRAM, Grant/Award Number: PHC-03-2015/667837-2; Ministero della Salute, Grant/Award Number: GR-2011-02346974; ERA NET, Grant/ Award Number: ER-2017-2364981; FISM Fondazione Italianana Sclerosi Multipla, Grant/Award Number: 2014/R/21 functional integrity of the BBB results in enhanced transmigration of leukocytes across the BBB that could contribute to an initiation of a neuroinflammatory re- sponse through activation of microglia. Using a humanized in vitro model of the BBB and T2DM patient post-mortem brains, we show the translatable applicabil- ity of our results. We find a leaky BBB phenotype in T2DM patients can be attrib- uted to a loss of junctional proteins through changes in inflammatory mediators and MMP/TIMP levels, resulting in increased leukocyte extravasation into the brain parenchyma. We further investigated therapeutic avenues to reduce and re- store the BBB damage caused by HFHS-feeding. Pharmacological treatment with recombinant annexin A1 (hrANXA1) or reversion from a high-fat high-sugar diet to a control chow diet (dietary intervention), attenuated T2DM development, re- duced inflammation, and restored BBB integrity in the animals. Given the rising incidence of diabetes worldwide, understanding metabolic-disease-associated brain microvessel damage is vital and the proposed therapeutic avenues could help alleviate the burden of these diseases. K E Y W O R D S basal lamina, blood-brain barrier, leukocytes migration, metabolic imbalance, MMPs, neuroinflammation K E Y W O R D S basal lamina, blood-brain barrier, leukocytes migration, metabolic imbalance, MMPs, neuroinflammation Funding information FASEB J. 2022;36:e22107. \| 1 of 27 https://doi.org/10.1096/fj.202101297R wileyonlinelibrary.com/journal/fsb2 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology. Abbreviations: ANXA1, annexin A1; FPR, formyl peptide receptor. SHEIKH et al. 2 of 27 2.3 \| Treatment with hrANXA1 and dietary reversion tered an oral bolus of glucose (2 g/kg in dH2O) and for the ITT, mice were administered a dose of insulin (1 unit/kg in PBS, i.p.). Blood glucose was measured via tail vein puncture at time 0, followed by measuring at 15-min intervals for a total duration of 120 min, using a glucometer (Accu-Chek Compact System; Roche Diagnostics, Basel, Switzerland). Non-fasting blood glucose values were obtained using a glucometer im- mediately after cardiac exsanguination. Commercially avail- able ELISA kits were used to measure serum levels of insulin (ThermoFisher Scientific), cholesterol (Abcam, Cambridge, UK), and triglycerides (Abcam, Cambridge, UK). The pharmacological intervention was provided by treating 10-week-old male mice fed a HFHS-fed diet for 10 weeks with hrANXA1. The pharmacological interven- tion was provided from weeks 5 to 10, mice were treated with 33 µg/kg body weight hrANXA1 in 100 µl of 50 mM Hepes, 140 mM NaCl, given i.p/mouse 5 days/week. A dose of 33 µg/kg body weight was chosen based on previ- ous studies.15 The hrANXA1 was produced and purified as previously published.16 Dietary intervention was provided by reverting mice fed a HFHS diet onto a standard chow diet. Ten-week-old male C57BL/6 mice were fed a HFHS diet for 10 weeks, after which mice were placed back on a chow diet for a further 5 weeks, the total dietary period for these mice was 15 weeks (Figure S1). All mice not treated with hrANXA1 were given a vehicle of 100 µl of 50 mM 2.4 Fresh food (150 g/cage) was also supplied at the begin- ning of each week and was regularly monitored through the week for color or consistency change. The diet from each cage was weighed before being changed to measure food (g/mouse/day) and calorie intake (kcal/mouse/day) using the following equations: For oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT), mice were fasted for 6 h. Testing was carried out at weeks 8 or 9, respectively, or weeks 14 or 15 for HFHS—chow reversion mice. For measuring OGTT, mice were adminis- Food intake = weight of diet at beginning of week −weight of diet at end of week (number of mice in cage × number of days) 2.1 \| Animal experiments This study was conducted in accordance to Arrive guide- lines using 10-week-old male, wild-type C57BL/6 mice purchased from Charles River (UK). Mice were housed in temperature (25 ± 2°C) and humidity (55%) controlled rooms. Mice were fed either a normal chow-based diet (5053, LabDiet Ltd) or a high-fat high-sugar diet (58R3, HFHS, AIN-76A TestDiet) for 10 weeks as schematized in Figure S1. All animal experimental procedures performed in this study were approved by the Animal Welfare Ethics Review Board (AWERB) of the Queen Mary University of London-UK. The study was performed under the Procedure Project License; PPL: 70/8350 issued by the home office. Animal welfare and performed protocols were conducted under the guidance of Operation of Animals (Scientific Procedures Act 1986) and the European Directive (2010/63/EU) on the protection of animals used for scientific purposes. All mice had access to food and water ad libitum. Assignment to diet and treatment was performed randomly and no blinding was performed. 2.2 \| Measuring body weight and feeding behavior All mice had free access to food and water, body weight was recorded weekly. The bodyweight of all animals was measured at the same time at the beginning of each week using the same balance for the whole experiment for the determination of body weight gain for measuring obesity. 1 \| BACKGROUND BBB dysfunction has been implicated in a growing number of CNS pathol- ogies including multiple sclerosis,4 Parkinson's disease,5 and Alzheimer's disease.2 Alteration of endothelial tight junctions (TJs) compromises the barrier function; im- pairing permeability, cell polarity, and transport systems and leading to secondary activation of astrocytes6 and microglia.7 Long-term BBB dysfunction can consequently cause neuronal dysfunction, injury, and degeneration.8,9 Neuro-imaging in obese patients has revealed atrophy of the frontal lobes, hippocampus, and the thalamus while post-mortem studies of diabetic patients' brains have SHEIKH et al. 3 of 27 2 \| MATERIALS AND METHODS Hepes, 140 mM NaCl, pH 7.4, daily (5 times/week) i.p. chow-fed, HFHS-fed, and HFHS-fed + hrANXA1 mice were 20 weeks of age at cull. HFHS—chow reversion mice were 25 weeks of age at cull. Animal studies were conducted in tandem for each group n = 55 for chow, HFHS, and HFHS + hrANXA1 mice. With the exception of HFHS—chow reversion mice with an n = 15, however, chow, HFHS, and HFHS + hrANXA1 were also collected alongside the reversion diet mice. In vitro permeability assays 2.9 Briefly, the meninges, white matter, and choroid plexus were removed from extracted brains. The remaining grey matter underwent two stages of enzymatic digestion to degrade the extracellular matrix. First, using Collagenase II (Sigma) and DNase1 (Sigma), followed by density-dependent cen- trifugation in 20% BSA to separate the heavier capillary fragments from the lighter myelin, neurons, and astrocytic components. The second enzymatic digest of microvesseles used collagenase-dispase (Roche), after which microves- sels were plated into Petri dishes coated in 0.2% rat tail col- lagen I (Corning). Cells were initially cultured in DMEM/ F12 medium (ThermoFisher Scientific) supplemented with 15% FBS, 1ng/ml bFGF (Roche), 100 µg heparin (Sigma), 1× insulin-transferrin-selenium (Gibco), 4 µg/ml puromycin (Sigma) to obtain pure monoculture and 100 units/ml gen- tamycin (Gibco). From day 3, cells were kept in 10% FBS, and from day 4, the medium was prepared without puromy- cin. Cultures were maintained at 37°C in 5% CO2 and pas- saged at 75% confluency. Primary brain endothelial cells, bEnd3 cells, or hCMEC/ D3 were grown on Transwell polycarbonate filters (pore size, 0.4 μm; Sigma-Aldrich, UK) coated with calfskin collagen type I followed by bovine plasma fibronectin for seven days. bEnd3 cells were stimulated overnight with 10% mouse serum. hCMEC/D3 cells were stimulated over- night with 20% human serum. Paracellular permeability was assessed using 55–77-kDa FITC-dextran (3 mg/ml) as previously described.19 Transendothelial electrical re- sistance (TEER) measurements were performed using the Epithelial Volt/Ohm (EVOM2) meter (World Precision Instruments, USA) cell-free insert resistance values were subtracted from values obtained in the presence of en- dothelial cells. 2.7 \| hCMEC/D3 culture After permeabilization with 0.5% Triton X-100 in PBS, free-floating sections were incubated with single or com- bined primary antibodies (Table S2) at 4°C ON, appropriate fluorophore-conjugated secondary antibodies (Table S2) for 45 min at room temperature, and counterstained with TO-PRO3™ (diluted 1:10k in PBS; Invitrogen). The sec- tions were collected on polylysine slides (Menzel-Glaser) and cover-slip with Vectashield® mounting medium (Vector Laboratories), and finally sealed with nail varnish. Negative controls were prepared by omitting the primary antibodies and mismatching the secondary antibodies. Sections were examined under a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems) using a sequential scan procedure. Confocal images were taken at 0.35 µm intervals through the x, y, and z axes of the sec- tion, with 40× and 63× oil immersion lenses. Immortalized human cerebrovascular endothelial cell line/clone D3, hCMEC/D3,18 were cultured in EBM-2 me- dium (Lonza) supplemented with 5% FBS and growth factors (hFGF, VEGF, R3-IGF-1, Ascorbic acid, hEGF, and gentamycin), as recommended by the manufacturer. Cultures were maintained at 37°C in 5% CO2 and passaged at 80%–90% confluency. 2.5 \| Primary murine brain endothelial cell culture Primary murine endothelial cells were isolated and cultured from brain capillary fragments as previously described.15 SHEIKH et al. 4 of 27 2.6 \| bEnd3 cell culture The animals were anesthetized using a ketamine/xylazine cocktail, 90 mg and 4.5 mg/kg, respectively, by intraperi- toneal injection and perfused with 2% paraformaldehyde (PFA) and 0.2% glutaraldehyde (GTA) in phosphate- buffered saline (PBS) solution given over 3 min. Whole brains were removed and post-fixed by immersion in the same fixative for 4 h at 4°C, then washed in PBS over- night (ON) at 4°C. Using a vibrating microtome (Leica Microsystems; Milton Keynes, UK), 30-μm sagittal sec- tions, evenly spaced at 200 µm intervals, were cut from each hemisphere. The sections were stored in 0.02% PFA in PBS at 4°C as free-floating sections. Immortalized mouse brain-derived endothelial cells, bEnd.317 were cultured in DMEM 1g/L D-glucose cul- ture medium (Gibco) supplemented with 10% FBS, 4 mM Glutamax (Gibco), 100 units/ml Gentamycin (Gibco), 50 µM β-Mercaptoethanol (Gibco), 1 mM Na-Pyruvate (Sigma-Aldrich), and 1× non-essential amino acids (Gibco). Cultures were maintained at 37°C in 5% CO2 and passaged at 80%–90% confluency. 2.14 The Mouse Cytokine Array XL Kit or Proteome Profiler Human XL Cytokine Array Kit (R&D Systems, Minneapolis, USA) was used to analyze different cytokines, interleu- kins, chemokines, and acute-phase proteins in mouse brain microvessels or hCMEC/D3 cell lysates stimulated with 20% human serum. The Human MMP Antibody array (Abcam) was used to analyze MMPs and TIMPs in human serum or hCMEC/D3 cell lysates stimulated with 20% human serum. These kits are membrane-based sand- wich immunoassays. Briefly, membranes were blocked with blocked buffer for 1 h followed by overnight incuba- tion with samples at 2–8°C. Membranes were incubated with a detection antibody cocktail. Bound antibodies were detected using Streptavidin-HRP and Chemi Reagent mix. Image Studio™ Lite (LI-COR Biosciences, USA) software was used to analyze the data. Mouse serum TIMP-1 lev- els were measured using a Quantikine ELISA kit (R&D Systems). Human serum IL-6 and TNF-α were measured 2.13 \| Zymography Gelatin zymography was used for the detection of gelati- nases MMP-1, MMP-2, and MMP-9 in brain microvessels. For each sample, 1 µg/lane was loaded for separation by non-reducing gel electrophoresis on a 7.5% acrylamide gel containing 1 mg/ml porcine skin gelatin (Sigma). Following electrophoresis, the gel was washed exten- sively with 50-mM Tris-HCL containing 2.5% Triton X- 100, 5-mM CaCl2, and 1-µM ZnCl2 to remove SDS and incubated overnight in activation buffer (50-mM Tris- HCl containing 1% Triton X-100, 5-mM CaCl2, and 1-µM ZnCl2) at 37°C. After incubation, the gel was stained in Coomassie brilliant blue, MMPs were detected as clear bands against a blue background of the undegraded sub- strate. Images were acquired with a ChemidocMP imag- ing system (Bio-Rad) and analyzed using ImageJ (NIH, USA). 2.8 In vivo permeability was assessed using Evans blue dye; 100 µl of 2% Evans blue dye (4 ml/kg) in 0.9% saline was injected intravenously into the tail vein.15 After 1 h, mice were sacrificed. Brain hemispheres were collected, mac- erated, and homogenized; samples were analyzed spec- trophotometrically, normalized to tissue weight, and expressed as a percentage of serum dye content. Quantification of fluorescence intensity was performed at 63× magnification on the mouse (n = 4 for each exper- imental group) and human (n = 6; T2DM n = 3, controls n = 3) samples (35 sections/sample, 10 randomly selected SHEIKH et al. 5 of 27 allow confluent monolayers to form. On the day of the assay, 600 µl of complete medium was added to the lower compartment of the transwells. Isolated and expanded mouse lymphocytes were added to the top compartment (1 × 106 cells/transwells) and incubated for 4 h at 37°C in 5% CO2. After 4 h, inserts were removed, and the en- tire volume of the lower compartment was collected to assess the migrated lymphocyte population. Lymphocytes adhered to monolayers were collected using 0.2% trypsin and served as the adhered population. Cells were stained and analyzed via flow cytometry. fields/section) using Cell F as image analysis software (Olympus Italia; Rozzano, Italy). Data plotting and sta- tistical analysis were performed on GraphPad. Statistical differences were evaluated using the Student's t-test. The values were expressed as mean ± SD. 2.11 \| Flow cytometry All samples were acquired by flow cytometry using a LSR Fortessa (BD Biosciences) and analyzed with FlowJo version 10. All antibodies are Biolegend unless other- wise stated. Once all staining was conducted, cell pellets were re-suspended in 200 µl of PBS+/+ ready for FACS analysis; tubes were stored at 4°C. Primary endothelial cells or lymphocytes were collected from experimental mice groups. For mouse sera samples, bEnd3 cells were stimulated overnight with 10% mouse serum. Cells were harvested, fixed in 2% PFA, and stained in FACS buffer. For cell surface staining of junctional proteins and ad- hesion molecules, the following antibodies were used: Occludin (ThermoFisher Scientific) with secondary an- tibody goat anti-mouse IgG-AF488 (Life Technologies), PECAM-1 AF488 (eBioscience), VE-Cadherin (Abcam) with secondary antibody goat anti-rabbit IgG-Cy3 (Abcam), ICAM-1-APC, VCAM-1-Pacific blue, and CD86-PE-Cy7. For mouse migration studies, the fol- lowing antibodies were used: CD45-APC-Cy7, CD4- PE-Cy7. Intracellular staining for G/F actin required permeabilization using the Mouse Foxp3 buffer kit (BD Pharmingen). Actin ratio was determined by staining cells with DNase1 AF488 (ThermoFisher) which recog- nized G-actin and with Phalloidin-568 (ThermoFisher) which recognized F-actin, the ratio of the MFI deter- mined the content of globular:fibrillar (G:F) actin ratio. For human samples, hCMEC/D3 cells were stimulated with HD or T2DM sera overnight, after which cells were harvested, fixed in 2% PFA, and stained using FACS buffer. In cell surface staining of junctional proteins and adhesion molecules, the following antibodies were used: Occludin (ThermoFisher Scientific) with secondary anti- body goat anti-mouse IgG-AF405, PECAM-1 AF488 (eBi- oscience), and VE-Cadherin (Abcam) with secondary antibody goat anti-rabbit IgG-Cy3 (Abcam) and ICAM- 1-APC (eBioscience). 2.12 \| Transmigration assay Polycarbonate transwell inserts (0.33 cm2 surface area, pore size 5 µm; Sigma-Aldrich, UK) were coated with laminin (50 µg/ml, Sigma). Cells were seeded on the top compartment of the transwells and cultured for 72 h to SHEIKH et al. 6 of 27 2.15 \| Human studies Blood samples were drawn from “healthy donors” (HD) and patients with T2DM of the population-based epidemiological “PLIC” (Progressione delle Lesioni Intimali Carotidee) study. Subjects gave their consent for participating in the study in accordance with the ethical approval (SEFAP/Pr0003F University of Milan 06/2/2001).20 Subjects were followed at the Societa Italiana per lo studio dell'aterosclerosi (SISA), Centre for the Study of Atherosclerosis, Bassini Hospital, Milan, Italy. All patients and healthy volunteers gave written informed consent in adherence to the Declaration of Helsinki. Complete information about clinical history was available from outpatient's registries and/oral hos- pital archives. HD were selected based on the follow- ing inclusion criteria: No personal and familial history of T2DM, no renal damage [urinary creatinine/albumin ratio as an average of three different measurements was collected to check for this parameter (not shown)] and absence of hepatic steatosis (ultrasound-based as previ- ously described).21 T2DM diagnosis was defined follow- ing the international guidelines,22 information about the disease duration as well as information about pharmaco- logical treatments, and regimes were gathered from the outpatients' registries. 2.17 \| Human brain immunohistochemistry using a multiplex Human Luminex Assay (R&D Systems) on the Luminex MAGPIX System (Luminex Corporation). using a multiplex Human Luminex Assay (R&D Systems) on the Luminex MAGPIX System (Luminex Corporation). For human brain post-mortem studies, the sampling and handling of the specimens were conformed to the ethical rules of the Department of Emergency and Organ Transplantation, Division of Pathology, University of Bari School of Medicine, Italy. The study was reviewed and approved by the Medical Ethics Committee of University Hospital of Bari, Italy in compliance with the principles stated in the Declaration of Helsinki. Human brain sam- ples (n = 6, subdivided into two groups: 3 with a known diagnosis of T2DM and 3 controls without a medical his- tory of relevant disorders) were taken during autopsy re- quired for legal purposes. Once arrived at the mortuary, the cadaver was analyzed and the anatomic-chronological parameters were reported, with a particular interest in the temperature, and a diary was compiled with the temper- ature at which the cadaver stayed, to ensure a uniform, maximized antigenicity among the samples selected ac- cording to routine histopathological evaluation. During the autopsy, the skull was generally the first body district to be analyzed for autopsy. The whole brain was removed, and two samples were taken from frontal and parietal lobes, which included both the cerebral cortex and the subcortical white matter. The samples were immediately fixed in 2% PFA and 0.2% GTA in PBS for 48 h at 4° C, washed in PBS overnight at 4°C, and then submitted to the same histological procedure as described for mouse brains. 2.16 Blood samples were drawn after overnight fasting (10 h at least) from antecubital vein and collected in EDTA tubes (BD Vacuette®). Blood samples were then centrifuged at 3000 rpm for 12 min in order to separate plasma for glu- cose quantification. Determination of lipid profile, glucose levels, and liver enzymes was performed by an enzymatic method (hexokinase reaction) through automatic sample analyzer (RX Daytona, Randox Laboratories Ltd®, Crumlin, UK). Low-density lipoprotein cholesterol (LDL-C) was de- rived from the Friedewald formula.23 Fatty liver index (FLI) was calculated from body mass index (BMI), waist circum- ference, gamma-glutamyltransferase (GGT), and triglycer- ide levels in the fasting condition.24 Patient characteristics are reported in Table S1. Serum was isolated from whole blood in serum tubes (Vacutainer) and stored at −80°C. The serum used for all biological tests was decomplemented at 56°C for 20 min. Twenty percent serum was used to stimulate hCMEC/D3 cells overnight prior to conducting experiments. All data are presented as mean ± standard error of mean (SEM) of n observations, where n denotes the number of animals studied and/or repeats. All statistical analysis was conducted using GraphPad Prism 8 (GraphPad Software, San Diego, California, USA). All data were tested for normality and analyzed by a Student's t-test or one-way ANOVA for multiple comparisons with post hoc analysis using Bonferroni's post hoc test. Results were considered significant at p < .05. 3 \| RESULTS We, therefore, investigated the potential of either a pharmacological intervention or a dietary inter- vention to reduce, repair, and restore the damage incurred at the BBB by T2DM. The pharmacological intervention was provided by hrANXA1, whereby mice were fed a HFHS diet for 10 weeks along with hrANXA1 treatment (0.35 µg/kg body weight, i.p. 5 times/week) from week 5 of the diet for 6 weeks (HFHS + hrANXA1 treated mice, Figure S1B). We have previously shown that the anti-inflammatory molecule annexin A1 (ANXA1) is an important regulator of TJ tightness15 and that prophylac- tic hrANXA1 treatment in the same diet-driven animal model of T2DM (HFHS-diet) prevented the development of peripheral microvascular complications (nephropathy and liver hepatosteatosis) through the restoration of insu- lin sensitivity.16 Alternatively, the dietary intervention was provided by reverting mice that had been fed a HFHS diet for 10 weeks back on to a normal chow diet for a further 5 weeks (HFHS—chow reversion mice, Figure S1C). In summary, it was seen that the HFHS-diet induced obesity and contributed to the development of MetS as noted by hyperglycemia, hyperinsulinemia, hypercho- lesterolemia, and hypertriglyceridemia. Generally, while both pharmacological and dietary interventions attenu- ate MetS development, treatment with hrANXA1 showed greater improvement across the majority of measures when compared to HFHS-fed mice, with a particular ef- fect on improving serum levels and glucose tolerance. Furthermore, results showed the levels/concentrations in HFHS-fed + hrANXA1-treated mice were largely similar to those in chow-fed mice, with the exception of weight gain. The reduction of weight gain, in comparison, was the strongest indicator of improvement in the HFHS— chow reversion diet group suggest that diet has an import- ant impact on obesity. Confirmation of metabolic disruption (obesity, hyper- glycemia, dyslipidemia, and insulin resistance) in this mouse model was conducted by measurements of weight gain, fat mass (epididymal and inguinal), fasting and non- fasting blood glucose, OGTT, and ITT as well as serum in- sulin, cholesterol, and triglyceride levels (Figure 1). At the end of the 10-week feeding period, mice fed a HFHS diet presented with significantly increased body weight and fat mass when compared to chow-fed mice (Figure 1A–D). Both pharmacological and dietary interventions signifi- cantly reduced the overall weight gain and epididymal fat mass compared to HFHS-fed mice, which was more prominent in the HFHS—chow diet reversion group; no changes were seen in the inguinal fat mass (Figure 1A–D). 3 \| RESULTS A number of different animals models exist for the inves- tigation of T2DM pathophysiology,25 some of which have demonstrated leakage of the BBB under a diabetic state.26 Here, we used our established mouse model of T2DM in- duced by feeding a HFHS diet for 10 weeks (Figure S1A)16 to investigate the effect of metabolic overload on the SHEIKH et al. 7 of 27 BBB. Diet-induced diabetic models work through caus- ing obesity from imbalanced food intake and low energy expenditure.25 Obesity is the single biggest risk factor for developing T2DM, through raising the levels of fatty acids, glucose, pro-inflammatory markers thereby alter- ing hormones and metabolism due to the accumulation of fat deposition, leading to the development of insulin resistance.27 The diet-induced models of diabetes are used to replicate the westernized lifestyle in humans, with in- creased intake of foods dense in fats and sugars along with a sedentary lifestyle. This was the rationale for using a diet that was high in both fats and sugars. compared to HFHS-fed diet mice, however, no changes were seen in the fasted blood glucose levels, with con- centrations significantly higher across all HFHS-fed groups when compared to chow-fed mice (Figure 1E,H). Insulin resistance was confirmed using OGTT and ITT; measurement of the area under the curve (AUC) in both tests found HFHS-fed mice to have an impaired insulin response compared to chow-fed mice, although this was not significant for the ITT (Figure 1F,G,I,J). While HFHS- fed + hrANXA1-treated mice had improved glucose toler- ance when compared to HFHS-fed mice, no changes were seen in HFHS-chow reversion mice (Figure 1F,G). While little changes were witnessed with the ITT, serum insulin levels were significantly elevated in HFHS-fed mice com- pared to chow-fed mice, that were reduced with both in- terventions, however, this was only statistically significant for HFHS-fed + hrANXA1 mice (Figure 1K). HFHS-fed mice were also found to have hyperlipidemia, as seen by elevated serum cholesterol and triglyceride levels com- pared to chow-fed mice that were significantly reduced with hrANXA1 treatment; HFHS—chow reversion mice showed trends toward improved lipid levels (Figure 1L,M). The rising incidence of obesity and T2DM and its asso- ciated macro- and micro-vascular complications requires the need for appropriate management of the disease that can be achieved through medical management or life- style changes. 3 \| RESULTS (F–I) Oral glucose tolerance test (OGTT, 15–38/group) or Insulin tolerance test (ITT, n = 6–15/group) was assessed over 120 min, after 6 h of fasting at week 8 or week 9, respectively. (G, J) The area under the curve (AUC) of OGTT and ITT was calculated and used for statistical analysis. (K) Serum insulin (n = 8–27/group), (L) cholesterol (n = 9–23/ group), and (M) triglycerides (n = 9–23/group) levels were measured in serum isolated from whole blood at harvest. Statistical analysis was performed by one-way ANOVA followed by a Bonferroni post hoc test. Data are expressed as mean ± SEM. p < .05, p < .01, p < .001, *p < .0001 versus chow; $p < .05, $$p < .01, $$$p < .001, $$$$p < .0001 versus HFHS. #p < .05, ####p < .0001 versus HFHS + hrANXA1 cells from HFHS-fed mice when compared to chow-fed mice (Figure 2B), concomitant with a decreased TEER (Figure 2C). Similar to in vivo BBB restoration, treatment with hrANXA1 in HFHS-fed mice or reversion to a chow diet restored BBB functionality in vitro through a reduc- tion in paracellular permeability and improved TEER when compared to HFHS-fed mice (Figure 2B,C). clear co-localization of laminin α2 at the astrocyte GFAP+ end-feet in chow-fed mice, suggestive of astrocytic end- feet embracing the microvessel structure to form an intact vessel, which is hardly distinguishable in HFHS-fed mice, where hypertrophic perivascular astrocytes showed a very low laminin α2 staining and detachment from the vessel structure (Figure 2H). Disrupted TJ structure induced by HFHS-feeding was restored upon hrANXA1 treatment as seen by reinstated levels of occludin and claudin-5, similar in staining pat- tern to those observed in chow-fed mice (Figure 2D,E). Moreover, treatment with hrANXA1 in HFHS-fed mice showed restoration of laminin subunits α2 and α4, when compared to HFHS diet-fed mice (Figure 1F,G) that al- lowed for reinstatement of co-localized laminin α2 with GFAP+ astrocyte perivascular end-feet to reform an intact- looking BBB (Figure 2H). In contrast, the reversion diet lacked any effect on BBB structure when compared to HFHS-fed mice, with no changes observed in claudin-5, occludin, endothelial laminin α4, or astrocytic laminin α2 staining, preventing reattachment of GFAP+ astrocytic end-feet to the vessel structure (Figure 2D–H). 3 \| RESULTS Tests for hyperglycemia found raised non-fasted in HFHS- fed mice when compared to chow-fed mice (Figure 1E). Treatment with hrANXA1 and reversion diet signifi- cantly reduced the non-fasted blood glucose levels when Following confirmation that HFHS-feeding causes metabolic disruption, we further determined that these mice exhibit brain microvascular disruption characterized by increased Evans blue dye leakage into the brain when compared to mice fed a standard chow diet (Figure 2A). Having shown that both treatments with hrANXA1 and dietary reversion could improve HFHS-induced met- abolic abnormalities, we investigated whether beneficial effects could be detected at the BBB. Indeed, both treat- ments improved BBB functionality as seen by a reduction in Evans blue dye leakage (Figure 2A). Further measure- ments of BBB integrity assessment were conducted in vitro using primary murine brain endothelial cells seeded on transwell inserts. Increased paracellular permeability to 55-77DA FITC-dextran tracer was seen in endothelial 27 \| SHEIK (A) (C) (E) (H) (K) (L) (M) (I) (J) (F) (G) (D) (B) SHEIKH et al. 8 of 27 (A) (A) (B) (A) (B) (C) (D) (C) (D) (E) (F) (G) (F) (G) (F) (G) (E) (G) (F) (E) (H) (I) (J) (J) (H) (I) (J) (K) (L) (M) (L) (M) (L) (K) (M) 9 of 27 SHEIKH et al. \| FIGURE 1 Treatment with hrANXA1 and a reversion diet attenuates the development of T2DM. C57BL/6 mice were fed either a standard diet (chow) or a high-fat high-sugar diet (HFHS) for 10 weeks. The pharmacological intervention was given by treating with human recombinant (hr) ANXA1 (1 μg/100 μl, i.p.) five times/week from weeks 5 to 10 (n = 55). Dietary intervention was provided by placing mice on a HFHS for 10 weeks, followed by a chow diet for 5 weeks (HFHS—chow reversion; n = 15). (A, B) Body weight gain of experimental mice groups over the 10- or 15-week diet period (n = 15–55/group). (C, D) Measurement of visceral fat (epididymal and inguinal) deposits as a percentage of overall body weight after 10 weeks of diet, as a measure of obesity (n = 15–55/group). (E) Basal non- fasted blood glucose was measured after 10 weeks of diet, one hour prior to harvest (n = 15–44/group). (H) Basal fasted blood glucose was measured after 6 h of fasting at week 8 (n = 15–39/group). 3.1 \| Changes to tight junction and vessel basil lamina components Structural BBB integrity depends on TJ effectiveness, therefore, we conducted immunohistochemistry on cor- tical mouse brain sections staining for TJ proteins clau- din-5 and occludin alongside the VBL content of laminin and its subunits α2 and α4. Significant reductions in both claudin-5 and occludin staining patterns were observed in the cerebral cortex of HFHS-fed mice, together with a reduced pan-laminin reactivity, when compared to chow- fed mice (Figure 2D,E). In HFHS-fed mice, the linear and continuous staining pattern, typical of structurally preserved TJ strands, appeared thinner, interrupted for both claudin-5, and occludin (Figure 2D,E). Endothelial cells and perivascular astrocytes contribute to provide a highly restrictive and regulated interface at the BBB via the expression of VBL molecules of basement membranes (BMs).28 Accordingly, we double immunostaining for pan-laminin/endothelial laminin α4 and laminin α4/as- trocytic laminin α2. We observed a significant reduction of laminin molecules in HFHS-fed mice when compared to chow-fed mice (Figure 2F–H), with a relatively higher reduction of laminin α2 (Figure 2G). In chow-fed mice, double laminin α4/laminin α2 immunostaining clearly showed the different distribution of laminin subunits,28,29 with laminin α2 primarily present in the astrocyte VBL layer (outer vessel surface) vs laminin α4 mainly restricted to the endothelial VBL layer (inner vessel surface), that is absent in HFHS-fed mice. Further double immunostaining with the astroglia marker GFAP and laminin α2 showed a 3.2 \| The balance of MMPs and TIMPs Quantification of staining shown as mean fluorescence intensity (arbitrary units) of markers (n = 4 mice/group, 35 section/animal, 10 randomly selected fields/sections). Statistical analysis was performed by one-way ANOVA followed by a Bonferroni post hoc test. Data are expressed as mean ± SEM. p < .05, p < .01, p < .001, **p < .0001 versus chow; $p < .05, $$p < .01, $$$p < .001 versus HFHS; #p < .05, ###p < .001 vs HFHS + hrANXA1 significantly reduced expression of MMP-9 (Figure 3A) and reduced activity of MMP-1, MMP-2, and MMP-9 (Figure 3B–E) in brain microvessels when compared to HFHS-fed mice, although statistical significance was not achieved when analyzing activity. Interestingly, despite the lack of BBB restoration, HFHS—chow reversion mice showed a significant reduction of MMP-1, MMP-2, and MMP-9 activity via the zymogram along with reduced ex- pression of MMP-9 in the brain microvessels, when com- pared to HFHS-fed mice, levels of proteolytic activity in the reversion mice were similar to those seen in chow-fed mice (Figure 3A–E). Analysis of isolated brain microvessel also revealed a disturbance in the levels of cytokines, chemok- ines, and interleukins in HFHS-fed mice compared to chow-fed mice (Figures 4B–E and S2), indicative of a raised inflammatory state at the BBB. Microvessels from HFHS-fed + hrANXA1-treated animals showed a general suppression of most inflammatory factors, a slight increase in both neutrophil/monocyte- and B/T-lymphocyte- directed chemokines but more lim- ited effects on pro- and anti-inflammatory cytokines (Figures 4B–E and S2). HFHS—chow reversion diet mice showed greater changes with suppression in in- flammatory factors, pro- and anti-inflammatory cyto- kines, and neutrophil/monocyte-directed chemokines, albeit with only minor effects on B/T-lymphocyte di- rected chemokines (Figures 4B–E and S2). In summary, both pharmacological and dietary intervention had gen- eralized anti-inflammatory effects, with diet reversion being the more potent of the two, perhaps accounting for the greater reduction in MMP activity seen with dietary reversion vs hrANXA1 treatment. MMP-2 and MMP-9 promoter regions contain transcription factor binding sites such as AP-1 and NF-kB34 that are regulated by the activation of signaling pathways such as ERK 1/2, JNK, and p38 MAP kinases (MAPK).35–38 To investigate whether enhanced inflammatory mediator presence in HFHS-fed mice could thus contribute to the observed MMP upregulation/activation at the brain microves- sels, western blot analysis of immortalized bEnd3 cells stimulated with sera from mice fed a chow or HFHS diet was conducted. 3.2 \| The balance of MMPs and TIMPs The role of matrix metalloproteases (MMPs) in TJ and VBL degradation is well established and could account for the observed loss of BBB integrity seen in HFHS-fed mice. Analysis of brain microvessels using a membrane- based sandwich immunoassay showed significantly in- creased levels of MMP-2 and MMP-9 in brain microvessels extracts of HFHS-fed mice when compared to chow-fed mice (Figure 3A). Confirmation of MMP proteolytic ac- tivity was conducted via zymography, where there was a significant increase in pro- and active MMP-2 and MMP-9 forms in HFHS-fed mice compared to chow-fed mice (Figure 3C–E). Restoration of BBB integrity in the HFHS-fed + hrANXA1 mice could, therefore, be accounted by the 10 of 27 \| (A) SHEIKH et al. 10 of 27 \| SHEIKH e (A) (D) (E) (F) (G) (H) (B) (C) 10 of 27 \| SHEIKH et (A) (D) (E) (F) (B) (C) (B) (C) (B) (C) (D) (D) (E) (E) (F) (F) (G) (H) (G) (G) (H) 11 of 27 SHEIKH et al. FIGURE 2 HFHS-feeding induces vascular leakage of the BBB due to loss of tight junction proteins and vessel basal lamina of endothelial cells and astrocytes, which is restored by pharmacological treatment with hrANXA1. C57BL/6 mice were fed either a standard diet (chow) or a high-fat high-sugar diet (HFHS) for 10 weeks. The pharmacological intervention was given by treating with human recombinant (hr) ANXA1 (1 μg/100 μl, i.p.) five times/week from weeks 5 to 10 (HFHS + hrANXA1). Dietary intervention was provided by placing mice on a HFHS for 10 weeks, followed by a chow diet for 5 weeks (HFHS—chow reversion). (A) In vivo assessment of BBB paracellular permeability via Evans blue dye extravasation (n = 6/group). (B) Measurement of paracellular permeability coefficients and (C) transendothelial electrical resistance (TEER) in primary mouse brain microvascular endothelial cells cultured from experimental mice groups using 70 kDA FITC-dextran and the Epithelial Volt/Ohm (EVOM2) Meter (World Precision Instruments, USA), respectively (n = 10 pooled/group, two technical replicates, performed as two-three independent experiments total n = 20–30). Confocal microscopy of cerebral cortical sections of mouse brains double immune-labeled with (D) claudin-5/pan-laminin, (E) occludin/pan-laminin, (F) endothelial laminin α4/pan-laminin, (G) astrocytic laminin α2/pan-laminin, and (H) astrocytic laminin α2/astrocytic marker glial fibrillary acidic protein (GFAP). Nuclei were labelled with TO-PRO3. Scale bar: 25 μm. 3.2 \| The balance of MMPs and TIMPs Results identified Akt, SAPK/JNK, and p38 MAPK as potential signal transduction pathways re- sponsible for MMP-mediated endothelial TJ dismantling and laminin degradation (Figure S3A–C), with all three proteins showing enhanced phosphorylation in brain endothelial cells, stimulated with sera from HFHS-fed mice. Inhibition of MMPs is mediated by the tissue inhibitor of metalloproteinases (TIMPs); through their binding to MMP domains.30,31 Measurement of TIMP-1 in the sera of the mice revealed significantly reduced expression of TIMP-1 in HFHS-fed mice compared to chow-fed mice (Figure 3F). Both hrANXA1 treatment and dietary rever- sion increased serum levels of TIMP-1, albeit not to the levels seen in chow-fed mice and this effect only reached statistical significance in HFHS-fed + hrANXA1-treated mice (Figure 3F), suggesting that perhaps the balance of MMP/TIMPs is crucial in BBB remodeling. On the contrary, MMP activity is regulated transcrip- tionally by reactive oxygen species (ROS), cytokines, growth factors, and hormones32–34; consequently, we mea- sured such factors in the sera and brain microvessels of our mice (Figure 4). These measurements showed increased CXCL12, IL-1α, and sICAM-1 expression with decreased IL-13 expression in sera of HFHS-fed mice compared to chow-fed mice (Figure 4A). Within the serum, treatment of HFHS-fed mice with hrANXA1 significantly reduced the levels of the majority of pro-inflammatory species investigated, including CXCL1, CXCL12, IL-1α, IL-16, s-ICAM-1, and TNF-α (Figures 4A and S2), although there was not a corresponding increase in anti-inflammatory markers such as IL-13. 12 of 27 \| SHEIKH et al. 003 003 003 $YHUDJH 3L[HO 'HQVLW\ $8 &KRZ +)+6 +)+6 KU$1;$ +)+6 &KRZ UHYHUVLRQ QWUDWLRQ (A) (B) &KRZ +)+6 +)+6 KU$1;$ +)+6 UHYHUVLRQ 003 003 003 (C) (E) (D) 3UR003 003 $YHUDJH 3L[HO 'HQVLW\ $8 3UR003 003 $YHUDJH 3L[HO 'HQVLW\ $8 &KRZ +)+6 0: 3UR003 003 3UR003 003 +)+6 KU$1;$ +)+6± &KRZ UHYHUVLRQ WƌŽͲDDWϭ DDWϭ 3UR003 003 $YHUDJH 3L[HO 'HQVLW\ $8 (F) SHEIKH et al. 12 of 27 \| SHEIKH et al. 3.2 \| The balance of MMPs and TIMPs Statistical analysis was performed by one-way ANOVA followed by a Bonferroni post hoc test. Data are expressed as mean ± SEM. p < .05, p < .01, p < .001. **p < .0001 versus chow; $p < .05, $$p < .01, $$$p < .001 versus HFHS; #p < .05, ##p < .01, ###p < .001 vs HFHS + hrANXA1 expression of both the adhesion molecules, whereas ex- pression was significantly increased in HFHS-fed mice (Figure 5A,B). Treatment with hrANXA1 in HFHS-fed mice and HFHS—chow reversion diet mice showed re- duced ICAM-1 and P-selectin expression, although this was only significant in HFHS-fed + hrANXA1 mice and the expression remained significantly higher than in chow-fed mice (Figure 5A,B). Further analysis of primary- isolated brain microvessels endothelial cells from chow- and HFHS-fed mice confirmed upregulated expression of adhesion molecules ICAM-1, P-selection, and VCAM-1 in HFHS-fed mice (Figure 5C); no changes were observed in the expression of E-selectin and co-stimulatory mol- ecule CD40 (Figure 5C). Both interventions significantly reduced ICAM-1 and VCAM-1 expression, when com- pared to HFHS-fed mice (Figure 4C); CD40, E-selectin, and P-selectin reduction in HFHS—chow reversion diet- fed mice cerebral microvessels were not significant when compared to HFHS-fed mice (Figure 5C). Further ex vivo histological analysis in mice brains, showed a threefold increase in inflammation-associated inducible nitric oxide synthase (iNOS) staining, primar- ily localized on the cortex microvessels, in HFHS-fed mice when compared to chow-fed mice (Figure 4F). Both pharmacological and dietary intervention reduced iNOS staining, indicating a role for circulating inflam- matory mediators in contributing to the damage of the BBB. The impact of enhanced inflammatory mediator pres- ence (locally and in the blood circulation) and the higher expression of iNOS was investigated at the microglia level. Microglia cells sense brain parenchyma alterations and are activated by circulating pro-inflammatory factors and hormones; and, therefore, a leaky BBB may also af- fect their functionality.39 Confocal microscopy of brain sections stained for Iba1, a microglia activation marker, showed activated microglia of the HFHS-fed mice when compared to chow-fed mice (Figure 4G), suggesting in- flammation and/or injury occurring within the brain. HFHS-fed + hrANXA1 mice and HFHS—chow reversion diet mice both presented with significantly reduced Iba1 immunostaining compared to HFHS-fed mice; notably, HFHS—chow reversion mice still had constitutively ac- tivated Iba1+ microglia in comparison to chow-fed and HFHS-fed + hrANXA1-treated mice (Figure 4G). 3.2 \| The balance of MMPs and TIMPs Next, we analyzed whether loss of BBB structure (TJ and astrocytic end-feet), increased inflammatory cyto- kine, and enhanced adhesion molecule expression cor- related with increased immune cell extravasation into the brain parenchyma, as is known to occur in the pe- ripheral system in T2DM. Cerebral cortex immunostain- ing for CD45 and pan-laminin demonstrated that CD45+ leukocytes, which were virtually absent in chow-fed mice were instead detected and relatively numerous and associated with cortex microvessels of HFHS-fed mice. In the latter, CD45+ cells were detectable both within the capillary lumen as well as enclosed in the two layers of the VBL or were seen in the act of moving toward the surrounding parenchyma (Figure 5D). Treatment with hrANXA1 resulted in no or rare CD45+ cells infiltration of the brain parenchyma (Figure 5D). However, diet re- version did not show any alterations in the CD45+ infil- tration when compared to HFHS-fed mice, with confocal imaging showing entrapped CD45+ cells within the VBL layers. 3.2 \| The balance of MMPs and TIMPs 003 003 003 $YHUDJH 3L[HO 'HQVLW\ $8 (A) &KRZ +)+6 +)+6 KU$1;$ +)+6 UHYHUVLRQ 003 003 003 (A) &KRZ +)+6 +)+6 KU$1;$ +)+6 UHYHUVLRQ 003 003 003 (A) 003 003 003 &KRZ +)+6 +)+6 KU$1;$ +)+6 &KRZ UHYHUVLRQ &KRZ +)+6 +)+6 KU$1;$ +)+6 &KRZ UHYHUVLRQ 7,03 FRQFHQWUDWLRQ SJPO (B) +) (C) (E) (D) 3UR003 003 $YHUDJH 3L[HO 'HQVLW\ $8 3UR003 003 $YHUDJH 3L[HO 'HQVLW\ $8 &KRZ +)+6 0: 3UR003 003 3UR003 003 +)+6 KU$1;$ +)+6± &KRZ UHYHUVLRQ WƌŽͲDDWϭ DDWϭ 3UR003 003 $YHUDJH 3L[HO 'HQVLW\ $8 (F) (B) &KRZ +)+6 0: 3UR003 003 3UR003 003 +)+6 KU$1;$ +)+6± &KRZ UHYHUVLRQ WƌŽͲDDWϭ DDWϭ (C) 3UR003 003 $YHUDJH 3L[HO 'HQVLW\ $8 (B) (C) (E) (D) 3UR003 003 3UR003 003 $YHUDJH 3L[HO 'HQVLW\ $8 3UR003 003 $YHUDJH 3L[HO 'HQVLW\ $8 (E) 3UR003 003 $YHUDJH 3L[HO 'HQVLW\ $8 (D) (E) &KRZ +)+6 +)+6 KU$1;$ +)+6 &KRZ UHYHUVLRQ 7,03 FRQFHQWUDWLRQ SJPO (F) &KRZ +)+6 +)+6 KU$1;$ +)+6 &KRZ UHYHUVLRQ 7,03 FRQFHQWUDWLRQ SJPO (F) (F) SHEIKH et al. 13 of 27 FIGURE 3 Increased MMP activity and loss of MMP/TIMP balance in HFHS-fed mice are responsible for the loss of the BBB structure. hrANXA1 treatment and dietary intervention reduce MMP activity at the BBB. (A) Expression of MMP-2, MMP-3, and MMP-9 was analyzed in brain microvessel extracts of chow-fed, HFHS-fed, HFHS-fed + hrANXA1-treated and HFHS—chow reversion diet mice using a Mouse Proteome Profiler, expressed as arbitrary units. (n = 10 pooled/group, two technical replicates per experiment, performed in duplicate total n = 20/group). (B) Proteolytic activity of MMP-2 and MMP-9 in brain microvessel extracts was determined via zymography, and (C–E) quantified panel (n = 5 pooled/group, representative gel of three independent experiments total n = 15), and (F) serum levels of tissue inhibitor of metalloproteinases (TIMP-1) were measured using a quantikine ELISA kit (n = 11/group). 3.4 \| Metabolic overload-induced inflammation is a key driver in the loss of BBB functionality Macro- and micro-vascular complications are attribut- able to endothelial dysfunction occurring as a result of the low-grade chronic inflammation induced by hyperglyce- mia, dyslipidemia, and hypertensive state. We showed in Figure 4 a raise in inflammatory mediators both within the serum and at the brain microvessels in HFHS-fed mice, we, therefore, hypothesized that systemic circulat- ing pro-inflammatory factors could contribute to the im- paired BBB state observed. We investigated the effect of sera on an in vitro model of the BBB, using bEnd3 cells, and first observed an increased production of NO by cells stimulated with HFHS-fed sera compared to cells stimu- lated with chow-fed mice serum (Figure 6A). Serum from HFHS-fed + hrANXA1-treated and HFHS—chow rever- sion diet mice both produced lower levels of NO when compared to HFHS-fed mice, although this was only sig- nificant for dietary reversion (Figure 6A). Measurement of parameters of permeability and TEER (Figure 6B,C) 3.3 \| A leaky BBB phenotype correlated with increased transmigration of leukocytes into the brain parenchyma p < .05, p < .01, p < .001 versus chow; $p < .05, $$p < .01 versus HFHS, #p < .05, ##p < .01, ###p < .001 versus HFHS + hrANXA1 showed disrupted BBB integrity from HFHS-fed mice serum when compared to chow-fed mice serum that was corrected with serum from HFHS-fed + hrANXA1 treated and HFHS—chow reversion mice. The in vitro results cor- related with a loss of TJ protein occludin (Figure 6E), ad- herens junction protein VE-cadherin (Figure 6E), and the actin cytoskeleton seen through an increased G:F actin ratio (Figure 6F) in HFHS-fed mice when compared to chow-fed. We have previously shown15 that hrANXA1 is able to restore TJ at the BBB through F-actin stabilization; here, we further confirmed our previous finding and fur- thermore showed that the reverse diet also restored the actin cytoskeleton through changes in the G:F ratio along with restored occludin and VE-cadherin (Figure 6D,E). Likewise, bEnd3 cells stimulated with HFHS-fed mice sera showed an increased presence of adhesion molecule ICAM-1 and co-stimulatory molecule CD86 (Figure 6F) that resulted in increased adherence/migratory capac- ity of CD45+ CD3+ leukocytes across the bEnd3 mon- olayer when compared to chow-fed mice (Figure 6G). Furthermore, reduction of ICAM-1 and CD86 in the inter- vention arms (Figure 6F), correlated with reduced adher- ence/migratory capacity of CD45+ CD3+ leukocytes when compared to HFHS-fed mice (Figure 6G). The results from these in vitro studies are comparable to the in vivo and ex vivo data shown measuring similar parameters, therefore, confirming the role played by metabolic overload-induced inflammation in BBB damage. they were placed in contact for 4 h with an in vitro model of the BBB, using bEnd3 cells plated on transwells to conduct a transmigration assay. The migrated cells rep- resented those, which had crossed the BBB, whereas the adhered cells were equivalent to the immune cells that had arrested on the endothelium but had not crossed the BBB. Both adhesion and migration of CD3+ CD45+ T-cells were greater with lymphocytes isolated from HFHS-fed mice than those from chow-fed animals (Figure 5E) sug- gesting that lymphocytes isolated from HFHS-fed mice have increased migratory activity. hrANXA1 treatment in HFHS-fed mice significantly reduced the number of CD45+ CD3+ adhered and migrated cells when compared to the HFHS-fed mice (Figure 5E). Intriguingly, HFHS— chow reversion diet did not abolish the higher adhered or migratory numbers of CD45+ CD3+ cells (Figure 5E). 3.3 \| A leaky BBB phenotype correlated with increased transmigration of leukocytes into the brain parenchyma Both in vitro and in vivo studies have linked hyperglyce- mia and hyperlipidemia in T2DM to endothelial activa- tion with enhanced expression of activation markers such as ICAM-1.40 We, therefore, examined whether a HFHS- diet exerted similar effects on the BBB and its interaction with immune cells. We first analyzed the expression of ICAM-1 and P-selectin in the cerebral cortex by confo- cal microscopy and immunofluorescence quantification. Major differences in expression of ICAM-1 (Figure 5A) and P-selectin (Figure 5B) were observable with chow- fed mice showing a very low constitutive endothelial Further assessments were performed using in vitro adhesion/migration experiments whereby isolated lym- phocytes from deep cervical lymph nodes were activated and expanded for 4 days using CD3 and CD28, after which 14 of 27 \| SHEIKH et al. (A) (D) (F) (G) (E) (B) (C) SHEIKH et al. 14 of 27 \| (A) 14 of 27 \| SHEIKH et al. (A) (D) (E) (B) (C) (B) (B) (A) (C) (D) (F) (G) (E) (E) (E) (E) (D) (E) (D) (D) (F) (F) (G) (G) SHEIKH et al. 15 of 27 FIGURE 4 Consumption of a HFHS-fed diet induces a low-grade chronic inflammatory state in the circulating serum and at the BBB. Pharmacological and dietary interventions have anti-inflammatory effects. Simultaneous measurement of multiple cytokines, chemokines, interleukins, other inflammatory markers, and metabolic markers in both serum and protein extract from brain microvessels of chow- fed, HFHS-fed, HFHS-fed + hrANXA1 treated, and HFHS—chow reversion diet mice using Mouse Cytokine Array Kits (R&D Systems). Membrane-based immunoassays detected the relative expression levels of analytes detected as arbitrary units. Analytes were measured in (A) serum (n = 10 pooled/group) or in (B–E) brain microvessels (n = 10 pooled/group, two technical replicates per experiment, performed in duplicate total n = 20/group) and categorized as (B) inflammatory factors, (B) chemokines, (D) pro-inflammatory interleukins, (E) anti-inflammatory interleukins. (F) Confocal microscopy of cerebral cortex sections immunostained stained for iNOS, or (G) IBA 1. Nuclei were labeled with TO-PRO3. Scale bar: 25 μm. Quantification of staining shown as mean fluorescence intensity (arbitrary units) of markers (n = 4 mice/group, 35 section/animal, 10 randomly selected fields/sections). Statistical analysis was performed by one-way ANOVA followed by a Bonferroni post hoc test (statistical analysis presented in Figure S3). Data are expressed as mean ± SEM. 3.5 \| Brain vascular leakage and immune cell infiltration correlate with increased MMP in T2DM patients To corroborate whether the observations made in the mouse model can be replicated in the human disease, we investigated post-mortem brains from T2DM patients (de- scribed in Materials and Methods). The autoptic human cerebral cortex was double-stained for VE-cadherin/CD31 (Figure 7A) and pan-laminin/CD45 (Figure 7B). Analysis 16 of 27 \| SHEIKH et al. ,&$0 :7 &+2: :7 +)+6 :7 +)+6$1;$ )OXRUHVFHQFH ,QWHQVLW\ +)+6&KRZUHYHUVLRQ +)+6KU$1;$ +)+6± &+2:UHYHUVLRQ &+2: +)+6 0 $ & , (A) SHEIKH et al. 16 of 27 \| SHEIKH et al. \| DISCUSSION 4 showed a slight but significant reduction of VE-cadherin and CD31 immunostaining in microvessels of T2DM patients, paralleled by laminin reduction in the VBL (Figure 7A,B). Additionally, CD45+ immune cells were more numerous and strongly immunoreactive in T2DM patients compared with healthy controls (Figure 7B). Together, these results parallel the disruption seen to tight junctions and vessel basal lamina components in mice that resulted in leukocyte transmigration into the mouse brain parenchyma (Figures 1 and 4). Endothelial cells serve as a key interface between the blood and organs; therefore, vascular health is a key determi- nant in disease progression. The endothelium of the brain in particular is exquisitely specialized to protect the brain from blood-borne inflammation and ensure brain homeo- stasis. Disruption of the BBB has been closely linked with cognitive impairment in a wide range of conditions, in- cluding Alzheimer's disease,40 Parkinson's disease,5 and T2DM.41 Here, we show that the metabolic overload and associated systemic, low-grade inflammation directly impairs BBB integrity, suggesting that the cerebral vas- culature is an important pathological target, even in the absence of overt disease. Notably, either dietary approach via a diet reversion or a pharmacological approach via ad- ministration of the anti-inflammatory protein hrANXA1 can in part reverse both the endothelial and inflammatory changes induced by a HFHS diet. As our mouse in vitro model showed that systemic cir- culating soluble factors can affect the integrity of the BBB, we conducted a similar investigating using a humanized in vitro BBB model with a human cerebral microvascular endothelial cell line, hCMEC/D3.4 Cells were stimulated with sera from healthy donors (HD) or T2DM patients (Table S1). Stimulation with T2DM sera resulted in a leaky BBB phenotype according to both analyzed parame- ters of paracellular permeability and TEER (Figure 7C,D); disruption to BBB function could be attributed to the de- creased expression of junctional proteins occludin and VE-cadherin (Figure 7E). Additionally, the expression of ICAM-1 was upregulated in cells stimulated with T2DM patient serum compared to HD serum, implicating the potential for the leukocyte transmigration (Figure 7F). Analysis of T2DM patient serum indicated raised lev- els of the pro-inflammatory cytokines, TNF-α and IL-6 (Figure 7G) that could be responsible for the observed change. Furthermore, stimulation of hCMEC/D3 cells with HD or T2DM donor sera resulted in an increase of MMP-9 and MMP-13 expression in T2DM patients ver- sus HD (Figure 7H). 3.5 \| Brain vascular leakage and immune cell infiltration correlate with increased MMP in T2DM patients (C) Relative expression of adhesion molecules E-selectin, ICAM-1, P-selection and VCAM-1 and co-stimulatory molecule CD40 in brain microvessel extracts of chow-fed, HFHS-fed, HFHS-fed + hrANXA1- treated, and HFHS—chow reversion diet mice (n = 10 pooled/group, two technical replicates per experiment, performed in duplicate total n = 20/group) using a membrane-based immunoassay (R&D Systems). (D) Double immune-cortical staining for leukocyte marker CD45 and pan-laminin. Nuclei were labeled with TO-PRO3. Scale bar: 25 μm. Quantification of staining shown as mean fluorescence intensity (arbitrary units) of markers (n = 4 mice/group, 35 section/animal, ten randomly selected fields/sections). (E) Isolated mouse lymphocytes from cervical lymph nodes of chow-fed, HFHS-fed, HFHS-fed + hrANXA1-treated, and HFHS—chow reversion diet-fed mice, were placed in contact on transwells with bEnd3 cells for 4 h (n = 5–9/group). Adhered populations of leukocytes were collected from the transwell, the upper compartment, and migrated cells were collected from the bottom compartment. Flow-activated cell staining was used to evaluate firm adhesion and transmigration of CD3+ T-cells expressing CD45+, plot gate and strategy are shown for both adhesion and migration. Statistical analysis was performed by one-way ANOVA followed by a Bonferroni post hoc test. Data are expressed as mean ± SEM. p < .05, p < .01, p < .001, **p < .0001 versus chow; $p < .01, $$p < .01, $$$p < .001, $$$$p < .0001 versus HFHS; #p < .01, ##p < .01, ###p < .001, ####p < .0001 versus HFHS + hrANXA1 3.5 \| Brain vascular leakage and immune cell infiltration correlate with increased MMP in T2DM patients &' (VHOHFWLQ ,&$0 3VHOHFWLQ 9&$0 $YHUDJH 3L[HO 'HQVLW\ $8 &KRZ +)+6 +)+6 KU$1;$ +)+6 &KRZ UHYHUVLRQ ,&$0 :7 &+2: :7 +)+6 :7 +)+6$1;$ )OXRUHVFHQFH ,QWHQVLW\ +)+6&KRZUHYHUVLRQ +)+6KU$1;$ +)+6± &+2:UHYHUVLRQ &+2: +)+6 0 $ & , 3 1 , 7 & ( / ( 6 +)+6KU$1;$ +)+6± &+2:UHYHUVLRQ &+2: +)+6 36HOHFWLQ &+2: +)+6 +)+6$1;$ )OXRUHVFHQFH ,QWHQVLW\ +)+6&KRZUHYHUVH 3DQODPLQLQ &' 0HDQ ,QWHQVLW\ RI IOXRUHVFHQFH $8 +)+6 KU$1;$ +)+6 &KRZ +)+6 &KRZ UHYHUVLRQ &KRZ +)+6 3$1 1 , 1 , 0 $ / &' +)+6KU$1;$ +)+6± &KRZUHYHUVLRQ 1R RI &' &' FHOOV (A) (B) (C) (D) (E) 3 1 , 7 & ( / ( 6 +)+6KU$1;$ +)+6± &+2:UHYHUVLRQ &+2: +)+6 3 6HOHFWLQ &+2: +)+6 +)+6$1;$ )OXRUHVFHQFH ,QWHQVLW\ +)+6&KRZUHYHUVH (B) (B) &' (VHOHFWLQ ,&$0 3VHOHFWLQ 9&$0 $YHUDJH 3L[HO 'HQVLW\ $8 &KRZ +)+6 +)+6 KU$1;$ +)+6 &KRZ UHYHUVLRQ 3 ( 6 36HOHFWLQ )OXRUHVFH (C) &' (VHOHFWLQ ,&$0 3VHOHFWLQ 9&$0 $YHUDJH 3L[HO 'HQVLW\ $8 &KRZ +)+6 +)+6 KU$1;$ +)+6 &KRZ UHYHUVLRQ (C) (C) +)+6 KU$1;$ +)+6 &KRZ UHYHUVLRQ &KRZ +)+6 3$1 1 , 1 , 0 $ / &' +)+6KU$1;$ +)+6± &KRZUHYH (D) 3DQODPLQLQ &' 0HDQ ,QWHQVLW\ RI IOXRUHVFHQFH $8 +)+6 KU$1;$ +)+6 &KRZ +)+6 &KRZ UHYHUVLRQ (D) $GKHUHG 0LJUDWHG 1R RI &' &' FHOOV &KRZ +)+6 +)+6 KU$1;$ +)+6 &KRZ UHYHUVLRQ (E) (E) SHEIKH et al. 17 of 27 FIGURE 5 HFHS feeding induces enhanced adhesion molecule expression on BBB endothelium correlating to increased transmigration of leukocytes into the brain parenchyma, this is restored with hrANXA1 treatment. Confocal microscopy of cerebral cortex immunostained for endothelial cell adhesion molecules (A) ICAM-1 and (B) P-selectin. \| DISCUSSION The activity of MMPs is further regulated by the presence and action of TIMPs33,34; hence, the main- tenance of efficient BBB function requires a balance be- tween MMP and TIMP expression. In HFHS-fed mice, TIMP-1 activity was significantly reduced in comparison to chow-fed mice, in combination with raised MMP-2 and MMP-9 activity, indicating a potential disruption in the balance and control of MMP activity. A critical component of T2DM is the low-grade chronic inflammation that is characterized by cytokine expression and immune cell infiltration that remains unresolved over time.42 In metabolic disorders, excessive nutrient consumption leading to hyperglycemia and dyslipidemia is accompanied by a plethora of inflammatory and meta- bolic responses in multiple cell types and is termed meta- flammation, i.e., metabolism-induced inflammation.43,44 A critical component of T2DM is the low-grade chronic inflammation that is characterized by cytokine expression and immune cell infiltration that remains unresolved over time.42 In metabolic disorders, excessive nutrient consumption leading to hyperglycemia and dyslipidemia is accompanied by a plethora of inflammatory and meta- bolic responses in multiple cell types and is termed meta- flammation, i.e., metabolism-induced inflammation.43,44 Although peripherally-originating leukocytes and den- dritic cells are occasionally detected in the perivascular compartment as components of the immune surveillance machinery, the number of these cells in the brain is typ- ically restricted compared to those seen in peripheral or- gans.57,58 The presence of few perivascular CD45+ cells (i.e., leukocytes) scattered in the cortex of HFHS-fed mice, may suggest a possible easier passage of immune cells into the brain parenchyma, and consequent activation of microglia cells as depicted by our Iba1 immunostaining. \| DISCUSSION Within the serum of T2DM patients, the TIMP-2 levels declined compared to HD (Figure 7I). Overall, the results from the human in vitro model showed similar disruption to BBB integrity with T2DM serum stimulation as was seen in HFHS-fed mice. Metabolic overload impairs BBB function by increas- ing paracellular permeability and decreasing TEER, changes attributable to the loss of TJ proteins occludin and claudin-5 and VBLs. It has been demonstrated that laminin α2 null mice have a defective BBB, with leakage of the injected exogenous tracers, and the lack of expres- sion of laminin 2 subunit is paralleled by molecular de- fects at the endothelium VBL layer.42 Under physiological conditions, laminin α2 is expressed by astrocytes and peri- cytes28,43,44 and its expression in astrocyte end-feet coin- cides with astrocyte-pericyte sites of interaction, which in turn are involved in cellular communication and BBB structural integrity.45 Considering that pericytes are also in charge of immune mediator release and regulation of leukocyte transmigration, and altered astrocyte-pericyte cross-talk may also participate in the neuroinflammatory processes.46,47 18 of 27 \| (A) 18 of 27 \| SHEIKH et al. (A) (B) (C) SHEIKH et al. 18 of 27 \| SHEIKH (A) (D) (E) (B) (C) (A) (D) (F) (G) (E) (B) (C) (B) (C) (A) (D) (D) (E) (E) (F) (G) (F) (G) (G) 19 of 27 SHEIKH et al. FIGURE 6 Mouse serum significantly disrupts BBB function in vitro¸ correlating to in vivo results. Sera (10%) from mice—chow-fed, HFHS-fed, HFHS-fed + hrANXA1 treated and HFHS—chow reversion diet fed, were used to stimulate mouse brain endothelial cells, bEnd3 monolayers for 16 h unless otherwise stated. (A) Production of nitric oxide (NO) from brain endothelial cells bEnd3 after stimulation for 6 h. Measurement of (B) paracellular permeability coefficients to 70 kDA FITC-dextran and (C) transendothelial electrical resistance (TEER) (n = 30/group, 10 pooled/group per independent experiments). Flow cytometric analysis of (D) junctional proteins occludin, PECAM-1 & VE-cadherin (E) G:F actin ratio and (F) adhesion molecules ICAM-1, VCAM-1, and CD86 (n = 6–26/group, pooled in pairs, 3 independent experiments) expressed as mean intensity of fluorescence values. Isolated mouse lymphocytes from cervical lymph nodes of chow mice were placed in contact on transwells with bEnd3 cells and stimulated with mouse serum for 4 h. Adhered cell populations were collected from the upper compartment, and migrated cells were collected from the bottom compartment. \| DISCUSSION (G) Flow activated cell staining was used to evaluate firm adhesion and transmigration of CD3+ CD45+ leukocytes (pool 10 mice sera/group, on 6 pooled cervical lymph nodes T cells expanded, see material and methods section). Statistical analysis was performed by one-way ANOVA followed by a Bonferroni post hoc test. Data are expressed as mean ± SEM. p < .05, p < .01. p < .01 versus chow; $p < .05, $$p < .01, $$$p < .001, versus HFHS; #p < .05 vs HFHS + hrANXA1 may enhance the levels of TIMP-1 in the brain microen- vironment to allow positive remodeling. However, under sustained inflammation, the levels of TIMP-1 cannot be maintained and, therefore, decline significantly. In a sim- ilar manner, in the diabetic mouse model where there is underlying chronic inflammation, the results show an im- balance of TIMP-1 and MMPs. In fact, a study of T2DM patients found serum concentrations of MMP-2 and MMP-9 to be lower in diabetic patients vs non-diabetic controls during short-lasting hyperglycemia induced by oral glucose tolerance test, whereas chronic hyperglyce- mia, reflected by glycated hemoglobin levels, correlated with higher MMP-9 levels in T2DM patients.56 Such pub- lished data are in line with our human serum studies, we find diabetic patients to have increased MMP-9. The laminin subunits of the endothelial VBL layer are regulated in part by MMP activity, helping to maintain normal tissue homeostasis, this process is known to be dysregulated in a number of CNS diseases, including MS, AD, stroke, and glioma.32,48 MMP activity is transcription- ally regulated by, among others, cytokines, and ROS,32–34 which may be relevant in the context of the low-grade, chronic, and non-resolving inflammation49 known to be present in T2DM and which was observed in HFHS- fed mice. The activity of MMPs is further regulated by the presence and action of TIMPs33,34; hence, the main- tenance of efficient BBB function requires a balance be- tween MMP and TIMP expression. In HFHS-fed mice, TIMP-1 activity was significantly reduced in comparison to chow-fed mice, in combination with raised MMP-2 and MMP-9 activity, indicating a potential disruption in the balance and control of MMP activity. which may be relevant in the context of the low-grade, chronic, and non-resolving inflammation49 known to be present in T2DM and which was observed in HFHS- fed mice. \| DISCUSSION However cellular source of hypothalamic macrophages accumulation has been reported in diet-induced obesity, though the mechanism is still unclear.59 Importantly, dis- ruption of the BBB alone is not sufficient to result in leu- kocyte transmigration into the CNS parenchyma,60 rather there appears to be a need for locally-derived cues, such as inflammatory activity.61 Several events are required to Numerous studies have reported pro-inflammatory IL-1α and TNF-α as upregulating TIMP-1 levels in both endothelial cells and astrocytes.50,51 In mouse mod- els of experimental encephalomyelitis (EAE), GFAP- expressing astrocytes increase TIMP-1 expression to aid remodeling of the brain ECM.52 Similarly, in the human T-lymphotropic virus, TH1 cells interact with astrocytes causing them to become reactive; reactive astrocytes se- crete pro-inflammatory cytokines and express TIMP-1 and TIMP-3.53 Although in our diabetic model, there is a substantial increase in the presence of T-cell inducing chemokines, the serum and brain microvessel protein lev- els of TIMP-1 are decreased. This contrast, between our results and other studies53 showing upregulated TIMP-1, may in fact represent the switch between a protective vs detrimental effect of MMPs/TIMPs. Although peripherally-originating leukocytes and den- dritic cells are occasionally detected in the perivascular compartment as components of the immune surveillance machinery, the number of these cells in the brain is typ- ically restricted compared to those seen in peripheral or- gans.57,58 The presence of few perivascular CD45+ cells (i.e., leukocytes) scattered in the cortex of HFHS-fed mice, may suggest a possible easier passage of immune cells into the brain parenchyma, and consequent activation of microglia cells as depicted by our Iba1 immunostaining. However cellular source of hypothalamic macrophages accumulation has been reported in diet-induced obesity, though the mechanism is still unclear.59 Importantly, dis- ruption of the BBB alone is not sufficient to result in leu- kocyte transmigration into the CNS parenchyma,60 rather there appears to be a need for locally-derived cues, such as inflammatory activity.61 Several events are required to However, differential regulation of TIMP-1, specifi- cally in astrocytes of AD patients, correlates with acute vs chronic inflammatory state of the disease.54,55 In inflam- mation, astrocytes initially become activated to serve as a protective mechanism, isolating the damaged area and facilitating brain circuit remodeling.6 As such, acute ac- tivation of astrocytes along with microglial activation of 27 \| (A) (B) SHEIKH et al. \| DISCUSSION of 27 \| SHEIKH et (A) (B) (C) (E) (H) (D) (F) (G) (I) (A) (B) (F) (D) (C) (F) (C) (D) (G) (E) (G) (G) (G) (E) (E) (I) (H) (I) (H) SHEIKH et al. 21 of 27 FIGURE 7 Patients with T2DM present with brain vascular leakage and immune cell infiltration and serum from T2DM patients also significantly disrupt BBB function. Post-mortem brain sections of health donor (HD) and T2DM patients immuno-stained for (A) VE- Cadherin/CD31 and (B) pan-laminin/CD45. Nuclei were labeled with TO-PRO3. Scale bar: 25 μm. Quantification of staining shown as mean fluorescence intensity (arbitrary units) of markers (n = 4 patients/group, 35 section/patient, 10 randomly selected fields/sections). Sera (20%) from healthy donors or T2DM patients was used to stimulate human brain endothelial cells hCMEC/D3 monolayers for 16 h. Measurement of (C) paracellular permeability coefficients and (D) transendothelial electrical resistance (TEER), (n = 12 donors/group, pooled in pairs matched in sex and age). (E) Flow cytometric analysis of junctional proteins occludin and VE-cadherin and (F) ICAM-1 (n = 12 donors/group, pooled in pairs matched in sex and age). (G) Concentration of TNF-α and IL-6 measured in the serum of HD or T2DM patients using a multiplex ELISA (n = 19–28/group, age and sex-matched). (H) Relative expression of MMPs measured in hCMEC/ D3 cells (n = 10 pooled/group) (I) Relative expression of TIMPs in sera of HD or T2DM patients (n = 10 pooled/group). Statistical analysis was performed by either an independent Student's t-test or Mann–Whitney U test. Data are expressed as mean ± SEM. p < .05, p < .01, p < .001, ***p < .0001 vs HD permit the migration of leukocytes across an endothe- lium barrier. First and foremost, chemokine signals in- duce the chemotaxis of immune cells, i.e., cell adhesion molecules such as ICAM-1, P-, and E-selectin is required in the brain microvasculature itself.59 Notably, we found that consumption of a HFHS diet enhanced expression of both chemokines and cell adhesion molecules in the ce- rebral microvasculature compared to chow-fed mice and that these changes were attenuated, although not fully reversed, by either diet reversion or hrANXA1 treatment, further emphasizing the ability of both interventions to protect the CNS environment through reduction of chronic inflammation and repair of the cerebral vasculature. \| DISCUSSION The findings in vivo/ex vivo correlates with the in vitro BBB model where bEND3 cells treated with HFHS-fed mice serum resulted in increased paracellular permea- bility and reduced TEER compared with chow-fed mice serum-stimulated cells, which can be accounted for the reduction of junctional proteins such as occludin and VE- cadherin as well as loss of the actin filamentous fibers. Moreover, the HFHS-fed mice serum activates the bEnd3 cells to upregulate adhesion molecule expression. In ad- dition, cells stimulated with HFHS-fed mice serum show an upregulation of co-stimulatory molecule CD86, indi- cating that the brain endothelial cells themselves act as antigen-presenting cells under inflammatory stimuli, as previously reported.45,46 This coincides with the increased adhesion and migratory profile of CD45+ leukocytes. Due to the tightly regulated nature of the brain micro- environment, the transmigration of leukocytes across the BBB into the brain parenchyma is in fact a two-step pro- cess.62–64 To cross into the brain parenchyma, the invad- ing cells must be able to cross both the inner endothelial BM and the outer astrocytic BM. In the HFHS-fed mice, double immunostaining with pan-laminin (a marker for both endothelial and astrocytic BM) and the general leu- kocyte marker CD45 shows clear entrapment of CD45+ leukocytes between the two BM layers, the perivascular space, with limited infiltration into the brain parenchyma itself, a feature not seen in chow-fed mice. Initial passage across the endothelial cells requires an integrin-adhesion molecules-mediated process. As noted, the number of adhered/migrated leukocytes was far greater in the HFHS-fed T2DM model indicating that these cells were interacting with adhesion molecules to allow the trans- migration cascade. Moreover, activated leukocytes also release MMPs to aid in passage across the BBB.48,65 The production of MMPs by tissue and immune-cell sources is believed to be strongly affected by cytokine levels. Work by Sorokin and colleagues show that the balance of TNFα vs IFNγ regulates activation of astrocytes and secretion of MMP-9.64 Interestingly, in the HFHS-diet model, there is an increase of both TNFα and IFNγ at the brain microves- sels; especially the relative expression of TNFα to IFNγ is much higher. Confirmation of the role of inflammatory mediators in inducing BBB damage, allowed us to corroborate our re- sults into a humanized in vitro BBB model using hCMED/3 cells stimulated with serum from T2DM patients or healthy donors. \| DISCUSSION A number of studies have reported correlations between high peripheral inflammation and risk of neu- rodegenerative diseases such as AD, PD, HD, ALS, and MS.47,48 Here, we showed raised TNF-α and IL-6 in T2DM patient sera resulted in junctional protein (occludin and VE-cadherin) loss contributing to a leaky BBB phenotype. Observations in human post-mortem brain confirmed the reduction of VE-cadherin and CD31 in microvessels of T2DM patients, paralleled by laminin reduction in the VBL. As with the mouse model, loss of the BBB structure corresponded with increased CD45+ leukocytes in the brain parenchyma of T2DM patients compared with healthy con- trols. BBB barrier disruption coupled with CNS leukocyte infiltration has been reported to drive CNS disorders of AD, MS, and PD.66 Furthermore epidemiological studies have long reported metabolic disorders to be a risk factor for cognitive decline67,68 and the development of demen- tias.51,52 Degenerating neurons have been observed in the cortex and hippocampal regions of rodents fed a high-fat and high-fructose diet for 24 weeks led to impaired spatial learning and memory, as tested using the Morris Water \| 22 of 27 SHEIKH et al. Maze test.69 In the past decade, there have been studies investigating the link between T2DM and AD pathology with similarities reported in the vascular pathologies be- tween mice fed a western diet and 3× transgenic AD mice for development of peripheral insulin resistance, accumu- lation of Aβ and phosphorylated tau, and behavioral and cognitive abnormalities.70–72 The results of our study impli- cate the pathophysiological changes occurring at the BBB arising from a peripheral metabolic disease that can lead to the development of these neurovascular diseases. The si- multaneous rise of pro- and anti-inflammatory mediators activate and alter multiple signaling cascades73–76 and it can be hypothesized that BBB breakdown in T2DM will alter the brain microenvironment to propagate a vicious cycle of self-propelling inflammation from brain endothelial cells, astrocytes, microglia and neurons that ultimately lead to chronic neuroinflammation and neurodegeneration.77 Investigation into neuronal loss is beyond the scope of this study; however, future studies would benefit from review- ing the effect on neurons and subsequent cognitive behav- iors, with particular focus on whether BBB improvement in pharmacological and dietary interventions can translate to the restoration of the cognitive abnormalities induced by high-fat feeding reported in other studies.78 α4 and laminin α2, respectively. \| DISCUSSION This allowed for the re- attachment of astrocytic end-feet to the brain endothelial cells to restore the full structure of the BBB in HFHS- fed + hrANXA1-treated mice that contributed to the re- duced infiltration of CD45+ into the brain parenchyma. Within the brain, ANXA1 has been shown to contribute to microglia surveillance and maintenance of brain homeo- stasis through reducing the phagocytosis of non-apoptotic neurons under inflammatory conditions and reducing the production of NO from microglia cells83 along with pro- moting the transition of activated microglia from an M1 to M2 phenotype through peptide-FPR engagement.74 In our study, we find hrANXA1 treatment in HFHS-feeding reduced Iba1+-activated microglia presence and reduced iNOS expression/NO production. It is well recognized that an improved diet can help control and manage T2DM and the development of CVDs84–88 and there is growing evidence of the role of Mediterranean and dietary approaches to stop hyperten- sion (DASH) diets in reducing cognitive decline and low- ering AD risk.89 However, the majority of these studies are large longitudinal observation studies or meta-analyses. To our knowledge, our study is the first to examine the effect of diet on the structural and functional changes of the brain microvasculature in a diabetic model induced by HFHS-feeding. From the results, it is clearly noticeable that dietary changes have a huge impact on BBB integrity with significantly reduced Evans blue dye extravasation, reduced paracellular permeability of FITC-dextran, and increased TEER compared to HFHS-fed mice. Reduced leakage can be accounted for the restoration of junctional proteins; although while occludin presence at junctional sites is beginning to be re-established, the laminin reac- tivity remains low preventing the ensheathment of astro- cytes. As a result of the lack of complete BBB restoration, in the HFHS—chow reversion diet mice CD45+ leuko- cytes are still present within the VBL layers and appear to show migration outwards into the brain parenchyma. These results together illustrate that brain endothelial cells contacts are repaired with dietary changes, however, the BBB vasculature in its entirety is not restored. This may be due to the length of time in which dietary rever- sion occurred (five weeks), which may not be sufficient to fully recover from the HFHS insult and further studies will be needed to address this. \| DISCUSSION Studies that have investi- gated dietary changes in diabetic rodents by food or cal- orie restriction,90–92 ketogenic diets,93 herbal formulas,94 reduced glycoxidation products,95 or addition of extra virgin olive oil96 report improved insulin sensitivity with feeding over a duration of 8–24 weeks on intervention. In our study, a 5-week intervention begins to improve insulin and lipid levels, however, optimal glycemic control has not been achieved, however, there is a significant reduction It has long been established that T2DM can cause mi- crovessel damage in the peripheral system resulting in secondary complications such as retinopathy and neurop- athy.79 The restoration of BBB integrity and functionality with hrANXA1 treatment or a dietary change implicates that damage the brain microvasculature is also a second- ary complication of T2DM-induced metaflammation. The benefits of ANXA1 as a therapeutic agent have long been investigated. We have previously demonstrated that ANXA1−/− mice fed a HFHS diet have a more se- vere diabetic phenotype compared to wild-type HFHS- fed mice, with severe dyslipidemia, hepatic steatosis, and renal dysfunction.16 Other studies using LDLR−/− mice have shown hrANXA1 treatment to reduce atheroscle- rotic plaque burden and improve plaque stability through reduced lipid accumulation,80 demonstrating the ther- apeutic benefit of ANXA1 in treating metabolic disease and its associated peripheral co-morbidities. Arguably, ANXA1's best therapeutic effects are known at the BBB. ANXA1 has been shown to improve tight junction stability through restoring F-actin organization via co-localization with β-actin,15 inhibition of leukocyte TEM through in- ducing L-selectin shedding from leukocytes to prevent tethering, rolling, and firm adhesion81 and through co- localization with α4β1 integrin on leukocytes to prevent binding to VCAM-1 on endothelium.82 Here, we extend our findings of the therapeutic effect of hrANXA1 treat- ment also on the essential components of the BM; with the clear restoration of endothelial and astrocytic laminin \| 23 of 27 SHEIKH et al. SHEIKH et al. FIGURE 8 Schematic representation of the effect of HFHS-feeding on BBB structure and functionality, leading to neuroinflammation. The traits of metabolic disorders—obesity, hyperglycemia, dyslipidemia, insulin resistance, and hypertension, create a low-grade chronic inflammatory state termed metaflammation which results over time in the development of T2DM. Chronic metaflammation is responsible for producing a number of cytokines, chemokines, and oxidative stress as well as activating the immune system. \| DISCUSSION The damage by inflammation and the immune system leads to a leaky BBB phenotype that can be correlated to the loss of TJs and AJs, the actin cytoskeleton, and the loss of basal lamina α4 on endothelial cells and α2 astrocytic end-feet. This breakdown is mediated by the imbalance of MMPs/TIMPs and results in complete loss of the BBB structure with the dissociation of the astrocytes to the blood vessel. The accumulation of inflammatory mediators and oxidative stress contributes to the production of these MMPs. Increased adhesion molecule expressions along with BBB dysfunction resulted in increased transendothelial migration of leukocytes into the brain parenchyma. Microglia become activated. It is hypothesized that the astrocytes, microglia, and neurons also sense changes in the brain microenvironment and are likely to respond to the increased presence of inflammatory mediator passage by further up-regulating the release of cytokines, chemokines, MMPs, and ROS production that triggers pro-inflammatory signaling pathways. These events together are, therefore, likely to create a self-destructing environment within the brain triggering neuroinflammation and subsequent neuronal destruction. Some objects/images have been extracted from the web Evaluating both interventions, it appears that ANXA1 has more direct effects on the BBB restoration, through its links with the actin cytoskeleton, basal lamina, and TEM prevention. On the contrary, the attributes of a healthy diet are through reduced inflammatory mediators, downstream this would result in the altering of signaling pathways associated with T2DM. It can, therefore, be hy- pothesized that a combination of hrANXA1 treatment with dietary reversion would result in greatly improved T2DM outcomes both in the peripheral and central systems. in metaflammation. The benefit of Mediterranean diets, rich in polyunsaturated fats, flavonoids, vitamins, and minerals (calcium, iron, zinc, etc.), are associated to the high presence of omega-3 fatty acids, antioxidants, and polyphenols that reduce free radical generation and cy- tokine production, thereby inhibiting pro-inflammatory signaling pathways97,98 that lead to improved cognition and learning by facilitating synaptic plasticity and/or en- hancing synaptic membrane fluidity through reduction of inflammation and oxidative stress.98–100 At this stage, we can speculate that dietary reversion, through its effect on inflammatory mediators, might benefit BBB function, but this remains a topic for future investigation. in metaflammation. ORCID Egle Solito https://orcid.org/0000-0001-5279-0049 Egle Solito https://orcid.org/0000-0001-5279-0049 DISCLOSURES The authors state that there are no conflicts of interest in connection with this article. 6. Pekny M, Nilsson M. Astrocyte activation and reactive gliosis. Glia. 2005;50:427-434. REFERENCES 1. Rochlani Y, Pothineni NV, Kovelamudi S, Mehta JL. Metabolic syndrome: pathophysiology, management, and modulation by natural compounds. Ther Adv Cardiovasc Dis. 2017;11: 215-225. 2. Panza F, Frisardi V, Capurso C, et al. Metabolic syndrome and cognitive impairment: current epidemiology and possible underlying mechanisms. J Alzheimer's Dis. 2010;21:691-724. 3. Frisardi V, Solfrizzi V, Capurso C, et al. Is insulin resistant brain state a central feature of the metabolic-cognitive syndrome? J Alzheimer's Dis. 2010;21(1):57-63. 4. Sheikh MH, Henson SM, Loiola RA, et al. Immuno-metabolic im- pact of the multiple sclerosis patients' sera on endothelial cells of the blood-brain barrier. J Neuroinflammation. 2020;17(153):1-19. 5. Janelidze S, Hertze J, Nägga K, et al. Increased blood-brain barrier permeability is associated with dementia and diabetes but not amyloid pathology or APOE genotype. Neurobiol Aging. 2017;51:104-112. \| DISCUSSION The benefit of Mediterranean diets, rich in polyunsaturated fats, flavonoids, vitamins, and minerals (calcium, iron, zinc, etc.), are associated to the high presence of omega-3 fatty acids, antioxidants, and polyphenols that reduce free radical generation and cy- tokine production, thereby inhibiting pro-inflammatory signaling pathways97,98 that lead to improved cognition and learning by facilitating synaptic plasticity and/or en- hancing synaptic membrane fluidity through reduction of inflammation and oxidative stress.98–100 At this stage, we can speculate that dietary reversion, through its effect on inflammatory mediators, might benefit BBB function, but this remains a topic for future investigation. In conclusion, our study demonstrates that a HFHS- dietary intake in mice or T2DM in patients causes mi- crovascular complications at the BBB (Figure 8). The SHEIKH et al. 24 of 27 biochemical data for the study and contributed to the dis- cussion of the manuscript; Davide Ferorelli, Alessandro Dell'Erba, and Eugenio Maiorano provided brain human samples; Magdi Yaqoob and Christoph Thiemermann provided assistance with the animal license. Federica Marelli-Berg contributed to results discussion and manu- script writing. Daniela Virgintino collected human brain samples and contributed to their analysis and discussion of the manuscript. Egle Solito designed the experiments, performed some experiments, interpreted, analyzed data, and wrote the manuscript. All authors reviewed and ap- proved the manuscript. growing obesity and T2DM epidemic, therefore, raise concerns for the potential pathogenesis of neurodegen- erative brain disorders and cognitive decline on top of the well-known macrovascular and microvascular com- plications T2DM. The need for innovative approaches to healthcare is paramount and the ability of hrANXA1 and dietary intervention to reduce and restore the meta- bolic triggered inflammation-induced BBB damage pro- vides hope for ways we can slow down T2DM-associated neuroinflammation. ACKNOWLEDGMENTS We would like to thank the following funding bodies for their support: the British Heart Foundation (Grant/Award number: FS 16/60/32739) to M.H.S.; Ministry of Education- CAPES, Brazil (Grant number: 7326/2014-09) to R.A.L.; “Cibo, Microbiota, Salute” by “Vini di Batasiolo S.p.A” AL-RIC19ABARA_01 to A.B.; “Post-Doctoral Fellowship 2020” by “Fondazione Umberto Veronesi” 2020-3318 to A.B.; Fondazione Cariplo 2016-0852 to G.D.N.; EFSD/ Lilly European Diabetes Research Programme 2018 to G.D.N.; Fondazione Telethon GGP19146 to G.D.N.; PRIN 2017K55HLC to G.D.N.; Fondazione Cariplo 2015-0524 and 2015-0564 to A.L.C.; H2020 REPROGRAM PHC- 03-2015/667837-2 to A.L.C.; Ministero della Salute GR- 2011-02346974 to A.L.C.; ERA NET ER-2017-2364981 to A.L.C.; PRIN 2017H5F943 to A.L.C.; FISM Fondazione Italianana Sclerosi Multipla (Cod. 2014/R/21) to E.S. 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Sheikh performed experiments as part of her PhD thesis, analyzed, and interpreted the data, and contrib- uted to the manuscript writing. Mariella Errede designed the experimental for confocal studies and contributed to confocal and morphometric data discussion. Antonio d'Amati performed experiments as part of his Medical de- gree thesis, executed, and analyzed immunostaining, and confocal microscopy, on both mouse and human brains, and morphometrically quantified the data. Noorafza Q. Khan performed the western blots and assisted with the zymography. Silvia Fanti performed transmigration exper- iments; Simon McArthur assisted with the zymography and contributed to the writing and discussion of the paper. Rodrigo A. Loiola and Gareth S. D. Purvis performed the Evans blue experiment. Caroline E. O'Riordan and Julius Kieswich provided technical assistance with animal work. Chis Reutelingsperger provided the purified hrANXA1. 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Different patterns of memory im- pairments accompany short- and longer-term maintenance on a high-energy diet. J Exp Psychol Anim Behav Process. 2010;36:313-319. SHEIKH et al. 25 of 27 32. Rosenberg GA. Matrix metalloproteinases in neuroinflamma- tion. Glia. 2002;39:279-291. 14. Murray AJ, Knight NS, Cochlin LE, et al. Deterioration of phys- ical performance and cognitive function in rats with short-term high-fat feeding. FASEB J. 2009;23:4353-4360. 33. Rosenberg GA. Matrix metalloproteinases and their multiple roles in neurodegenerative diseases. Lancet Neurol. 2009;8:205-216. 15. Cristante E, McArthur S, Mauro C, et al. AUTHOR CONTRIBUTIONS Bowman GL, Kaye JA, Moore M, Waichunas D, Carlson NE, Quinn JF. Blood-brain barrier impairment in Alzheimer disease: stability and functional significance. Neurology. 2007;68:1809-1814. 22. Association AD. Standards of medical care in diabetes-2011. Diabetes Care. 2011;34:S11. 23. Friedewald WT, Levy RI, Fredrickson DS. 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https://openalex.org/W4310442265	https://zenodo.org/records/7383106/files/%D0%90%D0%B1%D0%B4%D1%83%D1%80%D0%B0%D1%85%D0%BC%D0%BE%D0%BD%D0%BE%D0%B2%20%D0%90%D1%81%D1%80%D0%BE%D1%80%D0%B1%D0%B5%D0%BA%20%D0%A8%D0%BE%D0%BA%D0%B8%D1%80%D0%B6%D0%BE%D0%BD%20%D1%9E%D2%93%D0%BB%D0%B8.pdf	Russian	null	ҲУҚУҚБУЗАРЛИКЛАР ПРОФИЛАКТИКАСИ ВА ЖИНОЯТЧИЛИККА ҚАРШИ КУРАШИШ БЎЙИЧА ИДОРАЛАРАРО КОМИССИЯСИ ФАОЛИЯТИНИ ТАШКИЛ ЭТИШ	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	2,299	1 Шавкат Мирзиёев. Миллий тараққиёт йўлимизни қатъият билан давом эттириб, янги босқичга кўтарамиз. Т. 2017 йил Б. 312,313 Абдурахмонов Асрорбек Шокиржон ўғли ИИВ Академияси 3-босқич курсанти, сафдор Абдурахмонов Асрорбек Шокиржон ўғли ИИВ Академияси 3-босқич курсанти, сафдор Аннотация: Ушбу мақола олқали сиз, мавзуининг долзарблиги, хукубузарлилар профилатикаси ва жиноятчиликка қарши кураш бўйича республика идоралараро комиссияси фаолиятининг моҳияти, комиссия таркиби, тузилиши, асосий вазифалари, комиссия фаолиятини ташкил этиш тартиби, унинг ўзига хос хусусиятлари, ушбу фаолиятнинг бугунги кундаги холати, хукубузарликлар профилатикаси ва жиноятчиликка қарши кураш бўйича республика идоралараро комиссияси фаолиятини тартибга солувчи норматив- ҳуқуқий ҳужжатлар ҳамда ушбу соҳадаги муоммо ва камчиликлар ва уларни амалий ўрганилган ҳолда ушбу фаолиятни такомилаштириш йуналишлари ҳақида билиб олишингиз мумкин. Калит сўзлар: хукубузарлилар профилатикаси, республика идоралараро комиссияси, ўзига хос хусусиятлар, соҳадаги муоммо ва камчиликлар. https://doi.org/10.5281/zenodo.7383106 https://doi.org/10.5281/zenodo.7383106 2 Шавкат Мирзиёев “Ички ишлар органлари фаолияти, тизимда мавжуд муаммо ва камчиликлар, истиқболдаги вазифалар”га бағишланган йиғилишдаги маърузаси. 2017 йил 9 февраль. 3 Ўзбекистон Республикаси Президентининг 2017 йил 14 мартдаги “Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш тизимини янада такомиллаштириш чора-тадбирлари тўғрисида”ги ПҚ-2833-сон Қарори. КИРИШ Бугунги кунда мамлакатимизда юритилаётган давлат сиёсатининг асл моҳияти ва провард максади – халқимизнинг дарду ташвишлари , хаётий муаммо ва эҳтиёжларидан доимо хабардор бўлиб, унинг моддий фаровонлигини амалга ошириш, муносиб турмиш даражасини ва сифатини таьминлаш, тинч ҳаётни ҳимоя қилишдир. Энг муҳими халқимиз ўз ҳаётидан ва давлатидан рози бўлиб яшашини таминлаш бугун барчамизнинг бош вазифамиздир. Ушбу вазифаларни амалга оширишда “Ҳуқуқбузарликлар профилактикаси куни” ташкил этилганлиги бежизга эмасдир1. Ҳуқуқбузарликлар профилактикаси куни деб, профилактика инспектори томонидан барча соҳавий ҳизматлар ва жамоат ташкилотлари билан биргаликда, ҳуқуқбузарликлар профилактикаси тўғрисидаги қонунда белгилаб ўтилган умуммий, махсус, якка ва виктималогик тадбирлар ўткакзиш орқали фуқороларнинг дарди ва ташвишини ешитиш ва уларга амалий ёрдамлар бериш, фуқороларнинг ҳуқуқий маданиятини ошириш орқали ҳуқуқбузарлик ва жиноятчиликнинг олдини олиш фаолияти тизмига айтилади. Бу борада амалга оширилаётган ишлар ҳусусида ички ишлар органлари тизмида кўплаб ўзгаришлар жорий этилди. Президентимиз Шавкат Мирзиёев таькидлаганидек “Барча давлат органлари Шу билан бирга, ҳуқуқбузарликлар профилактикасини амалга оширувчи давлат иш шакли ва услублари, энг аввало ахборот-коммуникация технологияларидан етарли даражада фойдаланилмаётганлиги сабабли ҳозирги кун талабларига тўлиқ жавоб бермаганлиги” ҳақида алоҳида таькидлаб ўтди2. Давлат идоралари аксарият ҳолларда ҳуқуқбузарликлар профилактикасини фақатгина ҳуқуқни муҳофаза қилувчи органларнинг вазифаси сифатида баҳолаб, оқибатда ушбу фаолиятга лозим даражада эътибор қаратилмаган. МУХОКАМА ВА НАТИЖАЛАР Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш борасидаги чора-тадбирларнинг аниқ манзилга йўналтирилмаганлиги ва уларга комплекс ёндошилмаётганлиги, шунингдек ҳуқуқбузарликларнинг тизимли равишда содир этилишига доир сабаб ва шарт-шароитларни аниқлаш ва уларни бартараф этиш бўйича чора-тадбирларни ишлаб чиқиш самарадорлиги мавжуд эмаслиги кутилаётган натижаларни бермаётганлиги, ваколатли органларнинг етарли даражада ташаббус кўрсатмаётганлиги, лозим даражадаги идоралараро ҳамкорликнинг мавжуд эмаслиги, амалга оширилаётган чора-тадбирларнинг ўзаро номутаносиблиги ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш соҳасидаги фаолиятни тубдан такомиллаштиришни талаб этади. ҳ ў қ э п Бундан келиб чиқиб ҳафтанинг ҳар пайшанба куни «Ҳуқуқбузарликлар профилактикаси куни» этиб белгиланди ва ушбу кунда қуйидаги тадбирларни амалга оширишга алоҳида эътибор қаратиладиган масалар белгиланиб олинди ва алоҳида қоида сифатида жорий этилди3: Ҳуқуқбузарликларга муросасизлик маданиятини шакллантириш, ҳуқуқий нигилизмга барҳам бериш ва фуқароларнинг қонунга итоаткорлик хулқ- 2 Шавкат Мирзиёев “Ички ишлар органлари фаолияти, тизимда мавжуд муаммо ва камчиликлар, истиқболдаги вазифалар”га бағишланган йиғилишдаги маърузаси. 2017 йил 9 февраль. 3 Ўзбекистон Республикаси Президентининг 2017 йил 14 мартдаги “Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш тизимини янада такомиллаштириш чора-тадбирлари тўғрисида”ги ПҚ-2833-сон Қарори. 5 Ҳуқуқбузарликларнинг сабаб ва шароитларини аниқлаш, тахлил қилиш ва бартараф этиш. Ўқув қўлланма Т. 2017 йил Б-82 4 Ўзбекистон Республикаси Президентининг 2017 йил 14 мартдаги “Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш тизимини янада такомиллаштириш чора-тадбирлари тўғрисида”ги ПҚ-2833-сон Қарори. 6 Ҳуқуқбузарликларнинг сабаб ва шароитларини аниқлаш, тахлил қилиш ва бартараф этиш. Ўқув қўлланма Т. 2017. Б-82 атворини оммалаштиришга қаратилган, шу жумладан ички ишлар органлари таянч пунктлари, ҳуқуқни муҳофаза қилувчи ва бошқа давлат идоралари ва ташкилотларида «очиқ эшиклар куни»ни ташкил этиш орқали тизимли чора- тадбирларни амалга ошириш каби тадбирлар амалга оширилмоқда4. Ўзбекистон демократик ҳуқуқий давлатни куриш ва адолатли фуқоролик жамиятни шакиллантириш йўлида дадил бориб, жаҳон ҳамжамиятида мустаҳкам ўрин эгалламоқда. Барқаролик ва тартиб, миллатлароро тотувлик ва фуқоролар аҳиллиги туфайли ёш давлатимиз ишончи ва ҳурматига сазовор бўлишида профилактика инспектори томонидан ҳуқуқбузарликларга муросасизлик маданиятини шакллантириш, фуқароларнинг қонунга итоаткорлик хулқ- атворини оммалаштиришга қаратилган чора-тадбирлар ишлаб чиқиш, шу жумладан ички ишлар органлари таянч пунктлари иш фаолиятида, шунингдек, ҳуқуқни муҳофаза қилувчи ва бошқа давлат идоралари ва ташкилотларида тизимли чора-тадбирларни амалга ошириш орқали “Ҳуқуқбузарликлар профилактикаси куни” ўтказиш фаолиятини ташкил этади. Шуни таькидлаш жоизки, биз бугунги кунда жамиятда ҳуқуқий маданиятининг барча таркибий қисимлари мукаммал амалга оширилаяпти деб айта олмаймиз. Бу борада фуқороларнинг ҳуқуқий маданиятини шакиллантириш ва қонунга итаоткорлик рухида яшашга ундайдиган асосий хусусиятлар шакиллантирилиши керак: - ҳуқуқий нормалар мазмунини мукаммал билиши5; - ҳуқуқий нормалар мазмунини мукаммал билиши5; - қонунга ва ҳуқуқий талабларга чуқур ҳурматда бўлиши хиссини уйғотиш; - ҳуқуқий нормалар мазмунини мукаммал билиши; - қонунга ва ҳуқуқий талабларга чуқур ҳурматда бўлиши хиссини уйғотиш; у т - ҳуқуқий нормалар мазмунини мукаммал билиши; - қонунга ва ҳуқуқий талабларга чуқур ҳурматда бўлиши хиссини уйғотиш; - ҳуқуқий воқелик ва институтларни тўғри англаш ҳамда тўғри тушунтириш; - юксак ижтимоий ҳуқуқий фаоллик; - ҳуқуқни ижобий ижтимоий ўзгаришлар ва инсон манфаатларини муҳофазаловчи восита (қадрият) сифатада баҳолаш ва унга амал қилиш; - ҳуқуқий меёрлар талабига иҳтиёрий тарзда, ички эьтиқод амри билан риоя этиш ва бошқалар. 4 Ўзбекистон Республикаси Президентининг 2017 йил 14 мартдаги “Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш тизимини янада такомиллаштириш чора-тадбирлари тўғрисида”ги ПҚ-2833-сон Қарори. 5 Ҳуқуқбузарликларнинг сабаб ва шароитларини аниқлаш, тахлил қилиш ва бартараф этиш. Ўқув қўлланма Т. 2017 йил Б-82 5 Ҳуқуқбузарликларнинг сабаб ва шароитларини аниқлаш, тахлил қилиш ва бартараф этиш. Ўқув қўлланма Т. 2017 йил Б-82 7 Ҳуқуқбузарликларнинг сабаб ва шароитларини аниқлаш, тахлил қилиш ва бартараф этиш. Ўқув қўлланма Т. 2017 йил 8 Ички ишлар органлари тизимидаги туб ислохотлар – халқ манфаатларига хизмат қилишни таъминлаш кафолати. Ўқув қўлланма Т. 2017 йил Шахснинг ҳуқуқий маданиятини тавсифловчи барча хислатлар эса тўғридан тўғри жамият ҳуқуқий маданиятининг барча таркибий тасири остида шакилланади. Шуни ҳам қайд этиш лозимки, жамият ҳуқуқий маданиятининг таркибий қисимларидаги барча ижобий, шу билан бир қаторда, салбий ҳолатлар келажак авлод ҳуқуқий маданиятини шакиллантириш ва ўзгартиришга, албатта ўз таъсирини ўтказмасдан қолмайди. Шунинг учун ҳам келажак авлод ҳуқуқий маданиятини шакиллантириш ва юксалтириш аввалламбор, жамият ҳаёти ҳуқуқий маданиятининг ҳар бир таркибий қисмини мунтазам равишда такомиллаштириб боришни тақазо этади. Шундай экан,биз жамият ҳуқуқий маданиятининг ҳар бир таркибий қисмида мавжуд бўлган муаммоларини ўз вақтида аниқлашимиз ва ҳал этишимиз ҳамда уларни янада такомиллаштириш борасида мунтаззам равишда профилактика инспектори иш олиб бориши зарур6. Бугунги кунда келажак авлод ҳуқуқий маданиятини шакиллантириш ва юксалатиришнинг муаммоларига бой таркибий қисм ҳуқуқий онг ва уни янада юқори даражага кўтариш борасида ҳуқуқий саводхонликни янада ошириш ҳисобланади, десак хато қилмаймиз. Ёшларнинг ҳуқуқий саводҳонлиги шакиллантириш ва юксалтиришни биз шартли равишда қуйдаги уч босқичга ажратган ҳолда ўргандик : Ёшларнинг ҳуқуқий саводҳонлиги шакиллантириш ва юксалтиришни биз шартли равишда қуйдаги уч босқичга ажратган ҳолда ўргандик : биринчидан, шахс онгида илк ҳуқуқий маданиятнинг шакилланиши. Маьлумки шахс онгидаги илк ҳуқуқий тушунчалар биринчи навбатда, оилада ота-она ва оиланинг бошқа аьзолари, мактабгача таьлим муассасларида- тарбитячилар, мактабдан ташқари маданият муассасаларида эса у ерда ижод қилаётган театр актиёрлари, санаткорлар, парк ва боғлардаги турли кўрзмали воситалар орқали шакилланади;. биринчидан, шахс онгида илк ҳуқуқий маданиятнинг шакилланиши. Маьлумки шахс онгидаги илк ҳуқуқий тушунчалар биринчи навбатда, оилада ота-она ва оиланинг бошқа аьзолари, мактабгача таьлим муассасларида- тарбитячилар, мактабдан ташқари маданият муассасаларида эса у ерда ижод қилаётган театр актиёрлари, санаткорлар, парк ва боғлардаги турли кўрзмали воситалар орқали шакилланади;. иккинчидан, шахс онгида ҳуқуқий билими асосида ўзликсиз таьлим тизмининг бошланғич, ўрта таьлим макитабларида, академик лицейлар, касб- хунар коллежлари, олий ўқув юртларида билим олиш жараёнида, шунингдек жзамоат тузулмалари ҳамда профилактика инспектори томонидан ички ишлар органлари таянч пункитлари негизида фаолият олиб борувчи субьектлари томонидан амалга ошириладиган ҳуқуқий тарғибот-ташвиқот ишлари тасирида шакилланади ва бойиб борадаи ; учинчидан, шахс ҳуқий маданият саводхонлигни ошириб бориш. Шахс ҳуқуқий саводҳонлиги ўзлари фаолият олиб борувчи меҳнат жамолардаги, фаолияти амалдаги қонунлар билан тақиқланмаган норасмий гурухлардаги, ҳуқуқни муҳафаза қилувчи органлар фаолиятини амлаг ошириш жараёнидаги ижтимоий муносабатлар, шунингдек оммавий ахборат воситалари томонидан учинчидан, шахс ҳуқий маданият саводхонлигни ошириб бориш. Шахс ҳуқуқий саводҳонлиги ўзлари фаолият олиб борувчи меҳнат жамолардаги, фаолияти амалдаги қонунлар билан тақиқланмаган норасмий гурухлардаги, ҳуқуқни муҳафаза қилувчи органлар фаолиятини амлаг ошириш жараёнидаги ижтимоий муносабатлар, шунингдек оммавий ахборат воситалари томонидан Мазкур қарор билан ҳафтанинг ҳар пайшанба кунлари “Ҳуқуқбузарликлар профилактикси куни” деб эълон қилинди ҳамда ушбу кундаги фаолиятни ташкил этиш ва мувофиқлаштириш ички ишлар органлари ваколатига берилди. Энг муҳими халқимиз ўз ҳаётидан ва давлатидан рози бўлиб яшашини таминлаш бугун барчамизнинг бош вазифамиздир. Ушбу вазифаларни амалга оширишда “Ҳуқуқбузарликлар профилактикаси куни” ташкил этилганлиги бежизга эмасдир9. Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш борасидаги чора-тадбирларнинг аниқ манзилга йўналтирилмаганлиги ва уларга комплекс ёндошилмаётганлиги, шунингдек ҳуқуқбузарликларнинг тизимли равишда содир этилишига доир сабаб ва шарт-шароитларни аниқлаш ва уларни бартараф этиш бўйича чора-тадбирларни ишлаб чиқиш самарадорлиги мавжуд эмаслиги кутилаётган натижаларни бермаётганлиги, ваколатли органларнинг етарли даражада ташаббус кўрсатмаётганлиги, лозим даражадаги идоралараро ҳамкорликнинг мавжуд эмаслиги, амалга оширилаётган чора-тадбирларнинг ўзаро номутаносиблиги ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш соҳасидаги фаолиятни тубдан такомиллаштиришни талаб этади. амалга оширилаётган ҳуқуқий тарғибот ва ташвиқот ишлари таьсири натижасида шакилланади7. Юқоридагилардан кўриниб турибдики профилактика инспектори томонида фуқороларнинг ҳуқуқий маданиятирни шакиллантириш ва қонунга ҳурмат рухи асосида яшаш кўникмасини шакиллантириш ҳам “Ҳуқубузарликлар профилактикаси куни”да олиб бориладиган ишларнинг қисми сифатида олиб қарасак хато бўлмайди. “Ҳуқуқбузарликлар профилактикаси куни”ни ташкил этишдаги асосий талабларга қуйидагиларни киритиш мумкин. Жумладан, ички ишлар органлари таянч пунктлари, ҳуқуқни муҳофаза қилувчи ва бошқа давлат идоралари ва ташкилотларида «очиқ эшиклар куни»ни ташкил этиш орқали тизимли чора-тадбирларни амалга ошириш каби қоидалар белгиланган бўлиб, кенг қамровли мулоҳазани ташкил этади; профилактика инспектори ўз фаолиятини очиқ ва шаффоф тарзда, давлат органлари, фуқароларнинг ўзини ўзи бошқариш органлари, бошқа ташкилотлар ва фуқаролар, шунингдек, оммавий ахборот воситалари билан ҳамкорликда амалга оширади; жисмоний ва юридик шахслар қонун ҳужжатларида белгиланган тартибда ички ишлар органларининг профилактика хизмати фаолияти ҳақида, шунингдек, жисмоний ва юридик шахсларнинг ҳуқуқлари ҳамда қонуний манфаатларига бевосита дахлдор бўлган ҳаққоний ахборотни олиш ҳуқуқига эга, фойдаланилиши қонун билан чекланган ахборот бундан мустасно8. Ўзбекистон Республикаси Президентининг 2017 йил 14 мартдаги “Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш тизимини янада такомиллаштириш чора-тадбирлари тўғрисида”ги ПҚ- 2833-сон Қарори билан “Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш бўйича республика идоралараро комиссияси”нинг ташкил этилиши барча субъектларнинг ҳуқуқбузарликлар профилактикасида фаол иштирок этишини таъминлаш ва уларнинг имкониятларидан тўлиқ фойдаланиш, ҳамкорликда куч ва воситаларни тўғри тақсимлаган ҳолда самарали натижаларга эришиш, энг асосийси ҳуқуқбузарликларнинг барвақт олдини олиш механизмини яратиб берди. 9 Шавкат Мирзиёев. Миллий тараққиёт йўлимизни қатъият билан давом эттириб, янги босқичга кўтарамиз. Т. 2017 йил Б. 312,313 10 Ўзбекистон Республикаси Президентининг 2017 йил 14 мартдаги “Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш тизимини янада такомиллаштириш чора-тадбирлари тўғрисида”ги ПҚ-2833-сон Қарори. 9 Шавкат Мирзиёев. Миллий тараққиёт йўлимизни қатъият билан давом эттириб, янги босқичга кўтарамиз. Т. 2017 йил Б. 312,313 10 Ўзбекистон Республикаси Президентининг 2017 йил 14 мартдаги “Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш тизимини янада такомиллаштириш чора-тадбирлари тўғрисида”ги ПҚ-2833-сон Қарори. ХУЛОСА Ҳуқуқбузарликларга муросасизлик маданиятини шакллантириш, ҳуқуқий нигилизмга барҳам бериш ва фуқароларнинг қонунга итоаткорлик хулқ- атворини оммалаштиришга қаратилган, шу жумладан ички ишлар органлари таянч пунктлари, ҳуқуқни муҳофаза қилувчи ва бошқа давлат идоралари ва ташкилотларида «очиқ эшиклар куни»ни ташкил этиш орқали тизимли чора- тадбирларни амалга ошириш каби тадбирлар амалга оширилмоқда10. а т т т в Мустақиллик йилларида республикада ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш борасида комплекс чора-тадбирлар амалга оширилиб, мамлакатда ҳуқуқ-тартиботни таъминлашда ижобий натижаларга ҳамда криминоген вазиятни сезиларли даражада яхшилашга эришилди. Шу билан бирга, ҳуқуқбузарликлар профилактикасини амалга оширувчи давлат органларининг иш шакли ва услублари, энг аввало ахборот- коммуникация технологияларидан етарли даражада фойдаланилмаётганлиги сабабли ҳозирги кун талабларига тўлиқ жавоб бермайди. Давлат идоралари аксарият ҳолларда ҳуқуқбузарликлар профилактикасини фақатгина ҳуқуқни муҳофаза қилувчи органларнинг вазифаси сифатида баҳолаб, оқибатда ушбу фаолиятга лозим даражада эътибор қаратмаяпти11. Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш борасидаги чора-тадбирларнинг аниқ манзилга йўналтирилмаганлиги ва уларга комплекс ёндашилмаётганлиги, шунингдек ҳуқуқбузарликларнинг тизимли равишда содир этилишига доир сабаб ва шарт-шароитларни аниқлаш ва уларни бартараф этиш бўйича чора-тадбирларни ишлаб чиқиш самарадорлиги мавжуд эмаслиги кутилаётган натижаларни бермаяпти. Ваколатли органларнинг етарли даражада ташаббус кўрсатмаётганлиги, лозим даражадаги идоралараро ҳамкорликнинг мавжуд эмаслиги, амалга оширилаётган чора-тадбирларнинг ўзаро номутаносиблиги ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш соҳасидаги фаолиятни тубдан такомиллаштиришни талаб этади. Шу босидан, ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш бўйича фаолиятни мувофиқлаштиришнинг таъсирчан тизимини яратиш, қонун бузилишларининг олдини олиш ва уларни бартараф этишнинг замонавий ташкилий-ҳуқуқий механизмларини жорий этиш мақсадида биринчидан, давлат органларининг, шу жумладан ҳуқуқни муҳофаза қилувчи, маҳаллий давлат ҳокимияти органларининг ҳуқуқбузарликлар профилактикаси соҳасидаги ишлари қониқарсиз деб эътироф этилди, иккинчидан, ҳуқуқбузарликларнинг самарали профилактикасини амалга ошириш давлат органлари, шу жумладан ҳуқуқни муҳофаза қилувчи, маҳаллий давлат ҳокимияти органлари, бошқа давлат ташкилотлари, шунингдек хўжалик бошқаруви органларининг устувор вазифаси этиб белгиланди12. ФОЙДАЛАНИЛГАН АДАБИЁТЛАР РЎЙХАТИ: ЙХАТИ: 11 Ўзбекистон Республикаси Президентининг 2017 йил 14 мартдаги ПҚ-2833-сон «Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш тизимини янада такомиллаштириш чора-тадбирлари тўғрисида»ги Қарори // URL: http://www.lex.uz. 11 Ўзбекистон Республикаси Президентининг 2017 йил 14 мартдаги ПҚ-2833-сон «Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш тизимини янада такомиллаштириш чора-тадбирлари тўғрисида»ги Қарори // URL: http://www.lex.uz. 12 Ўзбекистон Республикаси Президентининг 2017 йил 14 мартдаги ПҚ-2833-сон «Ҳуқуқбузарликлар профилактикаси ва жиноятчиликка қарши курашиш тизимини янада такомиллаштириш чора-тадбирлари тўғрисида»ги Қарори // URL: http://www.lex.uz. 1.Одилқориев Ҳ. Т., Тультеев И. Т. ва бошқ. Давлат ва ҳуқуқ назарияси. –Т., 2009. 2009. 2.Зарипов С.З. ва бошқалар. Вояга етмаганлар ҳуқуқбузарликларининг олдини олишда ҳуқуқий тарбия воситаларининг аҳамияти. Ўқитувчилар учун қўлланма. Тошкент, 1997. 2.Зарипов С.З. ва бошқалар. Вояга етмаганлар ҳуқуқбузарликларининг олдини олишда ҳуқуқий тарбия воситаларининг аҳамияти. Ўқитувчилар учун қўлланма. Тошкент, 1997. 3.Мирзажанов Қ. Вояга етмаганлар ҳуқуқбузарликларининг олдини олишда ҳуқуқий тарбия воситаларининг аҳамияти. Тошкент 1997. 3.Мирзажанов Қ. Вояга етмаганлар ҳуқуқбузарликларининг олдини олишда ҳуқуқий тарбия воситаларининг аҳамияти. Тошкент 1997. 4.Абдурасулова Қ.Р. Вояга етмаганлар жиноятчилиги ва унинг олдини олиш муаммолари // Қонун ҳимоясида. –2001. –№ 1. –23–24-б. 5.“Ёшларга оид давлат сиёсатининг ҳуқуқий асосларини такомиллаштириш” мавзусидаги илмий-назарий конференция материаллари. – Т ТДЮИ ё 2008 253 б Т.: ТДЮИ нашриёти, 2008. 253 бет. 6.Исмоилов Н. Т., Жаҳонгиров И. С., Ў.Т. Хушвақтов Вояга етмаганлар ҳуқуқбузарликларининг якка тартибдаги профилактикаси: Ўқув-амалий қўлланма. – Т.: Ўзбекистон Республикаси ИИВ Академияси, 2010. – 114 б. 7.И.Исмаилов, М. Зиёдуллаев, Н.Исроилова, Ф. Азимова. вояга етмаганлар назоратсизлиги ва ҳуқуқбузарликларнинг олдини олиш: Ўқув-амалий қўлланма / Масъул муҳаррир И.Исмаилов. – Т.: Республика болалар ижтимоий мослашув маркази, 2011. – Б – 19. 8.Абдуганиев У. Личность несовершеннолетнего преступника: Учебно- практическое пособие. –Т., 1995. 8.Абдуганиев У. Личность несовершеннолетнего преступника: Учебно- практическое пособие. –Т., 1995. практическое пособие. Т., 1995. 9.Абызов Р. М. Кабулов Р.К. Прогнозирование и профилактика преступного поведения несовершеннолетних. –Т.,1991. 9.Абызов Р. М. Кабулов Р.К. Прогнозирование и профилактика преступного поведения несовершеннолетних. –Т.,1991. 9.Абызов Р. М. Кабулов Р.К. Прогнозирование и профилактика преступного поведения несовершеннолетних. –Т.,1991.
https://openalex.org/W3168925841	https://www.shs-conferences.org/articles/shsconf/pdf/2021/21/shsconf_icemt2021_01014.pdf	English	null	Prospects for Improving the Marketing Policy of Huawei in the Context of the Trade War between the United States and China	SHS web of conferences	2,021	cc-by	3,090	* Corresponding author: cvetlana.staryx.87@mail.ru SHS Web of Conferences 110, 01014 (2021) ICEMT 2021 SHS Web of Conferences 110, 01014 (2021) ICEMT 2021 https://doi.org/10.1051/shsconf/202111001014 © 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/). Prospects for Improving the Marketing Policy of Huawei in the Context of the Trade War between the United States and China Svetlana Staryh, Natalya Derkach, Svetlana Lavoshnikova, and Anastasia Chesnokova South-West State University, Kursk, Russia SHS Web of Conferences 110, 01014 (2021) ICEMT 2021 SHS Web of Conferences 110, 01014 (2021) https://doi.org/10.1051/shsconf/202111001014 Huawei, which significantly reduced their competitiveness and impeded the development of high-tech industries in China [3]. The administration of the new US President Joseph Biden has not yet taken decisive measures to eliminate the existing contradictions between the United States and China. At the same time, the media often speculates about the relaxation of trade conflicts after the revision of sanctions against Huawei, SMIC, and many other Chinese technology companies. As long as the sanctions against "Huawei" are not suspended, we will consider the topic of the trade war between the United States and China as relevant [4]. p [ ] During 2015 - 2019 China's GDP was approximately 2 times lower than that of the United States. At the same time, China's GDP grew by an average of 7% per year, while the same indicator for the United States was only 4% [5]. In 2018, China surpassed the United States in terms of GDP in terms of PPP in the following ratio: 25.2 trillion dollars from the PRC against 20.4 trillion dollars from the USA. At the same time, China's share in world GDP based on PPP increased from 2.3% in 1980 to about 18.3% in 2017, while the share of the United States in world GDP based on PPP fell from 24.3% to about 15.3% [6]. China's short-term economic superiority was impressive, especially given that in 1980, China's PPP GDP was only one-tenth of that of the United States. The IMF predicts that by 2024, China's economy will be 56% larger than the US economy based on PPP [6]. One of the central issues of the US economic relations with China for the US administration is the issue of the trade deficit. Despite tough measures from the United States to reduce the trade deficit with China, this figure has continued to rise in recent years [4]. The US trade balance with China at its peak in absolute terms in 2019 amounted to 48% of the US trade deficit (Table 1). Table 1. Prospects for Improving the Marketing Policy of Huawei in the Context of the Trade War between the United States and China Svetlana Staryh, Natalya Derkach, Svetlana Lavoshnikova, and Anastasia Chesnokova South-West State University, Kursk, Russia Abstract. The article examines the consequences of the trade war between the United States and China using the example of Apple and Huawei. Based on the calculation of the integral indicator, technical and economic parameters of the competitiveness of the products of these organizations were obtained. It was concluded that large-scale forms of competitive struggle are acquiring the character of a trade war, increasingly restraining the opponent with the help of the non-price factor of competition. In these conditions, the question of the prospects for further improving the marketing policy of Huawei is relevant. In recent decades, the growth of China's economic power has been accompanied by a decline in the US share of global production and international trade. This led to a change in the geopolitical landscape of the world and the emergence of the" Group of Two Countries", or G2 for short [1]. The escalation of mutual claims initiated by the Trump administration, which began in 2017, resulted in a war of tariffs and sanctions, which in turn continues to have a very negative impact on relations between the two countries [2]. y g p [ ] In this regard, the problem of transforming marketing policy by the protracted nature of the sanctions needs a practical justification. Recent achievements on the topic are devoted to the study of American-Chinese trade and economic relations at the turn of the presidency in the United States, consideration of digital marketing strategies of large technology companies. In this paper, we consider it necessary to analyze the competitiveness of smartphones and, based on the results obtained, consider the prospects for improving the marketing policy of Huawei. g p y The main purpose of this article is to study the marketing policy of Huawei in the context of the trade war between the United States and China. The main tasks of the work are to analyze the competitiveness of smartphones from large manufacturers of both countries, to identify the potential for promoting Huawei products in the international market. The last major criticism from the United States to China was an accusation of stealing hundreds of millions of dollars worth of American intellectual property. The consequences of this accusation were unilateral sanctions against the Chinese companies ZTE and © 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/). Prospects for Improving the Marketing Policy of Huawei in the Context of the Trade War between the United States and China Svetlana Staryh, Natalya Derkach, Svetlana Lavoshnikova, and Anastasia Chesnokova South-West State University, Kursk, Russia Trade balance, billions of dollars 2015 2016 2017 2018 2019 Trade balance of the USA and China -367,33 -346,83 -375,17 -418,95 -345,20 US trade balance -745,48 -735,33 -792,40 -872,04 -854,37 China's trade balance 678,7 610,6 489,2 382,4 429,6 2019 was the third consecutive year of declines in global sales (second in the analyzed period), even with the emergence of a new 5G market and the supply of smartphones with new technologies, including foldable models (Table 2). 2019 proved to be successful for Samsung, Xiaomi, and OPPO. The company "Huawei" holds a leading position in terms of growth rate throughout the entire period under review. In 2018, Samsung and Apple recorded a 7.7% and 1.7% drop in smartphone sales, respectively, while Huawei posted a 30.1% increase. In mid-June 2020, it became known that Huawei became the world's largest smartphone manufacturer for the first time and in April 2020 captured 21.4% of the global market [7]. Since the beginning of 2020, this Chinese company has increased its market share by 6%. During this time, the share of Samsung increased by 2%, and Apple decreased by 4%. Let us analyze the competitiveness of the products of these two companies in the conditions of comparable comparability of Apple and Huawei in the current economic conditions [8]. Table 1. Trade balance, billions of dollars 2015 2016 2017 2018 2019 Trade balance of the USA and China -367,33 -346,83 -375,17 -418,95 -345,20 US trade balance -745,48 -735,33 -792,40 -872,04 -854,37 China's trade balance 678,7 610,6 489,2 382,4 429,6 2019 was the third consecutive year of declines in global sales (second in the analyzed period), even with the emergence of a new 5G market and the supply of smartphones with new technologies, including foldable models (Table 2). 2019 proved to be successful for Samsung, Xiaomi, and OPPO. The company "Huawei" holds a leading position in terms of growth rate throughout the entire period under review. In 2018, Samsung and Apple recorded a 7.7% and 1.7% drop in smartphone sales, respectively, while Huawei posted a 30.1% increase. 2019 was the third consecutive year of declines in global sales (second in the analyzed period), even with the emergence of a new 5G market and the supply of smartphones with new technologies, including foldable models (Table 2). 2019 proved to be successful for Samsung, Xiaomi, and OPPO. g [ ] Let's define the group indicators of competitiveness: Let s define the group indicators of competitiveness: Prospects for Improving the Marketing Policy of Huawei in the Context of the Trade War between the United States and China Svetlana Staryh, Natalya Derkach, Svetlana Lavoshnikova, and Anastasia Chesnokova South-West State University, Kursk, Russia The company "Huawei" holds a leading position in terms of growth rate throughout the entire period under review. In 2018, Samsung and Apple recorded a 7.7% and 1.7% drop in smartphone sales, respectively, while Huawei posted a 30.1% increase. In mid-June 2020, it became known that Huawei became the world's largest smartphone manufacturer for the first time and in April 2020 captured 21.4% of the global market [7]. Since the beginning of 2020, this Chinese company has increased its market share by 6%. During this time, the share of Samsung increased by 2%, and Apple decreased by 4%. Let us analyze the competitiveness of the products of these two companies in the conditions of comparable comparability of Apple and Huawei in the current economic conditions [8]. 2 2 https://doi.org/10.1051/shsconf/202111001014 SHS Web of Conferences 110, 01014 (2021) ICEMT 2021 Table 2. Global smartphone sales 2019 2018 2017 Increment Sales, mln. Market share, % Sales, mln. Market share, % Sales, mln. Market share, % 2018- 2019 2017- 2018 Samsung 298,1 21,8 293,3 21,1 317,7 21,7 1,6% -7,7% Huawei 240,6 17,6 206,0 14,8 154,2 10,5 16,8% 33,6% Apple 198,1 14,5 212,2 15,3 215,8 14,7 -6,6% -1,7% Xiaomi 125,5 9,2 120,6 8,7 92,7 6,3 4,1% 30,1% Oppo 120,2 8,8 116,0 8,3 111,7 7,6 3,6% 3,8% Others 384,3 28,1 441,4 31,8 573,4 39,1 - 12,9% - 23,0% Total 1366,8 100,0 1389,5 100,0 1465,5 100,0 -1,6% -5,2% We will analyze the competitiveness of Apple and Huawei products using the example of smartphones in 2019 - iPhone 11 and Huawei P40. Since the leader in the field of smartphones is Samsung, the Samsung Galaxy S10 was taken as a sample, which, according to the ratings of several expert agencies, holds a leading position in the rating of smartphones in 2019 [9]. For the technical and economic indicators of comparison, the most important criteria were taken when choosing a smartphone. Expert assessments are the basis for determining the weight of each parameter in the general set. Table 3 Technical and economic indicators of smartphones We will analyze the competitiveness of Apple and Huawei products using the example of smartphones in 2019 - iPhone 11 and Huawei P40. Since the leader in the field of smartphones is Samsung, the Samsung Galaxy S10 was taken as a sample, which, according to the ratings of several expert agencies, holds a leading position in the rating of smartphones in 2019 [9]. To distribute the models shown in Table 3 by the level of competitiveness, it is first necessary to calculate the simple indicators for technical and economic parameters relative to the basic model "Samsung" [10]. Prospects for Improving the Marketing Policy of Huawei in the Context of the Trade War between the United States and China Svetlana Staryh, Natalya Derkach, Svetlana Lavoshnikova, and Anastasia Chesnokova South-West State University, Kursk, Russia Let's define the group indicators of competitiveness: 3 SHS Web of Conferences 110, 01014 (2021) SHS Web of Conferences 110, 01014 (2021) ICEMT 2021 https://doi.org/10.1051/shsconf/202111001014 By technical parameter: By technical parameter: GT1 = 0.91 0.5 + 1 * 0.2 + 0.75 * 0.2 + 1.24 * 0.1 = 0.93 (1) GT2 = 1.12 * 0.5 + 1 * 0.2 + 3.13 * 0.2 + 1.11 * 0.1 = 1.5 (2) By economic parameter: Ge1 = 1.01 * 0.7 + 0.93 * 0.3 = 0.99 (3) Ge2 = 0.74 * 0.7 + 1.01 * 0.3 = 0.82 (4) By technical parameter: GT1 = 0.91 * 0.5 + 1 * 0.2 + 0.75 * 0.2 + 1.24 * 0.1 = 0.93 (1) GT2 = 1.12 * 0.5 + 1 * 0.2 + 3.13 * 0.2 + 1.11 * 0.1 = 1.5 (2) By economic parameter: G 1 1 01 * 0 7 0 93 * 0 3 0 99 (3) By technical parameter: GT1 = 0.91 * 0.5 + 1 * 0.2 + 0.75 * 0.2 + 1.24 * 0.1 = 0.93 (1) GT2 = 1.12 * 0.5 + 1 * 0.2 + 3.13 * 0.2 + 1.11 * 0.1 = 1.5 (2) B i By economic parameter: Ge1 = 1.01 * 0.7 + 0.93 * 0.3 = 0.99 (3) Ge2 = 0.74 * 0.7 + 1.01 * 0.3 = 0.82 (4) (3) (4) The resulting group indicator Gтn characterizes the degree of compliance of a given product with the existing need for the entire set of technical parameters. Since the Huawei P40 has a higher figure, it generally satisfies the needs of consumers better than the iPhone 11. Calculations based on economic parameters show that the second model is more competitive, as it has the smallest group indicator equal to 0.82. Let's calculate the integral generall indicator of competitiveness (Table 4): I1 = Gt1 / Ge1 = 0.93 / 0.99 = 0.94 (5) I2 = Gt2 / Ge2 = 1.5 / 0.82 = 1.83 (6) (5) (6) ( ) (6) Table 4. Calculation of single indicators Table 4. Calculation of single indicators Single indicators of competitiveness (gi = pi / p0) Apple iPhone 11 (g1) Huawei P40 (g2) 1. Technical indicators 1.1 Battery capacity, mAh 0,91 1,12 1.2 Screen, inches 1,00 1,00 1.3 Camera (rear), MPix 0,75 3,13 1.4 Weight, g 1,24 1,11 2.Economic indicators 2.1 Price, rub. Prospects for Improving the Marketing Policy of Huawei in the Context of the Trade War between the United States and China Svetlana Staryh, Natalya Derkach, Svetlana Lavoshnikova, and Anastasia Chesnokova South-West State University, Kursk, Russia For the technical and economic indicators of comparison, the most important criteria were taken when choosing a smartphone. Expert assessments are the basis for determining the weight of each parameter in the general set. Table 3. Technical and economic indicators of smartphones Indicators Apple iPhone 11 Huawei P40 Sample Samsung Galaxy S10 Weight coefficient p1 p2 p0 Ai 1. Technical indicators 1.1 Battery capacity, mAh 3110 3800 3400 0,5 1.2 Screen, inches 6,1 6,1 6,1 0,2 1.3 Camera (rear), MPix 12 50 16 0,2 1.4 Weight, g 194 175 157 0,1 2.Economic indicators 2.1 Price, rub. 54490 39999 53990 0,7 2.2 Smartphone processor speed, MHz 2650 2860 2840 0,3 To distribute the models shown in Table 3 by the level of competitiveness, it is first necessary to calculate the simple indicators for technical and economic parameters relative to the basic model "Samsung" [10]. L t' d fi th i di t f titi Table 3. Technical and economic indicators of smartphones To distribute the models shown in Table 3 by the level of competitiveness, it is first necessary to calculate the simple indicators for technical and economic parameters relative to the basic model "Samsung" [10]. To distribute the models shown in Table 3 by the level of competitiveness, it is first necessary to calculate the simple indicators for technical and economic parameters relative to the basic model "Samsung" [10]. SHS Web of Conferences 110, 01014 (2021) ICEMT 2021 https://doi.org/10.1051/shsconf/202111001014 - withdrawal of new products from sanctions by selling subsidiaries; - developing a regulatory framework to provide access to their patents for 5G wireless communications [11]. - developing a regulatory framework to provide access to their patents for 5G wireless communications [11]. Thus, we can conclude that in the first half of 2020, Huawei continued to increase its share of the smartphone market, develop new high-tech products, attracting buyers to the market of Chinese developers. The above calculation of the integral indicator of competitiveness considers only technical and economic parameters. At the same time, the presence of Google services in them is a significant aspect in the choice of modern models of smartphones based on Android OS. This nuance reduces the demand for Huawei smartphones, allowing competitors to take leading positions by taking advantage of non- price competition. In turn, the United States, in the aggregate of all measures restraining the Chinese economy, uses this aspect to reduce the smartphone market share of a strong rival. Improved marketing policy should largely ease the sanctions, allowing the company to remain in the lead in the supply of smartphones. The research paper is written as part of the state grant for 2021 (№0851-2020-0034). Prospects for Improving the Marketing Policy of Huawei in the Context of the Trade War between the United States and China Svetlana Staryh, Natalya Derkach, Svetlana Lavoshnikova, and Anastasia Chesnokova South-West State University, Kursk, Russia 1,01 0,74 2.2 Smartphone processor speed, MHz 0,93 1,01 From the calculations of the integral indicator, it can be seen that the Huawei P40 smartphone is more competitive than the iPhone 11 since the value of this indicator is greater than 1. g Based on the conducted study, it can be concluded that today the company "Huawei" is a fairly efficiently functioning company. Despite serious sanctions restrictions, the foundation for the company's further sustainable development lies in the development of an effective digital marketing strategy. g g gy The prospects for improving Huawei's marketing policy are as follows: g g gy The prospects for improving Huawei's marketing policy are as follows: - the use of the ecosystem in the development and creation of new devices; - the use of the ecosystem in the development and creation of new devi - transition to its software, the attraction of specialists for the manufacture of competitive analogs of American components; - search for alternative ways to use Google services, such as the re-release of old rsions of smartphones; 4 SHS Web of Conferences 110, 01014 (2021) ICEMT 2021 References 1. W. M. Morrison, China’s Economic Rise: History, Trends, Challenges, Implications for the United States, CRS, 38 (2019) 2. N. Y. Chetyrkina, Features of regulation of competitive relations, VFU, 2, 50-55 (2017) 3. V. B. Supyan, U.S.-China Trade and Economic Relations: Causes of Crisis and Its Prospects, RFEB, 9, 23-32 (2019) 4. L. Pan, The state and problems of China's foreign trade activities in the face of a trade war, RFEB, 11, 112-118 (2019) 5. H. Schweitzer, The New Competition Tool: Its institutional set-up and procedural design, Pub. Office of the EU, 52 (2020) 6. А. А. Ryazanov, Evolution of Competition Theory, Mos. Witte Un. 2, 21-29 (2017). 7. А. О. Bychkova, Analysis of the Competitiveness of Smartphones, Alley.science, 15, 425-428 (2017) 8. Counterpoint (2020) // https://www.counterpointresearch.com/global-smartphone- share/ 9. N. S. Kuchma, US – China trade war: reaction of stock exchanges to the transformation of the foreign policy agenda, RUDN J. of Economics, 3, 415-428 (2019) 10. M.D. Seditov, N.E. Shashkov, Methods for assessing the competitiveness of the company, AASR, 80-84 (2020). 11. F. Kayin, Exploring Huawei's Digital Marketing Strategy, N. Sc. 16-20 (2020) 5 5
https://openalex.org/W4310340376	https://zenodo.org/records/7375805/files/AES-30(online023).pdf	Polish	null	Pluskwiaki różnoskrzydłe (Hemiptera: Heteroptera) w Muzeum Tatrzańskm im. dra Tytusa Chałubińskiego w Zakopanem	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	4,464	A c t a e n t o m o l o g i c a s i l e s i a n a A c t a e n t o m o l o g i c a s i l e s i a n a 023): 1–9 ISSN 1230-7777, ISSN 2353-1703 (online) SN 1230-7777, ISSN 2353-1703 (online) Vol. 30: (online 023): 1–9 ISSN 1230-7777, ISSN 2353-1703 (online) Vol. 30: (online 023): 1–9 Bytom, November 29, 2022 KEY WORDS: museum collection, faunistics, new data, first record, Poland, Slovakia. KEY WORDS: museum collection, faunistics, new data, first record, Poland, Slovakia. EY WORDS: museum collection, faunistics, new data, first record, Poland, Slovakia. Pluskwiaki różnoskrzydłe (Hemiptera: Heteroptera) w Muzeum Tatrzańskm im. dra Tytusa Chałubińskiego w Zakopanem http://doi.org/10.5281/zenodo.7375805 Grzegorz Gierlasiński1 , Marcin Warchałowski2 1 Zbiory Przyrodnicze, Wydział Biologii, Uniwersytet Adama Mickiewicza w Poznaniu, ul. Uniwersytetu Poznańskiego 6, 61-614 Poznań, e-mail: ggierlas@gmail.com, ORCID 0000-0002-2968-8553 2 Muzeum Tatrzańskie im. dra Tytusa Chałubińskiego w Zakopanem, ul. Krupówki 10, 34-500 Zakopane, 1 Zbiory Przyrodnicze, Wydział Biologii, Uniwersytet Adama Mickiewicza w Poznaniu, ul. Uniwersytetu Poznańskiego 6, 61-614 Poznań, e-mail: ggierlas@gmail.com, ORCID 0000-0002-2968-8553 1 Zbiory Przyrodnicze, Wydział Biologii, Uniwersytet Adama Mickiewicza w Poznaniu, ul. Uniwersytetu Poznańskiego 6, 61-614 Poznań, e-mail: ggierlas@gmail.com, ORCID 0000-0002-2968-8553 2 Muzeum Tatrzańskie im. dra Tytusa Chałubińskiego w Zakopanem, ul. Krupówki 10, 34-500 Zakopane, Polska, ORCID 0000-0001-5886-5697 2 Muzeum Tatrzańskie im. dra Tytusa Chałubińskiego w Zakopanem, ul. Krupówki 10, 34-500 Zakopane, Polska, ORCID 0000-0001-5886-5697 ABSTRACT. True bugs (Hemiptera: Heteroptera) in the collection of the Tatra Museum of Dr Tytus Chałubinski, Zakopane. The paper deals with the species of water and terrestrial true bugs stored in the collection of the Tatra Museum in Zakopane. A total 233 specimens of 39 species were identified and listed. Four species – Grypocoris sexguttatus, Harpocera thoracica, Lygus wagneri, and Himacerus mirmicoides – were recorded from Nowotarska Dale for the first time. The vast majority of the material collected in the years 1932-1983 was obtained in the Tatra Mountains and Podhale region. ABSTRACT. True bugs (Hemiptera: Heteroptera) in the collection of the Tatra Museum of Dr Tytus Chałubinski, Zakopane. The paper deals with the species of water and terrestrial true bugs stored in the collection of the Tatra Museum in Zakopane. A total 233 specimens of 39 species were identified and listed. Four species – Grypocoris sexguttatus, Harpocera thoracica, Lygus wagneri, and Himacerus mirmicoides – were recorded from Nowotarska Dale for the first time. The vast majority of the material collected in the years 1932-1983 was obtained in the Tatra Mountains and Podhale region. WSTĘP Rola muzeów przyrodniczych była w literaturze wielokrotnie dyskutowana (Lane 1996, Taszakowski et al. 2019). Poza najważniejszymi zadaniami obejmującymi między innymi pozyskiwanie nowych eksponatów, ich zabezpieczenie i naukowe opracowywanie, muzea historii naturalnej stanowią wyjątkową przestrzeń dla badań interdyscyplinarnych. Wszystkie razem, w dobie cyfryzacji, są czymś więcej niż tylko sumą swoich części. Umożliwiają zarówno rozpoznawanie problemów w skali globalnej (wynikające na przykład ze zmian klimatycznych), jak i znajdowanie odpowiedzi na nie (Bakker et al. 2020). Warto podkreślić, że wymienione wyżej działania są ściśle związane z zagadnieniem, które zwróciło uwagę badaczy na zbiory przyrodnicze zdeponowane w muzeach. Problemem tym jest obserwowany w ostatnim czasie spadek bioróżnorodności, nazywany często „szóstym wymieraniem” (Barnosky et al. 2011), spowodowany gwałtowną presją człowieka na środowisko (Tollefson 2019, Turvey & Crees 2019). Jednym z pierwszych zadań stawianych nowo powstałym muzeom jest dokumentacja zasobów naturalnych danego regionu (Lane 1996). Podobnie było również w przypadku Muzeum Tatrzańskiego (Trebunia-Staszel 2018). Początki tej instytucji sięgają końca XIX wieku, a idea jego założenia zrodziła się w gronie przyjaciół wybitnego lekarza i przyrodnika Tytusa Chałubińskiego. Najstarsze zbiory zgromadzone zostały drogą darów i kupna z rąk prywatnych zbieraczy. Wśród pierwszych kolekcji znajduje się zbiór dermoplastów Antoniego Kocyana oraz zielników Tytusa Chałubińskiego. Początkowo kolekcjonowano głównie materiały botaniczne, geologiczne i zoologiczne (Wójcik 1 1 Acta ent. siles. 30 (online 023) Bytom, November 29, 2022 2012), z czasem również etnograficzne (Trebunia-Staszel 2018). Mimo systematycznej rozbudowy kolekcji przez ostatnie dziesięciolecia, do dziś zbiory owadów są tylko częściowo opracowane (Cichocki & Cisło 1985, Lis J.A. 1990, Warchałowski & Pietraszko 2017, Pietraszko & Warchałowski 2018, Buszko et al. 2000). 2012), z czasem również etnograficzne (Trebunia-Staszel 2018). Mimo systematycznej rozbudowy kolekcji przez ostatnie dziesięciolecia, do dziś zbiory owadów są tylko częściowo opracowane (Cichocki & Cisło 1985, Lis J.A. 1990, Warchałowski & Pietraszko 2017, Pietraszko & Warchałowski 2018, Buszko et al. 2000). 2012), z czasem również etnograficzne (Trebunia-Staszel 2018). Mimo systematycznej rozbudowy kolekcji przez ostatnie dziesięciolecia, do dziś zbiory owadów są tylko częściowo opracowane (Cichocki & Cisło 1985, Lis J.A. 1990, Warchałowski & Pietraszko 2017, Pietraszko & Warchałowski 2018, Buszko et al. 2000). Obecnie w zbiorach Muzeum znajdują się także dwie kolekcje zawierające pluskwiaki różnoskrzydłe. Pierwsza z nich opiera się na materiale zgromadzonym przez Daphne Aubertin w latach 30. ubiegłego stulecia, druga z kolei, czterdzieści lat młodsza, zawiera owady zebrane przez Mieczysława Gałuszkę. WSTĘP Daphne Aubertin była asystentem kustosza w Katedrze Entomologii w londyńskim Muzeum Historii Naturalnej przez blisko osiem lat począwszy od lipca 1927 roku (Bionomia 2022, WorldCat 2022). Była także członkinią Towarzystwa Linneuszowskiego w Londynie (Wikidata 2022). W swojej działalności naukowej koncentrowała się przede wszystkim na zagadnieniach z malakologii i entomologii. W tej drugiej dziedzinie jej zainteresowania skupiały się na muchach, w szczególności na rodzinach Conopidae i Striatomyiidae (Aubertin 1930, Aubertin & Malloch 1933). Mieczysław Gałuszka był z zawodu pracownikiem bankowym, a entomologią zajmował się amatorsko. Swoje zbiory – głównie Hymenoptera i Coleoptera – gromadził w okolicach Krakowa i w Tatrach jeszcze po 90-tym roku życia (Cichocki & Cisło 1999). Celem niniejszej pracy jest opracowanie kolekcji Heteroptera znajdującej się w posiadaniu Muzeum Tatrzańskiego im. dra Tytusa Chałubińskiego. MATERIAŁ I METODY W niniejszej pracy przedstawiono niepublikowane wcześniej dane faunistyczne oparte w całości na zbiorach zdeponowanych w Muzeum Tatrzańskim im. dra Tytusa Chałubińskiego w Zakopanem. W pracy przyjęto granice regionów zoogeograficznych na podstawie Katalogu fauny Polski [KFP] (Burakowski et al. 1973). Klasyfikację gatunków przyjęto za Schuh & Weirauch 2020, nazewnictwo gatunków za „Catalogue of the Palaearctic Heteroptera” (Aukema 2022). Oznaczeń dokonano za pomocą następujących kluczy do oznaczania: Gierlasiński et al. (2019, 2020), Gorczyca (2004, 2007), Gorczyca & Wolski (2011), Jaczewski & Wróblewski (1976, 1978), Lis J.A. (2000), Lis B. et al. (2008), Lis J.A. et al. (2012), Péricart (1972, 1998a, b, c), Wagner & Weber (1964). Wykaz rodzin i gatunków przedstawiono w układzie alfabetycznym. Skróty zastosowane w pracy: DA – D. Aubertin, MG – M. Gałuszka. Brak ilości podanej po dacie w spisie danych oznacza jeden okaz. Przy stanowiskach leżący na terenie Polski w nawiasach kwadratowych podano współrzędne siatki UTM. Lygaeidae Lygaeus equestris (Linnaeus, 1758) Tatry: Dolina Kościeliska [DV15], 13.07.1967, 2 exx., leg. MG. Cydnidae Sehirus morio (Linnaeus, 1761) Tatry: Dolina Cicha [DV25], 26.06.1971, leg. MG. Sehirus morio (Linnaeus, 1761) Tatry: Dolina Cicha [DV25], 26.06.1971, leg. MG. Cymidae Cymus glandicolor Hahn, 1832 Kotlina Nowotarska: Zakopane [DV26], 28.06.1932, leg. DA. Słowacja: Ružomberok, 5.06.1932, leg. DA; Tatranská Polianka, 18.06.1932, 21.06.1932, 2 exx., leg. DA. Aradus aterrimus Fieber, 1864 Aradus aterrimus Fieber, 1864 Tatry: Dolina Pięciu Stawów [DV25], 27.07.1977, leg. MG. Uwaga: gatunek podany wcześniej ogólnikowo z Tatr (Lis J.A. 1990) na podstawie wymienionego wyżej okazu. Tatry: Dolina Pięciu Stawów [DV25], 27.07.1977, leg. MG. Uwaga: gatunek podany wcześniej ogólnikowo z Tatr (Lis J.A. 1990) na podstawie wymienionego wyżej okazu. Gerridae Gerris lacustris (Linnaeus, 1758) Tatry: Toporowy Staw [DV25], 25.08.1982, 10 exx., 9.06.1983, 6 exx., leg. MG. Gerris odontogaster (Zetterstedt, 1828) Tatry: Toporowy Staw [DV25], 23.07.1982, 2 exx., leg. MG. Gerris thoracicus Schummel, 1832 Tatry: Dolina Pięciu Stawów [DV25], 5.07.1932, leg. DA; Hala Gąsienicowa [DV25], 30.06.1932, 2 exx., leg. DA; Toporowy Staw [DV25], 9.08.1982, 23.07.1982, 5 exx., 22.08.1982, 3 exx., leg. MG. Coreidae Coreus marginatus (Linnaeus, 1758) Słowacja: Dobšiná, 1.06.1932, 2 exx., leg. DA; Tatranská Polianka, 12.06.1932, 2 exx., 28.05.1932, 2 exx., leg. DA. Anthocoridae Anthocoris nemorum (Linnaeus, 1761) Tatry: Dolina Małej Łąki [DV25], 14.06.1978, leg. MG; Dolina Olczyska [DV25], 27.07.1978, leg. MG; Hala Goryczkowa [DV25], 3.07.1977, leg. MG. Anthocoris nemorum (Linnaeus, 1761) Tatry: Dolina Małej Łąki [DV25], 14.06.1978, leg. MG; Dolina Olczyska [DV25], 27.07.1978, leg. MG; Hala Goryczkowa [DV25], 3.07.1977, leg. MG. 2 2 Acta ent. siles. 30 (online 023) Bytom, November 29, 2022 Closterotomus fulvomaculatus (De Geer, 1773) Cremnocephalus alpestris Wagner, 1941 Kotlina Nowotarska: Zakopane [DV26], 7.07.1979, leg. MG. Tatry: Kościelisko, Gronik [DV15], 21.07.1982, leg. MG; Grypocoris sexguttatus (Fabricius, 1777) Kotlina Nowotarska: Zakopane, Bystre [DV25], 19.07.1982, 4 exx., leg. MG; Tatry: Dolina Filipki [DV35], 21.08.1974, leg. MG; Dolina Małej Łąki [DV25], 3.08.1980, leg. MG; Dolina Suchej Wody [DV25], 6.08.1982; 2 exx., leg. MG; Polana Brzeziny [DV25], 20.07.1980, leg. MG; Dolina Roztoki [DV25], 17.07.1973, leg. MG. Gatunek nowy dla Kotliny Nowotarskiej. Grypocoris sexguttatus (Fabricius, 1777) Kotlina Nowotarska: Zakopane, Bystre [DV25], 19.07.1982, 4 exx., leg. MG; Tatry: Dolina Filipki [DV35], 21.08.1974, leg. MG; Dolina Małej Łąki [DV25], yp g ( , ) Kotlina Nowotarska: Zakopane, Bystre [DV25], 19.07.1982, 4 exx., leg. MG; Tatry: Dolina Filipki [DV35], 21.08.1974, leg. MG; Dolina Małej Łąki [DV25], 3.08.1980, leg. MG; Dolina Suchej Wody [DV25], 6.08.1982; 2 exx., leg. MG; Polana Brzeziny [DV25], 20.07.1980, leg. MG; Dolina Roztoki [DV25], 17.07.1973, leg. MG. Tatry: Dolina Filipki [DV35], 21.08.1974, leg. MG; Dolina Małej Łąki [DV25], 3.08.1980, leg. MG; Dolina Suchej Wody [DV25], 6.08.1982; 2 exx., leg. MG; Polana y p [ ], , g ; j ą [ 3.08.1980, leg. MG; Dolina Suchej Wody [DV25], 6.08.1982; 2 exx., leg. MG; Po Brzeziny [DV25], 20.07.1980, leg. MG; Dolina Roztoki [DV25], 17.07.1973, leg. MG. Gatunek nowy dla Kotliny Nowotarskiej. Harpocera thoracica (Fallén, 1807) Kotlina Nowotarska: Zakopane, Jaszczurówka [DV26], 29.07.1982, leg. MG. Gatunek nowy dla Kotliny Nowotarskiej. Leptopterna dolabrata (Linnaeus, 1758) Kotlina Nowotarska: Kościelisko, Bory [DV26], 13.08.1982, 7.08.1982, 2 exx., leg. MG; Zakopane, Bystre [DV25], 19.07.1982, 4 exx., leg. MG; Zakopane, Jaszczurówka l Leptopterna dolabrata (Linnaeus, 1758) Kotlina Nowotarska: Kościelisko, Bory [DV26], 13.08.1982, 7.08.1982, 2 exx., leg. MG; Zakopane, Bystre [DV25], 19.07.1982, 4 exx., leg. MG; Zakopane, Jaszczurówka [DV26], 29.07.1982, leg. MG; Tatry: Dolina Małej Łąki [DV25], 7.08.1980, leg. MG; Dolina Tomanowa [DV15], 11.07.1973, leg. MG; Kiry [DV15], 24.07.1982, 4 exx., leg. MG; Kościelisko, Gronik [DV15] 20 07 1982 leg MG; Nosal [DV25] 8 07 1982 4 e leg MG Leptopterna dolabrata (Linnaeus, 1758) Kotlina Nowotarska: Kościelisko, Bory [DV26], 13.08.1982, 7.08.1982, 2 exx., leg. MG; Zakopane, Bystre [DV25], 19.07.1982, 4 exx., leg. MG; Zakopane, Jaszczurówka [DV26], 29.07.1982, leg. MG; Tatry: Dolina Małej Łąki [DV25], 7.08.1980, leg. MG; Dolina Tomanowa [DV15], 11.07.1973, leg. MG; Kiry [DV15], 24.07.1982, 4 exx., leg. MG; Kościelisko, Gronik [DV15], 20.07.1982, leg. MG; Nosal [DV25], 8.07.1982, 4 exx., leg. MG. Lygocoris pabulinus (Linnaeus, 1761) Tatry: Dolina Strążyska [DV25], 27.08.1981, leg. MG; Polana Brzeziny [DV25], 30.08.1980, leg. Lygus wagneri Remane, 1955 Kotlina Nowotarska: Kościelisko, Bory [DV26], 13.08.1982, leg. MG; Zakopane [DV26], 28.06.1932, 3 exx., leg. DA; Zakopane, Bystre [DV25], 23.06.1978, leg. MG; Miridae Calocoris affinis (Herrich-Schaeffer, 1835) Kotlina Nowotarska: Zakopane, Bystre [DV25], 19.07.1982, 2 exx., leg. MG; Zakopane, Jaszczurówka [DV26], 29.07.1982, 4 exx, leg. MG. Tatry: Dolina Lejowa [DV15], 18.08.1982, leg. MG; Polana Brzeziny [DV25], 30 08 1980 leg MG; alocoris affinis (Herrich-Schaeffer, 1835) Kotlina Nowotarska: Zakopane, Bystre [DV25], 19.07.1982, 2 exx., leg. MG Calocoris affinis (Herrich-Schaeffer, 1835) Kotlina Nowotarska: Zakopane, Bystre [DV25], 19.07.1982, 2 exx., leg. MG; Zakopane, Jaszczurówka [DV26], 29.07.1982, 4 exx, leg. MG. a opa e, Jas c u ów a [ V 6], 9.07. 98 , e , eg. G. Tatry: Dolina Lejowa [DV15], 18.08.1982, leg. MG; Polana Brzeziny [DV25], 30.08.1980, leg. MG; 3 Acta ent. siles. 30 (online 023) Bytom, November 29, 2022 Calocoris alpestris (Meyer-Dür, 1843) Calocoris alpestris (Meyer-Dür, 1843) Tatry: Dolina Chochołowska [DV15], 14.07.1978, leg. MG; Dolina Małej Łąki [DV25], 24.07.1971, 17.08.1971, leg. MG; Dolina Tomanowa [DV15], 9.08.1973, 25.08.1978, 16.08.1980, 24.07.1982, 11.07.1982, leg. MG; Dolina Suchej Wody [DV25], 5.08.1977, 15.08.1978, leg. MG; Kościelisko, Gronik [DV15], 15.06.1982, 2 exx., leg. MG; Capsus ater (Linnaeus, 1758) Capsus ater (Linnaeus, 1758) Tatry: Dolina Chochołowska [DV15], 14.07.1978, leg. MG. Tatry: Dolina Chochołowska [DV15], 14.07.1978, leg. MG. Closterotomus fulvomaculatus (De Geer, 1773) Kotlina Nowotarska: Zakopane, Bystre [DV25], 18.07.1982, 2 exx., leg. MG; Zakopane, Pardołówka [DV26], 3.08.1976, 23.06.1979, leg. MG. Megaloceroea recticornis (Geoffroy, 1785) Kotlina Nowotarska: Kościelisko, Bory [DV26], 13.08.1982, 4 exx., 7.08.1982, 6 exx., leg. MG; Zakopane, Bystre [DV25], 15.07.1982, 19.07.1982, 4 exx., leg. MG. Psallus salicis (Kirschbaum, 1856) Tatry: Hala Gąsienicowa [DV25], 13.07.1972, leg. MG. Stenodema calcarata (Fallén, 1807) Kotlina Nowotarska: Zakopane, Bystre [DV25], 4.07.1978, leg. MG; Zakopane, Pardałówka [DV26], 23.06.1979, leg. MG. Stenodema holsata (Fabricius, 1787) Kotlina Nowotarska: Kościelisko, Bory [DV26], 7.08.1982, 6 exx., 13.08.1982, 10 exx., leg. MG; Zakopane, Bystre [DV25], 31.05.1978, 4.07.1978, 4 exx., leg. MG; Zakopane, Jaszczurówka [DV26], 22.08.1982, leg. MG; Zazadnia [DV36], 12.07.1978, leg. MG; Stenodema holsata (Fabricius, 1787) Kotlina Nowotarska: Kościelisko, Bory [DV26], 7.08.1982, 6 exx., 13.08.1982, 10 exx., leg. MG; Zakopane, Bystre [DV25], 31.05.1978, 4.07.1978, 4 exx., leg. MG; Zakopane, Jaszczurówka [DV26], 22.08.1982, leg. MG; Zazadnia [DV36], 12.07.1978, leg. MG; Tatry: Dolina Chochołowska [DV15], 27.06.1976, 23.07.1982, leg. MG; Dolina Kościeliska [DV15], 1.07.1982, leg. MG; Dolina Małej Łąki [DV25], 15.07.1980, 5.08.1980, 19.08.1982, leg. MG; Dolina Olczyska [DV25], 5.07.1980, 22.08.1982, leg. MG; Hala Goryczkowa [DV25], 3.07.1973, 3 exx., 25.06.1977, leg. MG; Kościelisko, Gronik [DV15], 15.06.1982, 3 exx., leg. MG; Kościelisko, Nędzówka [DV15], 3.07.1982, 2 exx., leg. MG; Myślenickie Turnie [DV25], 3.07.1973, leg. MG; Nosal [DV25], 8.07.1982, leg. MG; (ogólnie) [DV25], 6.07.1977, 4.06.1979, 4.07.1978, leg. MG; Tatry: Dolina Chochołowska [DV15], 27.06.1976, 23.07.1982, leg. MG; Dolina Kościeliska [DV15], 1.07.1982, leg. MG; Dolina Małej Łąki [DV25], 15.07.1980, 5.08.1980, 19.08.1982, leg. MG; Dolina Olczyska [DV25], 5.07.1980, 22.08.1982, leg. MG; Hala Goryczkowa [DV25], 3.07.1973, 3 exx., 25.06.1977, leg. MG; Kościelisko, Gronik [DV15], 15.06.1982, 3 exx., leg. MG; Kościelisko, Nędzówka [DV15], 3.07.1982, 2 exx., leg. MG; Myślenickie Turnie [DV25], 3.07.1973, leg. MG; Nosal [DV25], 8.07.1982, leg. MG; (ogólnie) [DV25], 6.07.1977, 4.06.1979, 4.07.1978, leg. MG; Słowacja: Tatranská Kotlina, 20.06.1932, 5 exx., leg. DA. Słowacja: Tatranská Kotlina, 20.06.1932, 5 exx., leg. DA. Closterotomus fulvomaculatus (De Geer, 1773) MG; (ogólnie) [DV25], 28.08.1981, leg. MG. y ą y g 30.08.1980, leg. MG; (ogólnie) [DV25], 28.08.1981, leg. MG. Lygus pratensis (Linnaeus, 1758) Słowacja: Tatranská Kotlina, 18.06.1932, leg. DA. Słowacja: Tatranská Kotlina, 18.06.1932, leg. DA. Lygus wagneri Remane, 1955 Kotlina Nowotarska: Kościelisko, Bory [DV26], 13.08.1982, leg. MG; Zakopane [DV26], 28.06.1932, 3 exx., leg. DA; Zakopane, Bystre [DV25], 23.06.1978, leg. MG; 4 Acta ent. siles. 30 (online 023) Bytom, November 29, 2022 Tatry: Dolina Małej Łąki [DV25], 19.06.1978, leg. MG; Dolina Suchej Wody [DV25], 21.06.1978, leg. MG; Słowacja: Tatranská Kotlina, 18.06.1932, 2 exx., leg. DA. Gatunek nowy dla Kotliny Nowotarskiej Tatry: Dolina Małej Łąki [DV25], 19.06.1978, leg. MG; Dolina Suchej Wody [DV25], 21.06.1978, leg. MG; Sł j T t ká K tli 18 06 1932 2 l DA Słowacja: Tatranská Kotlina, 18.06.1932, 2 exx., leg. DA. Gatunek nowy dla Kotliny Nowotarskiej. Megaloceroea recticornis (Geoffroy, 1785) Nabis limbatus Dahlbom, 1851 Nabis limbatus Dahlbom, 1851 Kotlina Nowotarska: Kościelisko, Bory [DV26], 7.08.1982, 13.08.1982, leg. MG; Zakopane, Bystre [DV25], 19.07.1982, leg. MG; Tatry: Polana Brzeziny [DV25], 30.07.1982, leg. MG. , Kotlina Nowotarska: Kościelisko, Bory [DV26], 7.08.1982, 13.08.1982, leg. MG akopane, Bystre [DV25], 19.07.1982, leg. MG; Zakopane, Bystre [DV25], 19.07.1982, leg. MG; Tatry: Polana Brzeziny [DV25], 30.07.1982, leg. MG. Tatry: Polana Brzeziny [DV25], 30.07.1982, leg. MG. Tatry: Polana Brzeziny [DV25], 30.07.1982, leg. MG. Notonectidae Notonecta glauca glauca Linnaeus, 1758 Kotlina Nowotarska: Zakopane, Jaszczurówka [DV26], 22.08.1982, leg. MG; Tatry: Polana Brzeziny [DV25], 2.08.1982, 5.08.1982, 2 exx., 22.08.1982, leg. MG; g g , Kotlina Nowotarska: Zakopane, Jaszczurówka [DV26], 22.08.1982, leg. MG; Tatry: Polana Brzeziny [DV25], 2.08.1982, 5.08.1982, 2 exx., 22.08.1982, leg. MG; Toporowy Staw [DV25], 9.06.1982, leg. MG. Tatry: Polana Brzeziny [DV25], 2.08.1982, 5.08.1982, 2 exx., 22.08.1982, leg. MG; Toporowy Staw [DV25], 9.06.1982, leg. MG. Notonecta reuteri reuteri Hungerford, 1928 Tatry: Polana Brzeziny [DV25], 23.07.1982, leg. MG. Dolycoris baccarum (Linnaeus, 1758) Dolycoris baccarum (Linnaeus, 1758) Tatry: Dolina Chochołowska [DV15], 11.06.1977, leg. MG; Waksmundzka Polana [DV35] 15 08 1978 leg MG; Tatry: Dolina Chochołowska [DV15], 11.06.1977, leg. MG; Waksmundzka Polana [DV35], 15.08.1978, leg. MG; Tatry: Dolina Chochołowska [DV15], 11.06.1977, leg. MG; Waksmundzka Polana [DV35], 15.08.1978, leg. MG; Słowacja: Dobšiná, 1.06.1932, 3 exx., leg. DA; Ružomberok, 2.06.1932, 2 exx., leg. DA; Tatranská Kotlina, 18.06.1932, 20.06.1932, leg. DA; Tatranská Polianka, 30.05.1932, 3 exx., leg. DA. [ ], , g ; Słowacja: Dobšiná, 1.06.1932, 3 exx., leg. DA; Ružomberok, 2.06.1932, 2 exx., leg. DA; Tatranská Kotlina, 18.06.1932, 20.06.1932, leg. DA; Tatranská Polianka, 30.05.1932, 3 exx., leg. DA. 30.05.1932, 3 exx., leg. DA. Eurydema oleracea (Linnaeus, 1758) Kotlina Nowotarska: Zakopane, Bystre [DV25], 4.07.1978, leg. MG. Pentatoma rufipes (Linnaeus, 1758) Kotlina Nowotarska: Bukowina Tatrzańska [DV36], 08.1978, leg. MG. Picromerus bidens (Linnaeus, 1758) Tatry: Dolina Suchej Wody [DV25], 30.08.1977, leg. MG. Rhacognathus punctatus (Linnaeus, 1758) Tatry: Dolina Kościeliska [DV15], 11.06.1975, 2 exx., leg. MG. Aelia acuminata (Linnaeus, 1758) Aelia acuminata (Linnaeus, 1758) Kotlina Nowotarska: Gubałówka [DV26], 6.06.1970, leg. MG. Kotlina Nowotarska: Gubałówka [DV26], 6.06.1970, leg. MG. Carpocoris fuscispinus (Boheman, 1850) Tatry: Dolina Roztoki [DV25], 10.07.1970, leg. MG; Słowacja: Tatranská Kotlina, 20.06.1932, leg. DA. Carpocoris fuscispinus (Boheman, 1850) Tatry: Dolina Roztoki [DV25], 10.07.1970, leg. MG; Słowacja: Tatranská Kotlina, 20.06.1932, leg. DA. Rhyparochromidae Acompus rufipes (Wolff, 1804) Tatry: Dolina Olczyska [DV25], 26.06.1932, leg. DA; Słowacja: Tatranská Kotlina, 20.06.1932, leg. DA. Nabidae Himacerus mirmicoides (O. Costa, 1834) Kotlina Nowotarska: Gubałówka [DV26], 8.06.1972, leg. MG. Gatunek nowy dla Kotliny Nowotarskiej. Nabis flavomarginatus Scholtz, 1847 Kotlina Nowotarska: Kościelisko, Bory [DV26], 13.08.1982, leg. MG; Zakopane [DV26], 19.07.1981, leg. MG; Zakopane, Bystre [DV25], 19.07.1982, 2 exx., leg. MG; Nabis flavomarginatus Scholtz, 1847 Kotlina Nowotarska: Kościelisko, Bory [DV26], 13.08.1982, leg. MG; Zakopane [DV26], 19.07.1981, leg. MG; Zakopane, Bystre [DV25], 19.07.1982, 2 exx., leg. MG; Z k P d łó k [DV26] 23 06 1975 l MG Zakopane, Pardałówka [DV26], 23.06.1975, leg. MG; Tatry: Dolina Miętusia [DV25], 17.07.1982, leg. MG; Dolina Olczyska [DV25], 30.08.1980, leg. MG; Dolina Tomanowa [DV15], 5.08.1972, leg. MG; Kościelisko, Gronik [DV15], 21.07.1982, leg. MG; Łysa Polana [DV35], 19.08.1982, leg. MG; Nosal [DV25], 8.07.1981, leg. MG; Zakopane, Krokiew [DV25], 23.07.1973, leg. MG. 5 Acta ent. siles. 30 (online 023) Bytom, November 29, 2022 DYSKUSJA Materiał przedstawiony w niniejszej pracy obejmuje 233 osobniki reprezentujące 39 gatunków z 13 rodzin Heteroptera, w tym cztery gatunki nowe dla Kotliny Nowotarskiej: Grypocoris sexguttatus, Harpocera thoracica, Lygus wagneri i Himacerus mirmicoides. Dane faunistyczne prezentowane w niniejszej pracy pochodzą z 38 stanowisk rozmieszczonych na terenie Polski (Kotlina Nowotarska, Tatry) i Słowacji. Opracowany materiał dostarczył danych faunistycznych, które pozwalają uzupełnić obraz składu fauny Heteroptera Tatr i okolic Zakopanego w latach 30. oraz 70. i 80. XX stulecia. Ponadto, w opisywanej kolekcji znajduje się okaz Aradus atterimus, na podstawie którego po raz pierwszy stwierdzono ten gatunek z Polski (Lis J.A. 1990). Warto w tym miejscu zaznaczyć, że dane faunistyczne zarówno z Tatr, jak i z Kotliny Nowotarskiej, w okresie 1932-1983 ograniczają się prawie wyłącznie do materiałów przedstawionych przez Smreczyńskiego (1954). Podał on 194 gatunki lądowych i wodnych Heteroptera z 209 wykazanych we wspomnianych krainach (Gierlasiński & Taszakowski 2022). Należy pamiętać, że kolekcje muzealne mogą stanowić podstawę do analizy trendów w rozmieszczeniu poszczególnych gatunków w świetle zmian klimatycznych czy też zmian w sposobie użytkowania terenów przekształconych przez człowieka (Page et al. 2015). Zbiory przyrodnicze dostarczają informacji o bioróżnorodności, a repozytoria te są tradycyjnie wykorzystywane do rozwiązywania podstawowych problemów z zakresu biogeografii, systematyki i ochrony przyrody. Porównanie okazów pochodzących z kolekcji historycznych z materiałami gromadzonymi współcześnie daje możliwość śledzenia między innymi zmian fenotypowych w czasie (Holmes et al. 2016). Wszystkie te zmiany mają bezpośredni wpływ na różnorodność owadów, w tym również pluskwiaków różnoskrzydłych. Z tego punktu widzenia istotne jest zachowanie wszelkich zbiorów owadów, nawet niekompletnych i częściowo zniszczonych i przekazywanie ich instytucjom o charakterze muzealnym w celu zabezpieczenia i przechowania do czasu, kiedy będą mogły być opracowane przez specjalistów. Muzea stanowią więc nieocenioną skarbnicę wiedzy opartej na okazach gromadzonych przez dziesiątki lat przez badaczy. Dane na etykietach często zawierają szereg dodatkowych informacji daleko wykraczających poza proste określenie miejsca zbioru okazów (Guralnick et al. 2016, Błoszyk & Konwerski 2017, Taszakowski et al. 2019). Tingidae Dictyla echii (Schrank, 1782) Słowacja: Ružomberok, 5.06.1932, 2 exx., leg. DA. Dictyla echii (Schrank, 1782) Słowacja: Ružomberok, 5.06.1932, 2 exx., leg. DA. Aubertin D. 1930. Stratiomyiidae. In: Diptera of Patagonia and South Chile. Part V. Fascicle 2. British Museum, London: 93–105. Aubertin D. 1932. Note on the name of a genus in the family Stratiomyiidae. In: Diptera of Patagonia and South Chile. Part V. Fascicle 3. British Museum, London: 284–285. Aubertin D., Malloch J.R. 1933. Conopidae. In: Diptera of Patagonia and Southern Chile. Part VI. Fascicle 3. British Museum, London: 172–175. Acompus rufipes (Wolff, 1804) 6 Acta ent. siles. 30 (online 023) Bytom, November 29, 2022 Aubertin D. 1932. Note on the name of a genus in the family Stratiomyiidae. In: Diptera of Patagonia and South Chile. Part V. Fascicle 3. British Museum, London: 284–285. Aubertin D., Malloch J.R. 1933. Conopidae. In: Diptera of Patagonia and Southern Chile. Part VI. Fascicle 3. British Museum, London: 172–175. PIŚMIENNICTWO 7 7 Acta ent. siles. 30 (online 023) Bytom, November 29, 2022 Bytom, November 29, 2022 Aukema B. (Ed.) 2022. Catalogue of the Palaearctic Heteroptera https://catpalhet.linnaeus.naturalis.nl/. Bakker F.T., Antonelli A., Clarke J.A., Cook J.A., Edwards S.V., Ericson P.G.P., Faurby S., Ferrand N., Gelang M., Gillespie R.G., Irestedt M., Lundin K., Larsson E., Matos-Maraví P., Müller J., von Proschwitz T., Roderick G.K., Schliep A., Wahlberg N., Wiedenhoeft J., Källersjö M. 2020. The Global Museum: natural history collections and the future of evolutionary science and public education. PeerJ 8:e8225. DOI: 10.7717/peerj.8225. Barnosky A.D., Matzke N., Tomiya S., Wogan G.O.U., Swartz B., Quental T.B., Marshall C, McGuire J.l., Lindsey E.L., Maguire K.C., Mersey B., Ferrer E.A. 2011. Has the Earth’s sixth mass extinction already arrived? Nature 471: 51–57. DOI:10.1038/nature09678. Bionomia 2022. https://bionomia.net/Q86636785. dostęp: 30.10.2022. Błoszyk J., Konwerski Sz. 2017. Zbiory Przyrodnicze Wydziału Biologii UAM kontynuatorem 160-letnich tradycji muzealnictwa przyrodniczego w Wielkopolsce, pp. 128–139, In: Muzealnictwo przyrodnicze w Polsce. Ośrodek Kultury Leśnej w Gołuchowie. Burakowski B., Mroczkowski M., Stefańska J. 1973. Chrząszcze Coleoptera. Biegaczowate – Carabidae, część 1. Katalog fauny Polski 23(2): 1–232. ę g f y ( ) Buszko J., Mikkola K., Nowacki J. 2000. Motyle (Lepidoptera) Tatr Polskich. Część I. Wstęp, przegląd gatunków, geneza fauny. Wiadomości entomologiczne 19, Suplement. Cichocki W., Cisło G. 1985. Zbiory zoologiczne Muzeum Tatrzańskiego im. Dra Tytusa Chałubińskiego w Zakopanem. Przegląd Zoologiczny 29(3) 369-374. y g w Zakopanem. Przegląd Zoologiczny 29(3) 369-374. p g g y ( ) Cichocki W., Cisło G. 1999. Przyrodnicy i ich zbiory w Muzeum Tatrzańskim. Zakopane: 63 pp Gierlasiński G., Lis. B., Kaszyca-Taszakowska N., Taszakowski A. 2020. 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https://openalex.org/W2086753242	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0018253&type=printable	English	null	Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors	PloS one	2,011	cc-by	12,051	Received November 29, 2010; Accepted February 28, 2011; Published May 25, 2011 Received November 29, 2010; Accepted February 28, 2011; Published May 25, 2011 Copyright: 2011 Knauer 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: Grant support: German Cancer Aid - http://www.krebshilfe.de/ (FKZ102362 to R.S. and R.M.), Head and Neck Cancer Foundation - http://www.stiftung- tumorforschung.de/ (to C.B.), Wilhelm-Sander Foundation - http://www.sanst.de, Funds of the Chemical Industry - http://fonds.vci.de, Stiftung Rheinland-Pfalz fu¨r Innovationen - http://www.stiftung-innovation.rlp.de, DFG KN973/1-1 and INST371/5-1FUGG, donation from R. Patzke, Alexander-Karl-Foundation - http://www. foerdern-und-stiften.uni-mainz.de/225.php, and the University Mainz Support Program. 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: bier@uni-mainz.de (CB); rstauber@uni-mainz.de (RHS) * E-mail: bier@uni-mainz.de (CB); rstauber@uni-mainz.de (RHS) . These authors contributed equally to this work. ¤ Current address: ETH Zurich, Institute of Pharmaceutical Sciences, Zurich, Switzerland protease signaling needs to be strictly regulated, and the deregulation of protease activity may contribute to various pathologies, including neoplastic diseases [1,2,4]. Shirley K. Knauer1., Verena Fetz2, Jens Rabenstein3, Sandra Friedl2, Bettina Hofmann4, Samaneh Sabiani3, Elisabeth Schro¨ der1, Lena Kunst1, Eugen Proschak4, Eckhard Thines5, Thomas Kindler6, Gisbert Schneider4¤, Rolf Marschalek3, Roland H. Stauber2, Carolin Bier2. 1 Institute for Molecular Biology, Centre for Medical Biotechnology (ZMB), University Duisburg-Essen, Essen, Germany, 2 Mainzer Screening Center (MSC), University Medical Center of the Johannes Gutenberg-University of Mainz, Mainz, Germany, 3 Institute of Pharmaceutical Biology/ZAFES, Goethe-University, Frankfurt/Main, Germany, 4 Institute Organic Chemistry and Chemical Biology/ZAFES, Goethe-University, Frankfurt/Main, Germany, 5 Institute of Biotechnology and Drug Research Kaiserslautern (IBWF), Kaiserslautern, Germany, 6 Department of Hematology/Oncology, University Medical Center of the Johannes Gutenberg-University of Mainz, Mainz, Germany Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors Shirley K. Knauer1., Verena Fetz2, Jens Rabenstein3, Sandra Friedl2, Bettina Hofmann4, Samaneh Sabiani3, Elisabeth Schro¨ der1, Lena Kunst1, Eugen Proschak4, Eckhard Thines5, Thomas Kindler6, Gisbert Schneider4¤, Rolf Marschalek3, Roland H. Stauber2, Carolin Bier2. Shirley K. Knauer1., Verena Fetz2, Jens Rabenstein3, Sandra Friedl2, Bettina Hofmann4, Samaneh Sabiani3, Elisabeth Schro¨ der1, Lena Kunst1, Eugen Proschak4, Eckhard Thines5, Thomas Kindler6, Gisbert Schneider4¤, Rolf Marschalek3, Roland H. Stauber2, Carolin Bier2. PLoS ONE \| www.plosone.org Abstract Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1’s (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high- throughput microscopy platform (Z’factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamide and 2- benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1’s function and its potential therapeutic relevance. Citation: Knauer SK, Fetz V, Rabenstein J, Friedl S, Hofmann B, et al. (2011) Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors. PLoS ONE 6(5): e18253. doi:10.1371/journal.pone.0018253 Citation: Knauer SK, Fetz V, Rabenstein J, Friedl S, Hofmann B, et al. (2011) Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors. PLoS ONE 6(5): e18253. doi:10.1371/journal.pone.0018253 Editor: Andy T. Y. Lau, University of Minnesota, United States of America Editor: Andy T. Y. Lau, University of Minnesota, United States of America Received November 29, 2010; Accepted February 28, 2011; Published May 25, 2011 Assays to study Taspase1 cleavage activity Assays to study Taspase1 cleavage activity To characterize the sequence and spatial requirements for efficient Taspase1 processing as well as to screen for potential Taspase1 inhibitors, we first tested an in vitro cleavage assay (Suppl. Figure S1B). Attempts to express and purify Taspase1 under native conditions as a GST-Taspase1-GFP fusion failed due to extensive protein aggregation, which was evident already in bacteria (Suppl. Figure S1A). Therefore, His-tagged Taspase1 (rTasp1) was purified by imidazol and nickel chelating affinity chromatography. Incubation of the substrate, GST-2Cl, containing the MLL cleavage sites 1 and 2 (MLL aa 2650–2808), with increasing amounts of rTasp1 resulted in the proteolytic cleavage of the substrate as well as in the autocatalytic processing of the proenzyme. However, cleavage occurred slowly, and a high ratio of enzyme/substrate was required for complete substrate cleavage (Suppl. Figure S1C and S1D). These results indicated the possibility that bacterially expressed Taspase1 displays only an attenuated catalytic activity. Taspase1 was first identified as the protease responsible for cleavage of the Mixed Lineage Leukemia (MLL) protein at conserved (Q3X2D1QG19) sites [5]. Proteolytic cleavage of MLL is considered to stabilize the MLL protein [7,8] as a crucial event for proper Hox gene expression and normal cell cycle [9,10]. However, MLL is also found as a translocation partner in a variety of acute leukemias [5,9,10,11,12]. Interestingly, we recently showed that only AF4NMLL but not the reciprocal translocation product, MLLNAF4, lacking the Taspase1 cleavage site, can cause proB ALL in a murine model [13]. To circumvent the limitations of the in vitro assay, we hence focused on the most relevant test tube, the living cell. As shown in our previous studies, translocation-based autofluorescent biosensors are powerful tools to assess protein-protein interac- tion as well as nucleo-cytoplasmic transport in vivo [20,21]. To generate a Taspase1 biosensor, we integrated the Taspase1 cleavage site from MLL (CS2; aa 2713KISQLDGVDD2722) into a biosensor backbone, composed of GST, GFP, the SV40 large T-antigen nuclear import signal (NLS) and a Myc-epitope- tagged nuclear export signal from the HIV-1 Rev protein (NESRev) (Figure 1A). The rationale of this specific modular set- up was that Taspase1-mediated cleavage of the biosensor should liberate the NESRev triggering nuclear accumulation of the fluorescent indicator protein. Indeed, the resulting NLS-GFP/ GST-CS2-NESRev fusion protein (TS-Cl2+) localizes predomi- nantly to the cytoplasm (Figure 1B), since the NES activity is dominant over the NLS. Assays to study Taspase1 cleavage activity Though, TS-Cl2+ is continuously shuttling between the nucleus and the cytoplasm, as confirmed by treatment with the export inhibitor LeptomycinB (LMB), which abrogates nuclear export leading to nuclear accumulation of the biosensor (Suppl. Figure S2C). Similar results were obtained for a biosensor containing the red fluorescent protein, mCherry (mCh), instead of GFP (NLS-mCh/GST-CST-NESRev = TS-Cl2+ R) (Figure 1C). Thus, proteolytic cleavage of MLL-fusion proteins by Taspase1 is considered a critical step for MLL-mediated tumorigenesis, although the molecular details are not yet resolved [5,9,10,11,12]. Besides Taspase1’s role in leukemogenesis the protease was suggested to be also overexpressed solid tumors [10]. In this respect, recent data indicate that also other regulatory proteins, such as the precursor of the Transcription Factor IIA (TFIIA) or Drosophila HCF [7,14], are Taspase1 targets. Hence, there is an increasing interest in defining novel Taspase1 targets. However, the molecular mechanisms how Taspase1 affects biological functions through site-specific proteolysis of its substrates and what other cellular programs are regulated by Taspase1’s degradome under normal or pathophysiological conditions is completely unknown. p y Besides genetic instruments, chemical decoys allowing the targeted inhibition/activation of proteins are powerful tools to dissect complex biological pathways. Small molecules that allow a chemical knock out of a cellular reaction or a cell phenotype can be selected by phenotypic screens, and used as molecular tools to identify previously uncharacterized proteins and/or molecular mechanisms. Hence, chemogenomics as studying the interaction of biological systems with exogenous small molecules, i.e., analyzing the intersection of biological and chemical spaces [15,16], seems an attractive approach to also dissect Taspase1 functions. Unfortunately, Taspase1’s catalytic activity is not affected by common protease inhibitors and no small molecule inhibitors for this enzyme are currently available to dissect Taspase1’s function in vivo [5,17]. Importantly, cotransfection of either the nuclear/nucleolar Taspase1-BFP (Tasp-BFP) or -mCherry (Tasp-mCh) results in the proteolytic cleavage of Myc-RevNES and the subsequent nuclear accumulation of TS-Cl2+ in various epithelial and liquid cancer cell lines (Figure 1B/C and Figure 2B). Cleavage which was already evident 24 h post transfection and similar results were obtained 48 h post transfection (data not shown). As a control, a construct containing a non-functional Taspase1 cleavage site (TS-Cl2+ mut; aa 2713KISQLAAVDD2722) or a cleavage site for Caspase3 (BS-Casp3; aa KRKGDEVDGVDE) remained cytoplas- mic not only under identical experimental conditions (Figure 1E), but also when cells were observed after 48 h (data not shown). Introduction Besides their critical role in intra- and intercellular ‘‘waste management’’, proteases are currently accepted as important signaling molecules involved in numerous biological and patho- logical functions [1,2]. These include metabolism, tissue remod- eling, apoptosis, cell proliferation and migration [1,3]. Thus, The human Threonine Aspartase 1/Taspase1 gene encodes a protein of 420 amino acids (aa), representing the proenzyme of the protease. In contrast to the other exclusively cis-active type 2 Asparaginases, only Taspase1 is also able to cleave other substrates PLoS ONE \| www.plosone.org 1 May 2011 \| Volume 6 \| Issue 5 \| e18253 Taspase1 Biosensors Taspase1 Biosensors in trans [5]. Therefore, Taspase1 represents a distinct class of proteolytic enzymes. Taspase1 mediates cleavage of proteins by recognizing a conserved peptide motif with an aspartate at the P1 position [5]. The N-terminal threonine (Thr234) is generated by autoproteolysis of the Taspase1 proenzyme (cis-cleavage) into the two subunits a and b, which appear to assemble into an asymmetric 28 kDa/22 kDa a2/b2-heterotetramer, the active protease [6]. The discovery of Taspase1 founded a new class of endopeptidases that utilize the N-terminal threonine of its mature b-subunit as the active site [5]. Mutation of this catalytic nucleophile, Thr234, abolishes Taspase1’s proteolytic activity [5,6]. PLoS ONE \| www.plosone.org Assays to study Taspase1 cleavage activity Also, no nuclear accumulation was observed upon coexpression of the catalytically inactive TaspT234V-BFP fusion, in which Thr234 was changed into Val (TaspT234V) [5,6] or of the nucleolar HIV-1 Rev-BFP protein, underpinning the assay’s specificity (Figure 1E). Proteolytic processing of the biosensor upon expression of untagged or tagged Taspase1 was independently confirmed by immunoblot analysis (Figure 1F and data not shown). Even coexpression of an unrelated protease, such as Caspase-3 or -9, did As biochemical data or potential drugs must be effective at the cellular level, reliable cell-based assays (CBA) for Taspase1 are urgently needed. Often, redistribution approaches, as cell-based assay technology that uses protein translocation as the primary readout have been used to study the activity of cellular signaling pathways [18,19]. Protein targets are labeled with autofluorescent proteins and are read using high-throughput, microscope-based instruments [18,19]. Although, protein translocation assays have the potential for high-content (HCS), high-throughput screening (HTS) applications, such assays are generally not used for proteases. Here, the spatial and functional division into the nucleus and the cytoplasm was exploited to design a translocation-based Taspase1-biosensor assay. The CBA was adapted on a HTS platform, employed to identify potential Taspase1 small molecule inhibitors, and was used to study Taspase1 function in living cells. PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e18253 2 Figure 1. Multi-color translocation biosensor assays to analyse Taspase1-mediated cleavage in living cells. A. In vivo assay. Schematic domain organization and principle of the TS-Cl2+ translocation sensor to probe Taspase1 activity. TS-Cl2+ is composed of GST, GFP, combinations of a nuclear import (NLS) and a Myc epitope-tagged export (NES) signal, combined with the Taspase1 cleavage site from MLL (Cl2+; aa 2713KISQLDGVDD2722). TS-Cl2+ localizes predominantly to the cytoplasm but is continuously shuttling between the nucleus and the cytoplasm. Co-expression of Taspase1-BFP (Tasp-BFP) results in the loss of the NES by proteolytic cleavage of the biosensor, triggering nuclear accumulation of the green fluorescent indicator protein. B. Expression of Taspase1 results in nuclear translocation of TS-Cl2+ in living HeLa transfectants. The autofluorescent biosensor is predominantly cytoplasmic (upper panel), whereas co-expression of Taspase1-BFP results in its proteolytic cleavage and Taspase1 Biosensors Taspase1 Biosensors Figure 1. Multi-color translocation biosensor assays to analyse Taspase1-mediated cleavage in living cells. A. In vivo assa domain organization and principle of the TS-Cl2+ translocation sensor to probe Taspase1 activity. Assays to study Taspase1 cleavage activity Cleavage-induced nuclear accumulation of the biosensor significantly increased already at a ratio of 1/10 (*: p,0.0001). Results from a representative experiment are shown. E. Biosensor assay specificity. Biosensors containing a non-functional Taspase1 cleavage site (TS-Cl2+ mut) or a cleavage site for Caspase3 (BS-Casp3) remained cytoplasmic upon co-expression of Tasp-BFP. No nuclear accumulation of TS-Cl2+ was observed upon coexpression of inactive TaspT234V-BFP or the nucleolar RevM10BL-BFP protein. F. Cleavage of TS-Cl2+, TS-Cl2+ mut or BS-Casp3 analyzed by immunoblot. 293T cells were transfected with the indicated biosensors together with the indicated Taspase1 expression plasmids, the empty vector (control), RevM10BL-BFP or the protease Caspase3. Expression of proteins and cleavage products in cell lysates was visualized using a-GST, -Taspase1, -GFP or -Casp3 Abs. GAPDH served as loading control. G. Ectopic expression of Caspase3 does not induce cleavage and nuclear translocation of TS- Cl2+. Caspase3 expression was visualized by IF using a-Casp3 antibody, its activation by a-ClevCasp3 Ab. GFP/BFP were visualized by fluorescence microscopy. Scale bars, 10 mm. doi:10 1371/journal pone 0018253 g001 py m doi:10.1371/journal.pone.0018253.g001 at a ratio of 1:10, was sufficient to catalyze efficient cleavage and nuclear accumulation of the biosensor (Figure 1D). Collectively, the results clearly underlined the practical advantages and biological relevance of the cellular assay to search for pharmacogenomic Taspase1 inhibitors. not affect the cytoplasmic localization of the biosensor (Figure 1G and data not shown). at a ratio of 1:10, was sufficient to catalyze efficient cleavage and nuclear accumulation of the biosensor (Figure 1D). Collectively, the results clearly underlined the practical advantages and biological relevance of the cellular assay to search for pharmacogenomic Taspase1 inhibitors. Notably, in contrast to the high amounts of rTasp1 required for cleavage in vitro (enzyme/substrate = 1:2), cotransfection of enzyme (Taspase1-BFP) and substrate (TS-Cl2+) expression plasmid even Collectively, the results clearly underlined the practical advantages and biological relevance of the cellular assay to search for pharmacogenomic Taspase1 inhibitors. Figure 2. Biosensor assay adaptation onto a HTS platform. A. Object selection parameters for nuclear (CIRC) and cytoplasmatic compartment (RING) analysis. B. Biosensor translocation assay analysis using the Cellomics ArrayscanH VTI platform. HeLa transfectants coexpressing TS-Cl2+ and active or inactive (TaspT234V) mCherry fusions were fixed 48 h post transfection and nuclei marked by Hoechst 33342. The Hoechst 33342, GFP, and mCherry signals were recorded in channels 1, 2 and 3, respectively. Overlay with the CIRC mask and RING region is outlined for GFP (right panel). nuclear accumulation (lower panel). Myc-NESRev was detected by indirect immunofluorescence using an a-myc-tag Ab. C. The translocation biosensor is functional not only in adherent but also in leukemia cell models. K562 cells were transfected with expression plasmids encoding the indicated proteins. Coexpression of Tasp-GFP but not of inactive TaspT234V-GFP resulted in proteolytic cleavage and nuclear accumulation of the red fluorescent biosensor, TS-Cl2+ R. Biosensor localization was analyzed 48 h post transfection in at least 200 fluorescent living cells, and representative images are shown. Dashed lines mark nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images. D. Quantitation of TS-Cl2+ processing in vivo. HeLa cells were transfected with the indicated ratios of enzyme (Taspase1-BFP) and substrate (TS-Cl2+). 48 h later, the percentage of cells showing cytoplasmic (C), cytoplasmic and nuclear (N/C) or nuclear (N) fluorescence was determined for at least 200 fluorescent cells. Cleavage-induced nuclear accumulation of the biosensor significantly increased already at a ratio of 1/10 (: p,0.0001). Results from a representative experiment are shown. E. Biosensor assay specificity. Biosensors containing a non-functional Taspase1 cleavage site (TS-Cl2+ mut) or a cleavage site for Caspase3 (BS-Casp3) remained cytoplasmic upon co-expression of Tasp-BFP. No nuclear accumulation of TS-Cl2+ was observed upon coexpression of inactive TaspT234V-BFP or the nucleolar RevM10BL-BFP protein. F. Cleavage of TS-Cl2+, TS-Cl2+ mut or BS-Casp3 analyzed by immunoblot. 293T cells were transfected with the indicated biosensors together with the indicated Taspase1 expression plasmids, the empty vector (control), RevM10BL-BFP or the protease Caspase3. Expression of proteins and cleavage products in cell lysates was visualized using a-GST, -Taspase1, -GFP or -Casp3 Abs. GAPDH served as loading control. G. Ectopic expression of Caspase3 does not induce cleavage and nuclear translocation of TS- Cl2+. Caspase3 expression was visualized by IF using a-Casp3 antibody, its activation by a-ClevCasp3 Ab. GFP/BFP were visualized by fluorescence microscopy. Scale bars, 10 mm. doi:10 1371/journal pone 0018253 g001 Assays to study Taspase1 cleavage activity TS-Cl2+ is composed of GST, GFP, comb nuclear import (NLS) and a Myc epitope-tagged export (NES) signal, combined with the Taspase1 cleavage site from M 2713KISQLDGVDD2722). TS-Cl2+ localizes predominantly to the cytoplasm but is continuously shuttling between the nucleus and th Co-expression of Taspase1-BFP (Tasp-BFP) results in the loss of the NES by proteolytic cleavage of the biosensor, triggering nuclear acc the green fluorescent indicator protein. B. Expression of Taspase1 results in nuclear translocation of TS-Cl2+ in living HeLa transf autofluorescent biosensor is predominantly cytoplasmic (upper panel), whereas co-expression of Taspase1-BFP results in its proteolytic c Figure 1. Multi-color translocation biosensor assays to analyse Taspase1-mediated cleavage in living cells. A. In vivo assay. Schematic domain organization and principle of the TS-Cl2+ translocation sensor to probe Taspase1 activity. TS-Cl2+ is composed of GST, GFP, combinations of a nuclear import (NLS) and a Myc epitope-tagged export (NES) signal, combined with the Taspase1 cleavage site from MLL (Cl2+; aa 2713KISQLDGVDD2722). TS-Cl2+ localizes predominantly to the cytoplasm but is continuously shuttling between the nucleus and the cytoplasm. Co-expression of Taspase1-BFP (Tasp-BFP) results in the loss of the NES by proteolytic cleavage of the biosensor, triggering nuclear accumulation of the green fluorescent indicator protein. B. Expression of Taspase1 results in nuclear translocation of TS-Cl2+ in living HeLa transfectants. The autofluorescent biosensor is predominantly cytoplasmic (upper panel), whereas co-expression of Taspase1-BFP results in its proteolytic cleavage and May 2011 \| Volume 6 \| Issue 5 \| e18253 PLoS ONE \| www.plosone.org 3 Taspase1 Biosensors Taspase1 Biosensors nuclear accumulation (lower panel). Myc-NESRev was detected by indirect immunofluorescence using an a-myc-tag Ab. C. The translocation biosensor is functional not only in adherent but also in leukemia cell models. K562 cells were transfected with expression plasmids encoding the indicated proteins. Coexpression of Tasp-GFP but not of inactive TaspT234V-GFP resulted in proteolytic cleavage and nuclear accumulation of the red fluorescent biosensor, TS-Cl2+ R. Biosensor localization was analyzed 48 h post transfection in at least 200 fluorescent living cells, and representative images are shown. Dashed lines mark nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images. D. Quantitation of TS-Cl2+ processing in vivo. HeLa cells were transfected with the indicated ratios of enzyme (Taspase1-BFP) and substrate (TS-Cl2+). 48 h later, the percentage of cells showing cytoplasmic (C), cytoplasmic and nuclear (N/C) or nuclear (N) fluorescence was determined for at least 200 fluorescent cells. Triple-color biosensor-based high content screening for Taspase1 inhibitors As we previously identified transport inhibitors by chemicogenomic screens [20], we first verified that CHC-A4 and DHC-C1 did not affect nuclear import of the biosensor rather than cleavage. Treatment with LMB resulted in nuclear accumulation of TS- Cl2+ R or TS-Cl2+ even in the presence of the inhibitors, excluding interference with nuclear import (Suppl. Figure S2C and data not shown). Taspase1 inhibition could be confirmed in other cell lines using a compound concentration of 50 mM, although no inhibition was detectable at a concentration of 5 mM (Figure 3D and data not shown). Factors contributing to the weak inhibitory activity observed may be compound instability and/or their inefficient cell entry. Hence, to circumvent these limitations, we directly microinjected both compounds into TS-Cl2+ R/Tasp-GFP express- ing transfectants. Compared to adding the compounds directly to the cell culture medium, cytoplasmic injection of both compounds resulted in improved Taspase1 inhibition reducing nuclear translocation of the biosensor in the majority of cells (Figure 3F). The coinjected fluorescent Ab allowed to select only healthy cells for the analysis showing no signs of damage due to the microinjection procedure. In order to allow a comparison of both experimental approaches, the cells were inspected after 48 h. The reason why inhibition did not occur in all injected cells is not known, indicating that rational chemical modification of the primary hits is required to improve their activity. Thus, we decided to use transiently expressing transfectants for the HTS assay. First, due to the low quantum yield of BFP, the high cellular and sometimes observed compound autofluorescence background upon UV-excitation, a green/red fluorescence dual- color screening assay was developed. Therefore, BFP was replaced by mCherry in the Taspase1 expression plasmids (Tasp-mCh). Similar to GFP fusions, Tasp-mCh was biologically fully active, whereas the inactive mutant TaspT234V-mCh did not cleave the GFP-based biosensor (Figure 2B) even after 48 h. ( g ) Second, for HCS the assay was adapted to the Cellomics ArrayscanHVTI platform. For this purpose, the molecular translocation assay was adjusted by modifying several parameters to ensure optimal object identification, including the adjustment of the background correction and to define the threshold of pixels derived from the Hoechst 33342 signal (Figure 2A). This calculation resulted in an optimized object identification, capable to automatically excluding ‘‘non cellular’’ irregular objects (too small/big, debris, compound aggregates, etc.) in channel 1. Triple-color biosensor-based high content screening for Taspase1 inhibitors Triple-color biosensor-based high content screening for Taspase1 inhibitors molecular weight (,500 Da) compounds were chosen (Suppl. Figure S3). The first group was named ‘‘deep hole compounds’’ (DHCs), as these substances were selected to hypothetically fit into the deep hydrophilic pocket of Taspase1 to prevent hydrolyzation of the substrate [6] (Figure 3A). The second class, ‘‘chloride hole compounds’’ (CHCs), should irreversibly occupy residues critical for chloride ion and substrate binding in Taspase1’s active centre (Figure 3B). In addition, as historically the majority of new drugs have been derived from natural products [24] we also tested lipophilic extracts prepared from 90 different types of fungi obtained from the culture collection at the IBWF (http://www. ibwf.de) [25]. The robust performance of the TS-Cl2+ CBA met critical requirements for high content screening: the biosensor was non- toxic, localized to the cytoplasm in the absence of ectopically expressed Taspase1, and efficiently accumulated in the nucleus following Taspase1-specific cleavage. Hence, we tested whether the assay can also be used on a high-throughput microscopy based screening platform. As cell lines inducibly expressing biosensors may facilitate certain HCS/HTS applications, we generated stable Tet-off TS- Cl2+ TRE cell lines (Suppl. Figure S2A/B). The tetracycline (doxycycline)-regulated system has been used successfully in various applications [22]. Whereas expression of TS-Cl2+ TRE was blocked in the presence of doxycycline (Dox), Dox removal induced TS-Cl2+ TRE expression (Suppl. Figure S2B). Cleavage of TS-Cl2+ TRE by the endogenous Taspase1 subsequently resulted in nuclear accumulation of the biosensor (Suppl. Figure S2B). Although this cell system circumvents the need for cotransfection of Taspase1, the levels of TS-Cl2+ TRE are low compared to transient expression, which we considered as a potential limitation for HTS applications. ) Compounds and extracts were tested in 293T cells, which not express detectable levels of endogenous Taspase1 (Figure 4B). Cells coexpressing TS-Cl2+ and Tasp-mCh or TS-Cl2+ R and Tasp-GFP (Figure 3C) were challenged in 96-well plates and analyzed 48 h after transfection to ensure that lack of inhibition is not due to slow intracellular entry rates of the substances. Although the majority of substances did not significantly affect Taspase1’s trans cleavage activity at a concentration of 50 mM, we identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2- yl)sulfanyl]ethyl]benzenesulfonamide (CHC-A4) and 2-benzyltria- zole-4,5-dicarboxylic acid (DHC-C1), partially inhibiting biosen- sor translocation (Figure 3E). In contrast, the tested fungal extracts did not show detectable inhibition in our assay, although we observed cytotoxicity for some extracts (data not shown). Assays to study Taspase1 cleavage activity Representative images are shown. Scale bar, 10 mm. C. Translocation index (Ti = CIRC:RING) plotted for coexpression of Tasp1-mCh variants on a single cell basis. Values were derived from analyzing ,400 cells/well. Mean from three wells is indicated. D. Ti was highly significantly increased upon coexpression of active compared to inactive Taspase1 (: p,0.0001). Columns, mean; bars, SD. doi:10.1371/journal.pone.0018253.g002 Figure 2. Biosensor assay adaptation onto a HTS platform. A. Object selection parameters for nuclear (CIRC) and cytoplasmatic compartment (RING) analysis. B. Biosensor translocation assay analysis using the Cellomics ArrayscanH VTI platform. HeLa transfectants coexpressing TS-Cl2+ and active or inactive (TaspT234V) mCherry fusions were fixed 48 h post transfection and nuclei marked by Hoechst 33342. The Hoechst 33342, GFP, and mCherry signals were recorded in channels 1, 2 and 3, respectively. Overlay with the CIRC mask and RING region is outlined for GFP (right panel). Representative images are shown. Scale bar, 10 mm. C. Translocation index (Ti = CIRC:RING) plotted for coexpression of Tasp1-mCh variants on a single cell basis. Values were derived from analyzing ,400 cells/well. Mean from three wells is indicated. D. Ti was highly significantly increased upon coexpression of active compared to inactive Taspase1 (*: p,0.0001). Columns, mean; bars, SD. doi:10.1371/journal.pone.0018253.g002 May 2011 \| Volume 6 \| Issue 5 \| e18253 PLoS ONE \| www.plosone.org 4 Taspase1 Biosensors Triple-color biosensor-based high content screening for Taspase1 inhibitors By adjusting object segmentation parameters, the fitting of the nuclear mask to the Hoechst 33342 signal was further optimized. Also for the channel 2 and 3, the background correction and values for the threshold of the GFP and mCherry signal were defined to exclude irregular and potentially false positive signals from the analysis. The translocation index (Ti) was calculated as the ratio of the nuclear to the cytoplasmic signal intensity (Ti = CIRC:RING) on a single cell basis (Figure 2A–C). As shown in Figure 2C/D, the Ti was highly significantly increased upon coexpression of Tasp- compared to inactive TaspT234V-mCh. Notably, compared to analyzing only GFP/Hoechst 33342 double-positive cells the inclusion of GFP/mCherry/Hoechst 33342 triple-positive cells resulted in an improved Ti. Based on the assay signal window and Z9-factor of 0.63, the criterion for the primary screen was set at a compound translocation index (Tic).2 (Tic = compound(CIR- C:RING)/DMSO(CIRC:RING)). Only valid objects, i.e., cells that pass the object selection criteria (Figure 2A) were included in the analysis. PLoS ONE \| www.plosone.org Biosensor-based probing of Taspase1 function doi:10.1371/journal.pone.0018253.g003 Figure 3. In vivo analysis of potential Taspase1 inhibitors. A./B. Pharmacophore-queries for virtual screening. A. Four-point MOE pharmacophore model based on the docked substrate QLDGVDD (shown as sticks coloured by element) together with the assigned pharmacophoric features (cyan: acceptor, green: hydrophobic, grey: acceptor and anion, left panel) B. Receptor-based SYBYL three-point pharmacophore model representing a negative charge (blue spheres) interacting with Lys57, hydrogen-bond donor (magenta) interacting with Ala50 and acceptor (green) interacting with Asn100 (right panel). The yellow surface indicates excluded volume. C. Expression of Tasp-GFP but not of inactive TaspT234V-GFP induces nuclear accumulation of the red fluorescent biosensor, TS-Cl2+ R, in HeLa transfectants. D. CHC-A4 and DHC-C1 partially inhibited TS-Cl2+ R translocation. HeLa cell transfectants coexpressing TS-Cl2+ R and Taspase1-GFP were treated with DMSO or compounds (50 mM final concentration), and analyzed 48 h later. Representative examples are shown. Scale bars, 10 mm. Dashed lines mark nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images. E. Structures and formulas of the respective compounds. F. Intracellular delivery of the compounds by microinjection resulted in enhanced inhibition. Vero cell transfectants coexpressing TS-Cl2+ R and Taspase1-GFP were either treated with DMSO or compounds (50 mM final concentration) or microinjected. 48 h later, the percentage of cells showing cytoplasmic (C), cytoplasmic and nuclear (N/C) or nuclear (N) fluorescence was determined for at least 100 sensor expressing cells. doi:10.1371/journal.pone.0018253.g003 chemically similar aa could not rescue cleavage, exempt the exchange of Leu for the also hydrophobic aa Ile or Val (Figure 5A/ B and Table 1). These results could be confirmed by immunoblot analysis (Figure 5D/E). Again, specificity of the assay was verified by cotransfection of inactive TaspT234V-mCh, which did neither result in cleavage nor nuclear accumulation of the biosensors (Suppl. Figure S2C). Nuclear accumulation of all the TS-Cl2+ CSmut variants upon LMB treatment further excluded the formal possibility that mutagenesis had affected import of the biosensors (Figure 5C). Collectively, these results underline the reliability and practical advantages of our visual cell based assay to probe Taspase1 function in living cells. Second, we analyzed the proteolytic acivity of Taspase1 mutants, in which the catalytic nucleophile, Thr234, was changed into Val or Ala (TaspT234V, TaspT234A) or Asp233 was mutated into Ala (TaspD233A). As shown in Figure 4C, coexpression of TaspT234V- or TaspT234A-GFP fusion did not result in cleavage and nuclear accumulation of TS-Cl2+ R confirming that both mutants are catalytically inactive [5]. Biosensor-based probing of Taspase1 function Besides their use in screening applications, we also exploited the biosensors as genetic tools to characterize Taspase1’s biological functions. First, we used the biosensor to probe expression and biological activity of endogenous Taspase1. As Taspase1 might also be relevant for solid tumors, we tested several cancer cell models. As depicted in Figure 4A/B, TS-Cl2+ remained cytoplasmic in cell lines with low endogenous Taspase1 levels, whereas partial or complete nuclear translocation was evident in cell lines expressing high Taspase1 levels already after 24 h (for summary see Suppl. Table S2). Later time points did not show a different localization. Next, we screened a focused compound library for potential Taspase1 inhibitors. As Taspase1 is not affected by general protease inhibitors [5,17], we used a pharmacophor screening based on Taspase1’s crystal structure and the model suggested by Khan et al. [6,23]. In an attempt to preclude the binding of the peptidic substrate to Taspase1’s active centre, two classes of low PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e18253 5 Taspase1 Biosensors Figure 3. In vivo analysis of potential Taspase1 inhibitors. A./B. Pharmacophore-queries for virtual screening. A. Four-point MOE pharmacophore model based on the docked substrate QLDGVDD (shown as sticks coloured by element) together with the assigned pharmacophoric features (cyan: acceptor, green: hydrophobic, grey: acceptor and anion, left panel) B. Receptor-based SYBYL three-point pharmacophore model representing a negative charge (blue spheres) interacting with Lys57, hydrogen-bond donor (magenta) interacting with Ala50 and acceptor (green) interacting with Asn100 (right panel). The yellow surface indicates excluded volume. C. Expression of Tasp-GFP but not of inactive TaspT234V-GFP induces nuclear accumulation of the red fluorescent biosensor, TS-Cl2+ R, in HeLa transfectants. D. CHC-A4 and DHC-C1 partially inhibited TS-Cl2+ R translocation. HeLa cell transfectants coexpressing TS-Cl2+ R and Taspase1-GFP were treated with DMSO or compounds (50 mM final concentration), and analyzed 48 h later. Representative examples are shown. Scale bars, 10 mm. Dashed lines mark nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images. E. Structures and formulas of the respective compounds. F. Intracellular delivery of the compounds by microinjection resulted in enhanced inhibition. Vero cell transfectants coexpressing TS-Cl2+ R and Taspase1-GFP were either treated with DMSO or compounds (50 mM final concentration) or microinjected. 48 h later, the percentage of cells showing cytoplasmic (C), cytoplasmic and nuclear (N/C) or nuclear (N) fluorescence was determined for at least 100 sensor expressing cells. Biosensor-based probing of Taspase1 function Notably, although in vitro studies reported a 1000-fold reduced activity for TaspD233A [5], the in vivo data indicated that TaspD233A-GFP was still able to recognize and process the biosensor albeit with a somehow attenuated efficacy (Figure 4C). Next, to uncover the sequence and spatial requirements for Taspase1 processing in vivo, we performed Ala scan mutagenesis of the MLL cleavage site (CS2; aa 2713KISQLDQGVDD2722) in the biosensor background. As depicted in Figure 5, coexpression of the TS-Cl2+ mutants (TS-Cl2+ CSmut) with Tasp-mCh resulted in proteolytic cleavage and nuclear accumulation of only those biosensors in which non-essential residues were mutated. In contrast, changing critical aa into Ala almost completely prevented cleavage and nuclear accumulation of the autofluorescent proteins, leading to the following consensus sequence: K6I5S4Q3L2D1Q G19V29D39D49 (essential aa in bold; see Table 1 for summarized results of targets). Notably, even replacing critical residues by Identification of novel human Taspase1 targets To bioinformatically identify novel human Taspase1 targets, we used the motifs Q3[I,L,V]2D1QG19X29X39D49 and Q3[I,L,V]2 D1QG19X29D39X49 obtained by our mutational analysis to scan the Swiss-Prot database. Besides the expected Taspase1 targets, MLL1 and MLL4, our analysis identified TF2A, the FERM Domain-Containing Protein 4B (FRM4B), the Tyrosine-Protein Phosphatase Zeta (PTRZ) and DNA Polymerase Zeta (DPOLZ) as putative Taspase1 substrates (see Table 2 for verified, Suppl. Table S3 for predicted targets). May 2011 \| Volume 6 \| Issue 5 \| e18253 PLoS ONE \| www.plosone.org 6 Taspase1 Biosensors Figure 4. Biosensor-based probing of Taspase1 function in vivo. A./B. Cleavage of the biosensor correlated with endogenous Taspase1 levels in adherent tumor cell lines. A. Indicated cell lines were transfected with equal amounts of TS-Cl2+ expression plasmid. 24 h later, localization of the biosensor was analyzed in at least 200 fluorescent cells displaying similar fluorescence intensity. Representative examples are shown. Cleavage- induced nuclear translocation differed significantly among tested cell lines. B. Endogenous Taspase1 levels were analysed by immunoblot using a- Tasp and -GAPDH Abs. C. Biosensor-based analysis of the proteolytic activity of Taspase1 variants in HeLa transfectants. Coexpression of TaspT234A- or TaspT234V-GFP fusion did not result in cleavage and nuclear accumulation of TS-Cl2+ R. TaspD233A-GFP displayed a reduced enzymatic activity compared to wt Taspase1-GFP. Scale bars, 10 mm. Dashed lines mark nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images. doi:10.1371/journal.pone.0018253.g004 Figure 4. Biosensor-based probing of Taspase1 function in vivo. A./B. Cleavage of the biosensor correlated with endogenous Taspase1 levels in adherent tumor cell lines. A. Indicated cell lines were transfected with equal amounts of TS-Cl2+ expression plasmid. 24 h later, localization of the biosensor was analyzed in at least 200 fluorescent cells displaying similar fluorescence intensity. Representative examples are shown. Cleavage- induced nuclear translocation differed significantly among tested cell lines. B. Endogenous Taspase1 levels were analysed by immunoblot using a- Tasp and -GAPDH Abs. C. Biosensor-based analysis of the proteolytic activity of Taspase1 variants in HeLa transfectants. Coexpression of TaspT234A- or TaspT234V-GFP fusion did not result in cleavage and nuclear accumulation of TS-Cl2+ R. TaspD233A-GFP displayed a reduced enzymatic activity compared to wt Taspase1-GFP. Scale bars, 10 mm. Dashed lines mark nuclear/cytoplasmic cell boundaries obtained from the corresponding phase contrast images. doi:10.1371/journal.pone.0018253.g004 [1,27]. Although Taspase1 was identified as the protease responsible for the cleavage of the MLL protein [5,8,10], relatively little is still known about its (patho)biological relevance. Identification of novel human Taspase1 targets This is in contrast to other disease relevant proteases, such as matrix metalloproteinases, which were the first protease targets consid- ered for combating cancer because of their role in extracellular matrix degradation [1,28]. Besides the complexity of (patho)bio- logical processes Taspase1 might be involved in ([5,8,10], this study), our knowledge is currently limited by the fact that neither efficient Taspase1 inhibitors nor assay systems applicable for the high-throughput identification of such chemical decoys are available. In order to successfully employ chemogenomics, cell based assays appear to be particularly relevant for investigating Taspase1. Previous in vitro cleavage assays were rather inefficient or operated with purified or in vitro translated enzyme, and thus are not amenable for high-throughput applications (this study, [6,17]). The reasons for the observed improved performance of the in vivo biosensor assay in this study may be multifold, including the possibility that Taspase1 produced in bacteria shows reduced catalytic activity due to partial denaturation. Also, a chloride ion, described to be interacting with the amino acids Gly49, Gln100 and To experimentally verify that these proteins represent most likely biologically relevant novel Taspase1 substrates, we first tested the cleavage sites of these targets in the biosensor system. Indeed, integration of the cleavage sites from FRM4B, PTRZ as well as DPOLZ resulted in cytoplasmic biosensors, which were efficiently recognized and processed by Taspase1 (Figure 6A–6D left panels) under the same experimental conditions as the developed TS-Cl2+ biosensor. Coexpression of the inactive TaspT234V mutant con- firmed the specificity of the assay (Figure 6A–6D right panels). Immunoblot analysis further demonstrated that also full length TFIIA-GFP was efficiently processed upon coexpression of Taspase1, underlining the in vivo relevance of the in silico prediction (Figure 6E). PLoS ONE \| www.plosone.org Discussion Identification of residues required for productive Taspase1 cleavage in living cells. A.–C. Nuclear translocation of the indicated biosensor cleavage site mutants (TS-Cl2+ CSmut) was analyzed in HeLa transfectants coexpressing the indicated biosensors together with Tasp- or inactive TaspT234V-mCherry. At least 200 fluorescent living cells were inspected, and representative examples are shown. Whereas substitution of Leu2 with Ile did not affect cleavage (A.), exchange of Asp1 with Ala completely abrogated cleavage (B.) LMB treatment verified that nuclear import of the variants was not affected. (C.) Scale bars, 10 mm. D./E. Cleavage of indicated cleavage site mutants by Tasp- (D.) or inactive TaspT234V-mCh (E.) analyzed by immunoblot. Notably, D19A, D39A and D49A mutants run lower in the gel, most likely due to the loss of the negative charge. Expression of Taspase1 proteins as well as of cleavage products in 293T cell lysates was visualized using a-GST or -Taspase1 Abs. GAPDH served as loading control. doi:10 1371/journal pone 0018253 g005 doi:10.1371/journal.pone.0018253.g005 cleavage activity in living cells. As expected, Asp at the P1 position was required for cleavage by this aspartase, and Gly at P19 did not even tolerate its replacement by Ala. Also, Gln at position P3 was critical for substrate recognition, as an exchange of this uncharged polar amino acid by the smaller hydrophobic residue Ala or even the similar but smaller amino acid Asn completely blocks cleavage. In contrast to previous studies [6], we found that albeit position P2 can hold hydrophobic residues of similar size (Leu, Ile, Val), other amino acids such as the smaller hydrophobic amino acid Ala were not tolerated. Hence, hydrophobicity in combination with certain size are likely to be structural requirements for productive cleavage. Position P29 was found to be flexible, whereas the amino acids at P39 and P49 seem to be interdependent. At least one of these residues needed to be Asp, although a small residue at the other position, like Gly or Ala, was tolerated. Glu at either position however impaired cleavage, indicating that not only charge but Thr234 of recombinant Taspase1 [6] may act as a competitive inhibitor under in vitro assay conditions. Although we are currently lacking experimental evidence it is suffice to speculate that eukaryotic post-translational modifications and/or co-factors may be required to render the enzyme fully active. Nevertheless, our results underlined the practical advantages and biological relevance of the cellular assay to investigate Taspase1 function. Discussion A critical requirement to understand the biological processes a protease participates in is to dissect the mechanisms of protease activity, as well as the biochemistry that relates their structure to function [2,26]. Various strategies including genetics, proteomics and in silico biology are currently pursued to achieve these goals PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e18253 7 Taspase1 Biosensors Figure 5. Identification of residues required for productive Taspase1 cleavage in living cells. A.–C. Nuclear translocation of the indicated biosensor cleavage site mutants (TS-Cl2+ CSmut) was analyzed in HeLa transfectants coexpressing the indicated biosensors together with Tasp- or inactive TaspT234V-mCherry. At least 200 fluorescent living cells were inspected, and representative examples are shown. Whereas substitution of Leu2 with Ile did not affect cleavage (A.), exchange of Asp1 with Ala completely abrogated cleavage (B.) LMB treatment verified that nuclear import of the variants was not affected. (C.) Scale bars, 10 mm. D./E. Cleavage of indicated cleavage site mutants by Tasp- (D.) or inactive TaspT234V-mCh (E.) analyzed by immunoblot. Notably, D19A, D39A and D49A mutants run lower in the gel, most likely due to the loss of the negative charge. Expression of Taspase1 proteins as well as of cleavage products in 293T cell lysates was visualized using a-GST or -Taspase1 Abs. GAPDH served as loading control. doi:10.1371/journal.pone.0018253.g005 Figure 5. Identification of residues required for productive Taspase1 cleavage in living cells. A.–C. Nuclear translocation of the indicated biosensor cleavage site mutants (TS-Cl2+ CSmut) was analyzed in HeLa transfectants coexpressing the indicated biosensors together with Tasp- or inactive TaspT234V-mCherry. At least 200 fluorescent living cells were inspected, and representative examples are shown. Whereas substitution of Leu2 with Ile did not affect cleavage (A.), exchange of Asp1 with Ala completely abrogated cleavage (B.) LMB treatment verified that nuclear import of the variants was not affected. (C.) Scale bars, 10 mm. D./E. Cleavage of indicated cleavage site mutants by Tasp- (D.) or inactive TaspT234V-mCh (E.) analyzed by immunoblot. Notably, D19A, D39A and D49A mutants run lower in the gel, most likely due to the loss of the negative charge. Expression of Taspase1 proteins as well as of cleavage products in 293T cell lysates was visualized using a-GST or -Taspase1 Abs. GAPDH served as loading control. doi:10.1371/journal.pone.0018253.g005 Figure 5. Identification of residues required for productive Taspase1 cleavage in living cells. A.–C. Nucl Figure 5. PLoS ONE \| www.plosone.org Discussion The reasons why other compounds were not active in our assay are versatile, including their potential inability to penetrate cell membranes. Also, the accuracy of virtual screening might have been flawed as details in the published crystal structure of Taspase1 are missing and the catalytic mechanism of Taspase1 is not yet resolved in detail. The first hit compound, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl) sulfanyl]ethyl]benzenesulfonamide (CHC-A4), was retrieved by SYBYL UNITY-Flex similarity searching (receptor-derived pharmacophore model). The second, 2-benzyltriazole-4,5-dicar- boxylic acid (DHC-C1), was selected based on the four-point substrate pharmacophore model using the software Molecular Operating Environment. Both compounds are small and polar, with a pronounced hydrogen-bonding potential, which can be readily explained by the requirements of the pharmacophore queries. Although we controlled that the compounds do not unspecifically act by blocking nuclear import of the biosensors, significant Taspase1 inhibition in vivo required relative high inhibitor concentrations (50 mM). Notably, we observed im- proved inhibition upon direct delivery of both compounds into the cells by microinjection, indicating that the weak inhibitory activity observed may be due to compound instability and/or their inefficient cell entry. Recently, Lee et al. [17] designed chemically modified peptidic derivates of a Taspase1 cleavage substrate. Although some of these compounds displayed mild inhibitory activity using in vitro Taspase1 assays (e.g., yzm18 IC5029.4 mM), these peptide-based inhibitors have not shown efficacy in living cells, in contrast to our low molecular weight inhibitors. also size is important for productive processing. Taken together, we defined the sequence motif Q3[I,L,V]2D1QG19V29D39D49 as an improved consensus recognition site for Taspase1. p g p Employing this motif, we bioinformatically identified not only known Taspase1 substrates, such as MLL1 and MLL4, but also proteins, which have not been considered as potential targets for this protease. These include the FERM Domain-Containing Protein 4B (FRM4B), the Tyrosine-Protein Phosphatase Zeta (PTRZ) and DNA Polymerase Zeta (DPOLZ), suggested to be relevant for various biological processes (Table 2). Although we are currently lacking experimental evidence how Taspase1-mediated processing of these targets contributes to their functional regulation, we could confirm that the cleavage sites of these proteins are recognized and processed by Taspase1 in vivo. The potential impact of Taspase1 for neoplastic diseases extrapolated from its processing of leukemia inducing MLL fusion proteins containing a functional Taspase1 cleavage site is further supported by our identification of these substrates. Discussion A key part of understanding protease signaling in both health and disease is to identify a protease’s physiological substrates. Although the sequence Q3X2D1QG19 has been proposed as a consensus cleavage site sequence for Taspase1 [6], employing this motif for the bioinformatic identification of novel Taspase1 targets is impractical, as more than 1000 putative substrates were predicted. To improve our understanding of Taspase1’s substrate specificity, we used our biosensor assay combined with positional scanning mutagenesis to identify residues essential for Taspase1 PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e18253 8 Taspase1 Biosensors Table 1. Cleavage-site residues critical for Taspase1 processing in vivo. Cleavage-site mutation In cell cleavage by Taspase1 K6A + I5A + S4A + Q3A 2 Q3N 2 L2A 2 L2I + D1A 2 G19A 2 V29A + D39A (2) D39E (2) D49A (2) D49E (2) Summary of results obtained from the biosensor-based mapping. Cleavage site aa residues: K6I5S4Q3L2D1QG19V29D39D49 (essential aa in bold). 2: no cleavage, (2): reduced cleavage, +: cleavage. doi:10.1371/journal.pone.0018253.t001 Nevertheless, there is increasing evidence that Taspase1 may be critically contributing to disease, underlining its pathobiological and potentially therapeutic relevance. However, we still do not comprehense the processes and molecular mechanisms Taspase1 might be involved in. Thus, besides genetic and biochemical approaches, small molecules allowing a (transient) chemical knockout of Taspase1 in a specific biological system or disease model would be highly valuable. These needs underline the relevance of the developed translocation biosensor for the identification and validation of inhibitors in living cells. Impor- tantly, the biosensors can operate with red or green autofluor- escent proteins, which can be optimally detected even by high- throughput fluorescence microscopy, and are not restricted to a specific cell type. The assay strictly depends on the presence of catalytically active Taspase1 and occurs with a high signal-to-noise ratio, allowing its use in HTS/HCS applications of large or focused compound libraries. As a proof of principle, we screened a collection of small molecules, which were chosen based on a pharmacophore screening relying on the published crystal structure of Taspase1 [6]. The low molecular weight compounds were selected by virtual screening to prevent substrate cleavage and/or arrest the enzyme in an inactive state. Noteworthy, we identified two substances showing inhibitory activity in living cells, which would represent a primary hit rate of 3%. Discussion We just showed that only AF4NMLL but not the reciprocal translocation product, MLLNAF4, lacking the Taspase1 cleavage site, can cause proB ALL in a murine model [13]. Albeit the exact biological relevance of PTRZ for disease and development is not yet resolved, this phosphatase was suggested as a therapeutic target for glioblastoma and glioblastoma-derived stem cells [29,30]. Likewise, although the function of FRM4B is unknown, other members of the protein 4.1 superfamily such as FRMD4A or FRMD3 have been implicated in oncogenic signaling [31,32,33]. Notably, DPOLZ is not only essential during embryogenesis but also important in defense against genotoxins. As recent evidence indicates that reduced DPOLZ levels enhance spontaneous tumorigenesis, it is tempting to speculate that Taspase1 might participate in controlling DPOLZ levels and thus, disease [34,35]. Notably, we found that Taspase1 is expressed in several solid tumor cell models. Whether the differences in Taspase1 expression levels detected have implications also on the (patho)biological charac- teristics of the tumor cell lines as well as for the primary disease remains to be investigated. Although natural products appear to interrogate a different area of chemical space than synthetic compounds [36], the tested lipophilic fungal extracts showed no inhibitory activity. Failure may be due to the fact that albeit such extracts contain a mixture of many different substances, the concentration of potentially active ingredients may be too low or outweighed by toxic effects of other components. Also, the numbers of samples which have to be screened in unfocussed natural product libraries are usually high, and hit rates are mostly below 0.01% [20,24]. Hence, as future strategies to identify potent Taspase1 inhibitors we suggest to focus on a rational synthesis of derivates based on the PLoS ONE \| www.ploson May 2011 \| Volume 6 \| Issue 5 \| e18253 PLoS ONE \| www.plosone.org 9 Taspase1 Biosensors Table 2. Characteristics of verified Taspase1 target genes according to their GO term classifications. Discussion Gene Locus GO : biological process GO : cellular component GO : molecular function ID description ID description ID description MLL1 0006366 transcription 0071339 nucleoplasm 0003680 DNA binding 0006461 protein complex assembly 0003700 nucleic acid binding 0006915 cell death 0003702 transcription regulator activity 0035162 developmental process 0008270 ion binding 0043984 histone acetylation 0042800 N-methyltransferase activity 0051568 histone methylation 0042803 protein homodimerization activity 0045322 transcription factor activity 0070577 histone binding MLL4 0006350 transcription 0035097 nucleoplasm 0005515 protein binding 0016568 chromatin modification 0008270 ion binding 0008284 cell proliferation 0010843 DNA binding 0033148 estrogen signalling 0018024 N-methyltransferase activity 0010552 transcription 0043627 estrogen signalling FRM4B - unknown 0005737 cytoplasm 0005488 binding 0005856 cytoskeleton PTRZ 0006470 metabolic process 0005887 integral to membrane 0005001 phosphatase activity 0007417 developmental process 0005515 protein binding 0008330 phosphatase activity TF2AA 0006367 transcription initiation 0005634 nucleus 0003677 DNA binding 0006368 RNA elongation 0005672 nucleoplasm 0003713 transcription regulator activity 0032568 transcription 0005737 cytoplasm 0016251 transcription regulator activity 0045449 transcription 0017025 TATA-binding protein binding 0046982 protein heterodimerization activity DPOLZ 0006261 DNA replication 0005634 nucleus 0000166 nucleotide binding 0006281 DNA repair 0003677 DNA polymerase activity 0003887 DNA polymerase activity 0008270 ion binding doi:10.1371/journal.pone.0018253.t002 doi:10.1371/journal.pone.0018253.t002 structures of our primary hits combined with HTS of large natural/synthetic compound libraries. stopped as soon as free glucose in the growth medium was depleted, and mycelia were separated by filtration. Lipophilic molecules were extracted from the culture broth with ethyl acetate. The extracts were dried in vacuo, redissolved in 25 mL DMSO, and aliquots of these extracts were used in the assays at a dilution of 1:2000. Materials and Methods Antibodies (Ab), reagents, compounds and fungal extracts Cell culture, microscopy and fluorescence imaging of cells Ab used: a-GST (sc-57753), a-Taspase1 (sc-85945), a-GAPDH (sc-47724) and a-GFP (sc-8334) (Santa Cruz Biotechnology, Heidelberg, Germany); a-myc-tag (NEB GmbH, Frankfurt am Main, Germany). Appropriate HRP-, Cy3- or AlexaDye-conju- gated secondary antibodies (Sigma Aldrich, Munich, Germany; Santa Cruz Biotechnology, Heidelberg, Germany) were used. Reagents were from Sigma Aldrich (Sigma Aldrich, Munich, Germany) unless stated otherwise. Cells were treated with leptomycin B (LMB) (10 nM) as described in [37]. Potential Taspase1 inhibitors (Suppl. Figure S3) were purchased from ASINEX Ltd (Moscow, Russia). Fungal extracts were obtained from submerged cultures of higher fungi, preferentially from asco- and basidiomycetes, deposited in the culture collection at the IBWF, as described [25]. Briefly, the fermentation of the fungi was Cell lines used in the study were maintained and transfected as described [20,37]. MEF3T3 stably expressing the Dox-inducible TS-Cl2+ TRE were established by G418- (800 mg/mL) and puro- mycin- (2 mg/mL) selection, and fluorescence activated cell sorting as reported [20]. Cells were cultured in medium containing 1 mg/ mL doxycycline (Dox) [20]. Twelve-bit black and white images were captured using a digital Axiocam CCD camera (Carl Zeiss, Jena, Germany). Quantitation, image analysis and presentation were performed as described [18,38]. The nuclear signal was similarly obtained by measuring the pixel intensity in the nucleus. Nuclei were marked by Hoechst 33258 staining as described [18,39]. To determine the average intracellular protein localiza- PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e18253 10 Taspase1 Biosensors Figure 6. Analysis of Taspase1-mediated cleavage of in silico predicted targets. A. HeLa cells were transfected with biosensors harboring full length TFIIA (TS-TF2A), the predicted cleavage site from DPOLZ (TS-DPOLZ), PTRZ (TS-PTRZ), or FRM4B (TS-FRM4B) together with the Tasp-mCh or inactive TaspT234V-mCh expression plasmid. 24 h later, trans-cleavage resulting in nuclear translocation of the biosensors was analyzed in at least 200 fluorescent living cells. Representative examples are shown. Productive cleavage was confirmed for all targets. Scale bars, 10 mm. B. Processing of full length TFIIA-GFP upon coexpression of Taspase1 but not of the inactive TaspT234V mutant shown by immunoblot analysis of transfected 293T cell lysates. Protein expression and cleavage was visualized by a-GFP and a-Taspase1 Ab. GAPDH served as loading control. doi:10.1371/journal.pone.0018253.g006 Figure 6. Analysis of Taspase1-mediated cleavage of in silico predicted targets. A. Cell culture, microscopy and fluorescence imaging of cells HeLa cells were transfected with biosensors harboring full length TFIIA (TS-TF2A), the predicted cleavage site from DPOLZ (TS-DPOLZ), PTRZ (TS-PTRZ), or FRM4B (TS-FRM4B) together with the Tasp-mCh or inactive TaspT234V-mCh expression plasmid. 24 h later, trans-cleavage resulting in nuclear translocation of the biosensors was analyzed in at least 200 fluorescent living cells. Representative examples are shown. Productive cleavage was confirmed for all targets. Scale bars, 10 mm. B. Processing of full length TFIIA-GFP upon coexpression of Taspase1 but not of the inactive TaspT234V mutant shown by immunoblot analysis of transfected 293T cell lysates. Protein expression and cleavage was visualized by a-GFP and a-Taspase1 Ab. GAPDH served as loading control. doi:10.1371/journal.pone.0018253.g006 5% CO2 and 95% humidity. Cells were transfected, and com- pounds (50 mM final concentration dissolved in DMSO) were added 4 h later. For each experiment two wells were drug treated, and each experiment was performed in duplicates. DMSO was used as a control. 48 h later, cells were fixed by the addition of 50 mL 4% PFA, and nuclei were stained by addition of Hoechst 33342 at a final concentration of 40 mM for 10 min. After a final wash with PBS, 50 mL PBS were left in each well and the plates were sealed and stored at 4uC. Images were acquired and analyzed on the Cellomics ArrayScanH VTI Imaging Platform as described [20]. Briefly, binary image masks were created for GFP, mCherry and Hoechst 33342 positive staining to define regions of interest (ROI) for analysis. For this purpose, we applyed a median filter (363 pixel radius) to remove noise and to approximate the distribution of staining intensity to a median value. Automatic thresholding using the Isodata algorithm was used to convert the image to a binary mask that included all fluorescence data above background [20]. The Hoechst 33342 staining (channel 1) mask was used to define the nuclear ROI. Subsequently, the Hoechst 33342 mask was subtracted from the GFP mask (channel 2) to create a staining mask defining the cytoplasmic ROI. Scans were performed sequentially with settings to give sub-saturating fluorescence intensity, and a minimum of 400 valid objects per well was recorded. tion, at least 200 fluorescent cells from three separate images were examined in three independent experiments. Cell culture, microscopy and fluorescence imaging of cells The number of cells exhibiting cytoplasmic (C, cytoplasmic signal .75% of the total cellular signal), cytoplasmic and nuclear (C/N), or nuclear (N, nuclear signal .75% of the total cellular signal) fluorescence was counted. Computer-assisted microinjection Vero cells were transfected with TS-Cl2+ R and Taspase1-GFP expression plasmids (1 mg each). 4 h after transfection, DMSO or compounds (200 mM concentration in DMSO) were microinjected into the cytoplasm as described in detail [40]. An Alexa350- labelled a-IgG Ab (0.5 mg/mL) served to mark injected cells. 48 h later, the percentage of cells showing cytoplasmic (C), cytoplasmic and nuclear (N/C) or nuclear (N) fluorescence was determined for at least 100 GFP/mCherry-positive and injected cells. PLoS ONE \| www.plosone.org Supporting Information Figure S1 In vitro Taspase1 cleavage assay. A. Extensive aggregation of GST-Tasp1-GFP expressed in BL21 bacteria visualized by fluorescence microscopy. In contrast, GST-GFP showed no aggregation and was efficiently expressed. Images were taken with identical CCD camera settings. Scale bars, 1 mm. B. Schematic representation of expression constructs for His-tagged Taspase1 (rTasp) and the substrate GST-2Cl, containing the MLL cleavage sites CS1 and CS2 (MLL aa 2650–2808). Molecular weight of the expected cleavage products is indicated. C. Concentration dependent processing of GST-2Cl by recombinant Taspase1 (rTasp). GST-2Cl (5 mM) was incubated with increasing amounts of rTasp (lane1: 2.5 mM, 2: 1.25 mM, 3: 0.63 mM, 4: 0.32 mM, 5: 0.16 mM, 6: 0.08 mM, 7: 0.04 mM, 8: 0.02 mM) for 60 min. Cleavage was visualized by SDS-PAGE and Coomassie staining. Uncleaved and cleaved proteins are indicated. D. Time dependent processing of GST-2Cl by recombinant Taspase1. GST-2Cl (5 mM) was incubated with 2.5 mM Taspase1, and cleavage was monitored over time. Cleavage was visualized by Plasmids were verified by sequence analysis as described [47]. Oligonucleotides used for PCR amplification and cloning are listed in Suppl. Table S1. Virtual screening and database searches An X-ray structure of the inactive autocatalytically processed Taspase 1 dimer (Protein Data Bank ID 2a8j, 1.9 A˚ resolution [6] served as the basis for pharmacophore model generation and computer-based similarity searching in a commercial screening compound collection (Asinex Gold collection nov. 2005: 231,812 compounds; ASINEX Ltd, Moscow, Russia) [23]. Briefly, screening compounds were reduced to ‘‘druglike’’ compounds (clogS.4, no rule-of-five violation) using Molecular Operating Environment (MOE) 2005.06 (Chemical Computing Group, Mon- treal, Canada), and for the remaining 181,403 substances single conformers were computed using CORINA 3.20 (Molecular Networks GmbH, Erlangen, Germany). Bases were de-protonated, acid groups were protonated (‘‘wash’’ function in MOE). Two types of pharmacophore hypotheses were generated: (i) ‘‘ligand- based’’ models from hypothetical binding mode of the Taspase1 cleavage site substrate QLDQGVDD [5], (pre-docking of the substrate by GOLD 3.0.1; force-field relaxation using AMBER99 in MOE; manual assignment of potential pharmacophoric points in MOE; similarity searching with MOE), and (ii) a ‘‘receptor- based’’ model of a hypothetical ligand pharmacophore using SYBYL 7.1 (Tripos Inc, Missouri, U.S.A.), with UNITY-Flex search option. The resulting ligand-based pharmacophore models yielded a total of 62 perfect matches in the screening compound collection, and the receptor-based model retrieved 209 perfect matches. From these hits, compounds were selected for testing. The Taspase1 or TFIIA coding sequence was amplified from cDNA obtained from a human head and neck tumor. mRNA preparation and cDNA synthesis from tumor tissue was performed as described [42]. Cloning of the Taspase1 coding sequence into expression vectors pc3, pc3-GFP, pc3-BFP, and pc3-mCherry using BamHI/EcoRI- or BamHI/NheI-restriction sites, respectively, allowed the expression of Taspase1, alone or as a fusion with fluorescent proteins as described [21,43]. Plasmid p_TaspT234V- GFP, p_TaspT234A-GFP, p_TaspD233A-GFP and p_TaspT234V- mCherry or –BFP encoding the catalytically inactive Taspase1 mutant, were generated by splice overlap extension polymerase chain reaction as reported [18,44]. p_TFIIA-GFP, encoding a TFIIA-GFP fusion, was generated by PCR amplification and cloning into pc3-GFP as reported [45]. pc3_RevM10BL-BFP, encoding a mutant HIV-1 Rev protein, was described [40]. For the bioinformatic identification of potential human Taspase1 targets, ScanProsite searches were performed in the human taxon of the UniProtKB/SwissProt database using the patterns Q- [IVL]-D-G-X-D-D, Q-[IVL]-D-G-X-X-D and Q-[IVL]-D-G-X- D-X as queries. Cellomics ArrayScanH VTI-based HCS Automated analysis of the molecular translocation assay was performed using the Cellomics ArrayScanH VTI Imaging Platform (Thermo Fisher Scientific Inc., Berkshire, UK). Cells were seeded with an electronic multichannel pipette (Eppendorf, Hamburg, Germany) into black-walled 96-well thin bottom Greiner mclearH plates (Greiner, Frickenhausen, Germany) and incubated at 37uC, PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e18253 11 Taspase1 Biosensors MgCl2, 40 mM KCl, 20% Saccharose, 10 mM DTT). Trans- cleavage assays were performed in cleavage buffer adding recombinant Taspase1 to 5 mM of GST-2Cl. Analysis of cleavage was carried out by SDS page followed by Coomassie staining as outlined in [49]. Statistical analysis Bacterial expression plasmid pGEX_GST-Tasp1-GFP encod- ing a GST-Tasp1-GFP fusion protein and pET22-Tasp, encoding a His-tagged Taspase1 protein, were generated by inserting the Taspase1 coding sequence into pGEX-GFP or pET22b+, respectively, as reported [46]. The coding sequence for the MLL aa 2650–2808 (2Cl) was inserted into vector pGEX5T to generate pGEX5T_GST-2Cl, encoding a His-tagged-GST-2Cl fusion protein. For experiments stating p-values, a paired Student’s t-test was performed. Unless stated otherwise, p-values represent data obtained from three independent experiments done in triplicate. p-values,0.05 were considered as significant. Plasmids To generate plasmid p_NLS-GFP/GST-CS2-NESRev (p_TS- Cl2+), encoding a fusion composed of the SV40 large T-antigen NLS, GST, GFP, the Taspase1 cleavage site from MLL (CS2; aa 2713KISQLDGVDD2722), and a Myc-epitope-tagged NES from the HIV-1 Rev protein (NESRev) [41], the CS2 coding sequence was inserted into vector pNLS-GFP/GST-CS3-RevNES (p_BS- Casp3), replacing CS3. p_BS-Casp3 encodes a biosensor harbor- ing the cleavage site for Caspase3 (CS3: aa KRKGDEVDGVDE) [18]. p_TS-Cl2+ R encodes a red fluorescent biosensor (NLS- mCherry/GST-CS2-NESRev), in which GFP was replaced by mCherry [18]. Expression plasmids for TS-Cl2+ variants, in which CS2 was mutated (p_TS-Cl2+ mut; see Table 2, and Suppl. Table S1 for oligonucleotides used) were generated by oligonucleotide- annealing and cloning into the NotI/XhoI-restriction sites of p_TS- Cl2+ as described [18]. Likewise, the coding sequence for full length TFIIA (p_TS-TF2A), or the cleavage sites from DPOLZ (p_TS-DPOLZ), PTRZ (p_TS-PTRZ) or FRM4B (p_TS-FRM4B) were inserted into p_TS-Cl2+, thereby replacing the CS2. pTRE- NLS-GFP/GST-CS2-NESRev (p_TS-Cl2+ TRE) allows the inducible expression of the biosensor (‘‘tet-off’’) and was constructed by inserting the NLS-GFP/GST-CS2-NESRev coding sequence into pTRE-NLS-GFP/GST-NESRev [18]. Protein extraction, immunoblot analysis and immunofluorescence Preparation of whole cell lysates and immunoblotting were carried out as described [39,48]. Immunofluorescence was performed as reported in detail [18,49]. References 18. Knauer SK, Moodt S, Berg T, Liebel U, Pepperkok R, et al. 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Wrote the paper: SKK GS RM RHS CB. Analyzed the data: SKK VF JR EP GS RM RHS CB. Contributed reagents/materials/analysis tools: ET TK GS RM. Wrote the paper: SKK GS RM RHS CB. Analyzed the data: SKK VF JR EP GS RM RHS CB. Contributed reagents/materials/analysis tools: ET TK GS RM. Wrote the paper: SKK GS RM RHS CB. In vitro Taspase1 cleavage assay p g y His-tagged Taspase1, GST-Taspase1-GFP and His-tagged GST-2Cl substrates were expressed in BL21 bacteria and purified by nickel chelating or glutathione affinity chromatography as described [18,46]. Fractions were eluted (50 mM NaH2PO4, 300 mM NaCl, 250 mM Imidazol, pH 8.0) and dialyzed against Taspase1 cleavage buffer (200 mM Hepes/KOH pH 7.9, 10 mM PLoS ONE \| www.plosone.org May 2011 \| Volume 6 \| Issue 5 \| e18253 12 Taspase1 Biosensors SDS-PAGE and Coomassie staining. Uncleaved and cleaved proteins are indicated. (CVX) Figure S3 Chloride and deep hole compounds analyzed in HCS assay. Chemical structures and formulas are shown. Abbreviations: CHC, chloride hole compound; DHC, deep hole compound. (TIF) Figure S2 In vivo screening for inhibitors of Taspase1 activity. A. Principle of the inducible biosensor system, Tet-off TS-Cl2+ TRE. Dox interacts with tTA preventing its binding and thus activation of the TRE-containing CMV promoter. Removal of Dox allows tTA binding, triggering transcriptional activation and expression of the shuttling biosensor, which predominately localizes to the cytoplasm. Dox, Doxycylin; pCMV/pCMVmin, (minimal) CMV promoter; TRE, Tetracycline-responsive promot- er element; tTA, Tet-controlled transactivator. B. Dox-induced biosensor expression. MEF3T3 cells stably expressing TS-Cl2+ TRE were cultured in the presence or absence of Dox. In presence of Dox, no expression of the biosensor is detectable. Three days after Dox removal, expression of cytoplasmic TS-Cl2+ TRE is visible (2Dox 3d), and cleavage by endogenous Taspase1 results in its nuclear accumulation 24 h later (2Dox 4d). Living cells were analyzed by fluorescence microscopy and images taken with identical CCD camera settings. Scale bars, 10 mm. C. CHC-A4 or DHC-C1 do not interfere with nuclear import of the biosensor. HeLa transfectants were treated with DMSO or compounds (50 mM final concentration) for 12 h. Treatment with the export inhibitor LMB (10 nM, 2 h) resulted in efficient nuclear accumulation of TS-Cl2+R even in the presence of the compounds. Localization of TS-Cl2+R was analyzed in at least 200 fluorescent cells. Representative examples are shown. Scale bars, 10 mm. (TIF) Table S2 Taspase1 expression levels and proteolytic activity in solid cancer cell line models. (DOC) Table S2 Taspase1 expression levels and proteolytic activity in solid cancer cell line models. (DOC) Table S3 List of potential human Taspase1 targets predicted by ScanProsite. References Lee JT, Chen DY, Yang Z, Ramos AD, Hsieh JJ, et al. (2009) Design, syntheses, and evaluation of Taspase1 inhibitors. 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Knauer SK, Heinrich U-R, Habtemichael N, Docter D, Helling K, et al. (2010) An otoprotective role for the apoptosis inhibitor protein survivin. Cell Death and Disease;doi:10.1038/cddis.2010.25. 46. Ru¨cker E, Schneider G, Steinha¨user K, Lo¨wer R, Hauber J, et al. (2001) Rapid evaluation and optimization of recombinant protein production using GFP tagging. Protein Expression and Purification 21: 220–223. 38. Habtemichael N, Heinrich UR, Knauer SK, Schmidtmann I, Bier C, et al. References (2010) Expression analysis suggests a potential cytoprotective role of Birc5 in the inner ear. Mol Cell Neurosci 45: 297–305. 47. Habtemichael N, Wunsch D, Bier C, Tillmann S, Unruhe B, et al. (2010) Cloning and functional characterization of the guinea pig apoptosis inhibitor protein Survivin. Gene 469: 9–17. 39. Engels K, Knauer SK, Loibl S, Fetz V, Harter P, et al. (2008) NO signaling confers cytoprotectivity through the survivin network in ovarian carcinomas. Cancer Res 68: 5159–5166. p 48. 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https://openalex.org/W3181349335	https://www.mdpi.com/2075-5309/11/7/294/pdf?version=1626694209	English	null	Prospects of Developing Prefabricated Masonry Walling Systems in Australia	Buildings	2,021	cc-by	15,901	Received: 3 June 2021 Accepted: 2 July 2021 Published: 6 July 2021 Keywords: prefabrication; masonry; connections; life cycle analysis; design; reinforced masonry; post-tensioned masonry; thin layered mortared masonry 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. buildings buildings buildings an Thamboo 1 , Tatheer Zahra 2 , Satheeskumar Navaratnam 3,* , Mohammad Asad 2 and erthan Poologanathan 4 https:// doi.org/10.3390/buildings11070294 Academic Editor: Elena Ferretti Received: 3 June 2021 Accepted: 2 July 2021 Published: 6 July 2021 Citation: Thamboo, J.; Zahra, T.; Navaratnam, S.; Asad, M.; Poologanathan, K. Prospects of Developing Prefabricated Masonry Walling Systems in Australia. Buildings 2021, 11, 294. https:// doi.org/10.3390/buildings11070294 Article Prospects of Developing Prefabricated Masonry Walling Systems in Australia Julian Thamboo 1 , Tatheer Zahra 2 , Satheeskumar Navaratnam 3,* , Mohammad Asad 2 and Keerthan Poologanathan 4 an Thamboo 1 , Tatheer Zahra 2 , Satheeskumar Navaratnam 3,* , Mohammad Asad 2 and erthan Poologanathan 4 1 Department of Civil Engineering, South Eastern University of Sri Lanka, Oluvil 32360, Sri Lanka; jathamboo@seu.ac.lk jathamboo@seu.ac.lk 2 School of Civil Engineering and Built Environment, Queensland University of Technology, Brisbane 4000, Australia; t.zahra@qut.edu.au (T.Z.); m.asad@qut.edu.au (M.A.) 3 School of Engineering, RMIT University, Melbourne 3000, Australia 4 Faculty of Engineering and Environment, Northumbria University, Newcastle upon Tyne NE1 8ST, UK; keerthan.poologanathan@northumbria.ac.uk * Correspondence: sathees.nava@rmit.edu.au; Tel.: +61-4525-08931 j 2 School of Civil Engineering and Built Environment, Queensland University Australia; t.zahra@qut.edu.au (T.Z.); m.asad@qut.edu.au (M.A.) 3 School of Engineering, RMIT University, Melbourne 3000, Australia g g y 4 Faculty of Engineering and Environment, Northumbria University, Newcastle upon Tyne NE1 8ST, UK; keerthan.poologanathan@northumbria.ac.uk keerthan.poologanathan@northumbria.ac.uk * Correspondence: sathees.nava@rmit.edu.au; Tel.: +61-4525-08931 Abstract: Prefabrication has been shown to be an effective way of construction in the modern- day context. Although much progress has been made in developing reinforced concrete (RC), timber and steel prefabricated elements/structures, prefabrication of masonry walling systems has received limited attention in the past. Conventional masonry construction is labour-intensive and time-consuming; therefore, prefabrication can be an effective solution to accelerate the masonry construction to make it more cost-effective. Therefore, in this paper, an attempt has been made to evaluate the effectiveness of prefabricated masonry systems (PMS) in terms of their structural characteristics and sustainability perspectives in an Australian context. Subsequently, the available studies related to PMS and the prospects of developing prefabricated masonry walling systems were appraised and reported. In order to assess the applicability of PMS, a case study was carried out by designing four types of prospective prefabricated masonry walling systems for a typical housing unit in Australia. It was shown that the reinforced (RM), post-tensioned (PT) and thin layered mortared (TLM) masonry systems are better suited for prefabrication. Later, in order to assess the sustainability of the considered masonry walling systems, life cycle energy analyses were carried using the Environmental Performance in Construction (EPIC) database. It was found that there can be nearly 30% and 15% savings, respectively, in terms of energy saving and CO2 emissions in prefabricated construction than the conventional masonry construction. Finally, the prospects of developing PMS and the need for future research studies on these systems are highlighted. Citation: Thamboo, J.; Zahra, T.; Navaratnam, S.; Asad, M.; Poologanathan, K. Prospects of Developing Prefabricated Masonry Walling Systems in Australia. Buildings 2021, 11, 294. 1. Introduction Subsequently, in Australia, the prefabricated construction system has been promoted as one of the eight key “visions” for improving the efﬁciency and performance of the Australian construction industry in their Construction vision 2020 [17]. Accordingly, concrete, steel and timber industries have made substantial progress in establishing prefabricated systems and their developments are well supported by system- atic research studies [18–24]. Subsequently, plenty of studies been carried out to assess the performance of concrete, steel and wood based prefabricated construction systems [25–30]. In relative terms, attention given to develop prefabricated masonry walling systems is minimal. While various forms of prefabricated masonry systems (PMS) were developed in many parts of the world with adequate design requirements, these have not yet reached the criteria required for widespread mass production [31]. Figure 1 shows a prefabricated masonry façade walling panel. The reasons for limited development in establishing fully- ﬂedged PMS varies across different countries. The economy-driven criteria of selecting a structural system primarily play a signiﬁcant role in developing prefabricated masonry. Further, limited awareness among the architects and engineers on the performance, beneﬁts, method and knowledge of PMS also restricts its extensive application. g pp Moreover, systematic research studies to investigate the structural performance of PMS are limited [32–37]. It can be implied that most of the developed PMS in the past were proprietary in nature with insufﬁcient details provided on materials, structural design, fabrication and erection methods. Any PMS with connecting components should be able to withstand loads induced by the occupancy of the structure as well as the external loads exerted due to wind, ﬁre and earthquake. In this regard, minimal research studies were deliberated in the past, which is a major hindrance in conﬁdent uptake of PMS by the industry. Furthermore, the life cycle energy and life cycle cost (LCE and LCC) of the PMS are not well accounted to highlight the beneﬁts of the prefabricated masonry to the developers and end users. Comparing LCE and LCC of prefabricated concrete, steel and timber systems with their corresponding conventional construction methods show that they are more or less similar. The prefabricated systems are considered superior in the individual aspects of the life cycle analyses, such as construction time, wastage and reusability [38,39]. Samani et al. [40] analysed the LCC of a prefabricated ﬁbre reinforced composite walling system and conventional masonry buildings in the US context. 1. Introduction Masonry is one of the oldest construction materials in the world; nevertheless, it is still a preferred material for construction due to its simple construction method, relatively good loadbearing capacity, better ﬁre and acoustic properties and aesthetic appeal. How- ever, the conventional masonry construction method is slow and labour intensive; thus, the masonry construction industries at present are dealing with challenges against the depleting skilled labour force, time bound economy driven nature of modern construction and new-generation materials/walling systems. Subsequently, many alternative masonry construction systems were developed to meet the demands and reduce the labour intensive- ness of masonry construction. These alternative masonry construction techniques include larger and lighter units (e.g., Aerated Autoclaved Concrete units), thin layer mortaring (TLM) and mortarless masonry systems [1–3]. Further, to improve the structural capacities 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/). https://www.mdpi.com/journal/buildings Buildings 2021, 11, 294. https://doi.org/10.3390/buildings11070294 Buildings 2021, 11, 294 2 of 22 and ductility of the masonry, core grouting techniques, reinforcing, prestressing and surface rendering with composites have been incorporated in the past [4–7]. and ductility of the masonry, core grouting techniques, reinforcing, prestressing and surface rendering with composites have been incorporated in the past [4–7]. On the other hand, increasing population, depleting skill labourers and thereby rising labour costs, and the requirement for rapid construction of infrastructures have encouraged the embrace of prefabricated or modular construction techniques [8–11]. In addition, damages to the infrastructures due to ever increasing disasters, particularly in Australia, such as bushﬁres, ﬂoods and cyclones, demand rapid reconstruction and favour prefabricated construction techniques. The prefabricated construction systems comprise modular panels that are typically manufactured off-site in a quality-controlled environment with architectural ﬁnishes and services. These modules are then transported and installed on-site as load-resisting structural elements of the building [12]. Prefabrication enables a speedy construction, high volume output and consistent quality at a competitive cost. It also provides environmental beneﬁts, such as the reduction of construction wastes, CO2 emissions, and less constraints at the construction site by minimising on-site waste, noise and dust [13,14]. These advantages drive many countries to adopt prefabricated building systems and Australia is no exception to this scenario [15,16]. 1. Introduction The results showed that the composite prefabricated walling systems consumed higher maintenance and lower demolition cost compared to the conventional masonry. It can be hypothesised that the PMS would consume less maintenance cost and energy than other prefabricated systems due to the good inherent thermal and acoustic insulation characteristics. Therefore, the overall LCE and LCC of the prefabricated masonry can be less than the other prefabricated systems. 3 of 22 3 of 22 Buildings 2021, 11, 294 Buildings 2021, 11, x FO Figure 1. Lifting of a prefabricated façade masonry walling system. Figure 1. Lifting of a prefabricated façade masonry walling system. Figure 1. Lifting of a prefabricated façade masonry walling system Figure 1. Lifting of a prefabricated façade masonry walling system. Moreover, systematic research studies to investigate the structural performance of PMS are limited [32–37]. It can be implied that most of the developed PMS in the past were proprietary in nature with insufficient details provided on materials, structural de- sign, fabrication and erection methods. Any PMS with connecting components should be able to withstand loads induced by the occupancy of the structure as well as the external loads exerted due to wind, fire and earthquake. In this regard, minimal research studies were deliberated in the past, which is a major hindrance in confident uptake of PMS by the industry. Furthermore, the life cycle energy and life cycle cost (LCE and LCC) of the PMS are not well accounted to highlight the benefits of the prefabricated masonry to the develop- ers and end users. Comparing LCE and LCC of prefabricated concrete, steel and timber systems with their corresponding conventional construction methods show that they are more or less similar. The prefabricated systems are considered superior in the individual In summary, the development and practice of prefabricated masonry construction are not well taken by the industry due to limited research and industrial manufacturing facilities. Although the prefabricated masonry can be an attractive solution to accelerate the labour intensive conventional masonry construction, especially for the low-rise buildings, the uptake is hindered by a lack of understanding of the structural and sustainable charac- teristics of PMS. Therefore, in this paper, an attempt has been made to critically analyse the status quo of the PMS with their future prospects in the Australian context. Initially, the PMS developed in the past are outlined in terms of their construction type and structural performance. 1. Introduction Thereafter, the available design guidelines and erection methodologies of prefabricated systems have been applied to design a typical Australian housing unit with various kinds of PMSs as a case study. Further, for the selected masonry prefabricated masonry walling systems, LCC and LCE were analysed to verify the economic beneﬁt or limitation of the system. 2.1. Reinforced Masonry The reinforced masonry (RM) walls are preferred over unreinforced masonry where substantial lateral load resistance is required due to seismic and wind load effects. The introduction of reinforcement in masonry improves the tensile resistance and ductility of the masonry. The reinforcing of the masonry wall is carried out by placing the steel bars in the hollow vertical cores of the masonry blocks and grouting of cores. The vertical bars are also restrained horizontally by the steel bars provided in the bed joints. Additionally, depending on the design requirement, the walls can be fully or partially grouted and reinforced. In masonry building design and construction, the RM walls are considered as a counterpart for reinforced concrete (RC) walls. While reinforcing helps to enhance the structural performance of masonry, it also facilitates its use as a prefabricated system, where the reinforcement acts as an integral component for handing walls during the prefabrication, transportation and assemblage. p p g Few research studies have been reported on prefabricated reinforced masonry systems. Braun et al. [43] developed a prefabricated reinforced masonry system in Switzerland which was made of dry stacked hollow blocks with no mortar joints. The walling system was fully reinforced with a vertical reinforcement inserted into each hollow vertical core with grouting and horizontal bars placed in all bed joints. This study focused on investigating the suitable connection details between prefabricated wall and foundation through quasi- static cyclic in-plane shear tests in which two types of connection details between wall and foundation were considered. The dowel thickness and anchorage lengths were differed in both of these connections as shown in Figure 2. The in-plane cyclic shear testing of both walls revealed similar behaviour and thus are recommended for the wall to foundation connection. Conventionally, RM walls require starter bars at foundation/ﬂoor levels; thus, it can be said that using dowels to connect the prefabricated walls at foundation/ﬂoor level would not be an additional effort or cost. VIEW 5 of 22 wall to foundation connection. Conventionally, RM walls require starter bars at founda- tion/floor levels; thus, it can be said that using dowels to connect the prefabricated walls at foundation/floor level would not be an additional effort or cost. Figure 2. Prefabricated reinforced masonry: wall to foundation connection, Braun et al. [43] Further a team of researchers from Spain Portugal and Italy have developed sem Figure 2. aspects of the life cycle analy [38,39]. Samani et al. [40] analy 2. Review of the Existing PMS y p p walling system and conventional masonry buildings in the US context. The results showed that the composite prefabricated walling systems consumed higher maintenance and lower demolition cost compared to the conventional masonry. It can be hypothesised that the PMS would consume less maintenance cost and energy than other prefabricated systems due to the good inherent thermal and acoustic insulation characteristics. There- fore, the overall LCE and LCC of the prefabricated masonry can be less than the other prefabricated systems. In summary, the development and practice of prefabricated masonry construction are not well taken by the industry due to limited research and industrial manufacturing facilities. Although the prefabricated masonry can be an attractive solution to accelerate In order to comprehend the characteristics of prefabricated masonry, some of the PMS reported in the available literature were reviewed and outlined in this section. It must be mentioned that there are some patented PMS which were not evolved into successful or widely used applications, and therefore are not considered in this review. Similarly, prefabricated composite or reinforced concrete walling systems, where pre-cut masonry slips were provided to give masonry a façade appearance [41], are not considered in this review as these are not truly a masonry system which should consist of discrete bricks/blocks and mortar joints. It is commonly understood that the prefabrication of conventional unreinforced masonry system is not feasible, as the bond between the units (bricks or blocks) and conventional cement mortar is relatively weak, and thus would Buildings 2021, 11, 294 4 of 22 crack during the transportation and erection stages. To encounter this limitation, mainly the reinforced, post-tensioned and high bond strength masonry walling systems were adopted for prefabrication [42]. Accordingly, some of those systems are brieﬂy summarised and discussed in this section for the prospect of developing prefabricated masonry in the Australian context. 2.1. Reinforced Masonry Prefabricated reinforced masonry: wall to foundation connection, Braun et al. [43]. Further a team of researchers from Spain Portugal and Italy have developed sem Figure 2. Prefabricated reinforced masonry: wall to foundation connection, Braun et al. [43] Figure 2. Prefabricated reinforced masonry: wall to foundation connection, Braun et al. [43]. Further, a team of researchers from Spain, Portugal and Italy have developed semi- prefabricated reinforced brick masonry light-weight vaults [44,45]. The construction of the lt i t d f t t (1) i f b i t d t l b i k h t d li d t Further, a team of researchers from Spain, Portugal and Italy have developed semi- prefabricated reinforced brick masonry light-weight vaults [44,45]. The construction of the Buildings 2021, 11, 294 5 of 22 5 of 22 vault consisted of two stages: (1) semi prefabricated steel-brick sheets were delivered at site and (2) construction was completed by ﬁlling and spraying the joints and top portion of the vaults. The structural performance of the semi-prefabricated vault was tested under instantaneous and sustain loading conditions to determine the load capacity, ductility and creep behaviour. Additionally, a predeﬁned support displacement and instantaneous line loading were applied to verify the ﬂexibility of the supporting members. The structural testing have revealed that the load capacity, ductility and joint integrity of the developed semi-prefabricated system were adequate. Recently, Muirhead et al. [46] patented a prefabricated reinforced masonry system in the USA. Hollow concrete blocks with slits on the face shells were used to fabricate the walls, where a provisional reinforcement is embedded in the slits, as shown in Figure 3a. The purpose of providing provisional reinforcement is to increase the tensile resistance of the walls during the erection. Therefore, 3 mm FRP bars were embedded in the slits with epoxy grout bonding. Further, U shaped blocks were laid on top and bottom of the wall with provisional reinforcement and grouted. Similar arrangements were proposed where the openings are required for lintels as shown in Figure 3b. Further anchor slings as indicated in Figure 3b were used to lift and place the walls during the transportation. Subsequently, the prefabricated reinforced masonry walls were transported to the site and the required vertical reinforcement was applied. 6 of 22 Figure 3. Prefabricated RM system developed by Muirhead et al. 2.1. Reinforced Masonry [46] (a) Sectional view of the pre- fabricated wall and (b) lifting position of the walls. Figure 3. Prefabricated RM system developed by Muirhead et al. [46] (a) Sectional view of the prefabricated wall and (b) lifting position of the walls. Figure 3. Prefabricated RM system developed by Muirhead et al. [46] (a) Sectional view of the pre- fabricated wall and (b) lifting position of the walls. Figure 3. Prefabricated RM system developed by Muirhead et al. [46] (a) Sectional view of the prefabricated wall and (b) ifting position of the walls Figure 3. Prefabricated RM system developed by Muirhead et al. [46] (a) Sectional view of the pre- fabricated wall and (b) lifting position of the walls. Figure 3. Prefabricated RM system developed by Muirhead et al. [46] (a) Sectional view of the prefabricated wall and (b) lifting position of the walls. et al. [47] has reported a prefabricated reinforced masonry system for which gated the in-plane shear characteristics by varying horizontal reinforcement compressive stress and vertical joint detailing. The detail of the proposed ver- angement is shown in Figure 4, where the vertical joints were designed at the walls to avoid a complex reinforcement arrangement at the joints. The horizon- ment bars were embedded into the joints to act as shear key in the vertical n-plane shear test results revealed that the failure modes of the vertically were similar to that of conventional cast in-situ walls. Based on this research, d that with the connection details available (wall to foundation and wall to M can be an effective system to establish prefabricated masonry walls. Zhang et al. [47] has reported a prefabricated reinforced masonry system for which they investigated the in-plane shear characteristics by varying horizontal reinforcement detail, axial compressive stress and vertical joint detailing. The detail of the proposed vertical joint arrangement is shown in Figure 4, where the vertical joints were designed at the web of the walls to avoid a complex reinforcement arrangement at the joints. The horizontal reinforcement bars were embedded into the joints to act as shear key in the vertical joints. The in-plane shear test results revealed that the failure modes of the vertically jointed wall were similar to that of conventional cast in-situ walls. 2.1. Reinforced Masonry Based on this research, it can be said that with the connection details available (wall to foundation and wall to wall), the RM can be an effective system to establish prefabricated masonry walls. 6 of 22 search, wall to Buildings 2021, 11, 294 Figure 4. Proposed vertical joints for prefabricated RM walls by Zhang et al. [47] 200 mm 400 mm 100 mm Horizontal Reinforcement Horizontal Reinforcement Overlap Vertical Reinforcement Stirrups Figure 4. Proposed vertical joints for prefabricated RM walls by Zhang et al. [47]. 400 mm 200 mm 100 mm Horizontal Reinforcement Horizontal Reinforcement Overlap Stirrups Vertical Reinforcement Figure 4. Proposed vertical joints for prefabricated RM walls by Zhang et al. [47] Figure 4. Proposed vertical joints for prefabricated RM walls by Zhang et al. [47]. 2.2. Post-Tensioned Masonry 2.2. Post-Tensioned Masonry Prefabricated PT masonry bridge decks used (Caine): [53] (a) positioning of prefabricated masonry deck and (b) cross sectional view of the deck. Figure 5. Prefabricated PT masonry bridge decks used (Caine): [53] (a) positioning of prefabricated masonry deck and (b) cross sectional view of the deck. OR PEER REVIEW 8 of 22 and erection, and the tendons can be later released once the walls are positioned and con- nected. This might provide a cost-effective prefabrication solution for masonry with more research studies on this aspect in the future. 8 of 22 n, and the tendons can be later released once the walls are positioned and con- i ht id t ff ti f b i ti l ti f ith er rele i er rele Figure 5. Prefabricated PT masonry bridge decks used (Caine): [53] (a) positioning of prefabricated masonry deck and (b) cross sectional view of the deck Figure 5. Prefabricated PT masonry bridge decks used (Caine): [53] (a) positioning of prefabricated masonry deck and (b) cross sectional view of the deck nected. This might provide a cost-effective prefabrication solution for masonry with more research studies on this aspect in the future. In absence of research details specific to prefabricated PT masonry, a similar analogy between RM and PT masonry can be drawn. Wight et al. [54] have outlined the application of PT masonry system for a single storey house in New Zealand. The walling system was designed to resist seismic action as per NZS 4203 [55]. Figure 6a,b provides typical wall to floor/foundation detailing of the PT wall and control joint detail between two wall panels. While detailing was specified for on-site construction, a similar technique could be devel- oped to connect prefabricated PT masonry panels. It must be highlighted that the PT walling system reported in Wight et al. [54] used dry-stack concrete blocks, where no mortar was used in erecting the walls. Subsequently, this technique would facilitate to construct the walls quicker than the conventional block- works with the required bending and shear resistance provided by the PT. A similar dry- stack PT system was reported in Ota [56], where it was referred to as a Bolt-A-Blok wall system. The key feature of the system is the usage of bolts and treaded bars as the PT component in every layer of the blockwork. 2.2. Post-Tensioned Masonry 2.2. Post-Tensioned Masonry y Similar to prefabricated RM walling system, pre-stressed/post-tensioned masonry systems are also a prospective solution for prefabrication of masonry. However, not much research efforts have been invested in assessing the performance of prefabricated pre- stressed masonries. Nevertheless, many studies were dedicated to investigating the in- plane and out of plane response of the cast in-situ prestressed masonry in the past [48– 52]. Subsequently, rational design rules are available in the masonry design standards. Similar to prefabricated RM walling system, pre-stressed/post-tensioned masonry sys- tems are also a prospective solution for prefabrication of masonry. However, not much re- search efforts have been invested in assessing the performance of prefabricated prestressed masonries. Nevertheless, many studies were dedicated to investigating the in-plane and out of plane response of the cast in-situ prestressed masonry in the past [48–52]. Subse- quently, rational design rules are available in the masonry design standards. Caine [53] outlined some of past projects that utilised prefabricated post-tensioned (PT) masonry ele- ments in UK. It was highlighted that by using the PT method, horizontal masonry elements similar to bridge decks were prefabricated for pedestrian bridges. Figure 5 shows the schematic diagram of the prefabricated PT masonry deck used in Tring Bridge as outlined in Caine [47]. It was mentioned that the prefabricated panels were built vertical and then positioned horizontally as shown in Figure 5a. Adequate camber for the prestressed section was designed to match the required eccentricity of the pre-stressing force to resist the bending and shear actions. Figure 5b shows the cross-section view of the constructed foot bridge using the prefabricated masonry deck. g g p y In absence of research details speciﬁc to prefabricated PT masonry, a similar analogy between RM and PT masonry can be drawn. Wight et al. [54] have outlined the application of PT masonry system for a single storey house in New Zealand. The walling system was designed to resist seismic action as per NZS 4203 [55]. Figure 6a,b provides typical wall to ﬂoor/foundation detailing of the PT wall and control joint detail between two wall panels. While detailing was speciﬁed for on-site construction, a similar technique could be developed to connect prefabricated PT masonry panels. 7 of 22 Buildings 2021, 11, 294 cal str u e oo i ge u i g e p e a i a e a o y e Figure 5. 2.2. Post-Tensioned Masonry 2.2. Post-Tensioned Masonry walling system reported R 16 lapping bars 800 mm long debonded one side with plastic sleeve ust be highlighted D 12 trim vertical beyond ed bend reported 1/HD 12 Top steel as a Bolt-A-Blok w Horizontal wall reinforcement e it was referred sage of bolts an Filler strip and Sealant to control joint d treaded bars as t e plenty of studies Vertical wall reinforcement to both sides of control joint can d f (b) mason ff it (a) unbonded tendons off-site during the fabrication, which would facilitate transportation Figure 6. Connection detailing of PT masonry wall [54]: (a) wall to floor/foundation and (b) wall to Figure 6. Connection detailing of PT masonry wall [54]: (a) wall to ﬂoor/foundation and (b) wall to wall with control joint. bonded tendons off site during the fabrication, which would facilitate transportation ure 6. Connection detailing of PT masonry wall [54]: (a) wall to floor/foundation and (b) wall to ction detailing of PT masonry wall [54]: (a) wall to ﬂoor/foundation and (b) wall to wall with control joint. unbonded tendons off site during the fabrication, which would facilitate transpor Figure 6. Connection detailing of PT masonry wall [54]: (a) wall to floor/foundation and (b) w Figure 6. Connection detailing of PT masonry wall [54]: (a) wall to ﬂoor/foundation and (b) wall to wall w trol joint. ered Mortared Masonry yered mortared (TLM) masonry is another construction technique that could erection of prefabricated masonry walls [61,62]. The main difference between d the conventional masonry is the composition and thickness of mortar used Normally in TLM, the mortar thickness of 0.5 mm to 3 mm is adopted, which the dimensional tolerance of units used. Typically, proprietary mortar mixes ere the constitutive materials (mainly sand) are much finer than those of con- It must be highlighted that the PT walling system reported in Wight et al. [54] used dry- stack concrete blocks, where no mortar was used in erecting the walls. Subsequently, this technique would facilitate to construct the walls quicker than the conventional blockworks with the required bending and shear resistance provided by the PT. A similar dry-stack PT system was reported in Ota [56], where it was referred to as a Bolt-A-Blok wall system. The key feature of the system is the usage of bolts and treaded bars as the PT component in every layer of the blockwork. 2.2. Post-Tensioned Masonry 2.2. Post-Tensioned Masonry Moreover, there are plenty of studies on PT masonry walls with unbonded tendons [57–60], where unbonded tendons were mainly used to ease the requirement of grouting and as well as self-centring action during the lateral loading situation. Thus, it can be hypothesised that this technique can also be used in prefabrication of masonry walls, where the post-tensioning can be applied using the unbonded tendons off-site during the fabrication, which would facilitate transportation Figure 6. Connection detailing of PT masonry wall [54]: (a) wall to floor/foundation and (b) wall to 85 mm 200 mm 300 mm 665 HRC mesh on 2 chairs per pod 2/HD 12 1/HD 12 Rib steel 1/HD 12 Top steel D 12 trim does not extend into edge beam D 12 trim vertical beyond D 12 trim horizontal below all openings Typical Lintel 2/D16 along, R6 at 140 centre to centre R 16 lapping bars 800 mm long debonded one side with plastic sleeve This side debonded, with Plastic sleeve Filler strip and Sealant to control joint Vertical wall reinforcement to both sides of control joint Horizontal wall reinforcement (a) (b) Figure 6. Connection detailing of PT masonry wall [54]: (a) wall to ﬂoor/foundation and (b) wall to wall with control joint. and control joint detail between two wall panels construction, a similar technique could be devel y panels. walling system reported in Wight et al. [54] used ar was used in erecting the walls. Subsequently ct the walls quicker than the conventional block ear resistance provided by the PT. A similar dry ], where it was referred to as a Bolt-A-Blok wal s the usage of bolts and treaded bars as the PT ork. Moreover, there are plenty of studies on PT [57–60], where unbonded tendons were mainly g and as well as self-centring action during the ypothesised that this technique can also be used re the post-tensioning can be applied using the brication, which would facilitate transportation R 16 lapping bars 800 mm long debonded one side with plastic sleeve This side debonded, with Plastic sleeve Filler strip and Sealant to control joint Vertical wall reinforcement to both sides of control joint Horizontal wall reinforcement (b) nique could be de This side debonded, with Plastic sleeve construction, a similar te y panels. 2.3. Thin Layered Mortared Masonry Thin layered mortared (TLM) masonry is another construction technique that could facilitate the erection of prefabricated masonry walls [61,62]. The main difference between the TLM and the conventional masonry is the composition and thickness of mortar used in the joints. Normally in TLM, the mortar thickness of 0.5 mm to 3 mm is adopted, which depends on the dimensional tolerance of units used. Typically, proprietary mortar mixes are used, where the constitutive materials (mainly sand) are much ﬁner than those of conventional mortar mixes. Subsequently mortar application on unit layers in TLM masonry is carried out using mortar spreaders rather than traditional trowels, which make the TLM masonry construction relatively faster than the conventional construction with less wastage at site [63]. It was generally reported that the TLM construction is about 2–3 times faster than the conventional masonry construction [64]. Other than the European standards (EN 1996-1 [65]), other masonry design standards such as Australian standards AS 3700 [66], Canadian standards (CSA S304.1-04 [67]) and American standards (MSJC [68]) outline TLM mortar application with only Autoclaved aerated concrete (AAC) blocks. pp y ( ) It was concluded in the previous studies that some of the proprietary mortars used in TLM masonry provided relatively higher bond strength than the conventional mor- tar [69,70], which could provide better resistance against the transportation and erection actions, if it is to be used as prefabricated systems. Additionally, if the bond strength of TLM masonry is not adequate to resist the erection forces, the system can be compen- sated with nominal reinforcement or prestressing. Comparatively fewer studies have been conducted on TLM masonry under various stress-states or investigating TLM masonry’s behaviour at a structural scale. Da Porto et al. [71] have reported that the TLM masonry shear walls made of perforated clay blocks portrayed moderately higher in-plane shear resistance and less deformity due to relatively higher bond strength characteristics between unit and mortar. Dhanasekar et al. [72] studied a high bond strength TLM masonry walling system to develop a prefabricated masonry walling system as shown in Figure 7. The developed system was demonstrated to withstand the handling actions as well as the in-plane shear and out of plane ﬂexural actions. Further, Dhanasekar et al. 2.2. Post-Tensioned Masonry 2.2. Post-Tensioned Masonry Moreover, there are plenty of studies on PT masonry walls with unbonded tendons [57–60], where unbonded tendons were mainly used to ease Buildings 2021, 11, 294 8 of 22 8 of 22 the requirement of grouting and as well as self-centring action during the lateral loading situation. Thus, it can be hypothesised that this technique can also be used in prefabrication of masonry walls, where the post-tensioning can be applied using the unbonded tendons off-site during the fabrication, which would facilitate transportation and erection, and the tendons can be later released once the walls are positioned and connected. This might provide a cost-effective prefabrication solution for masonry with more research studies on this aspect in the future. 2.3. Thin Layered Mortared Masonry In-plane shear wall testing of TLM masonry walls [72]: (a) in-plane shear testing arra ment and (b) sliding failure of a TLM wall. In addition Ven der Meer et al [74] have investigated creep and shrinkage cha (a) (b) Figure 8. In-plane shear wall testing of TLM masonry walls [72]: (a) in-plane shear testing arrange- ment and (b) sliding failure of a TLM wall. Figure 8. In-plane shear wall testing of TLM masonry walls [72]: (a) in-plane shear testing arrangement and (b) sliding failure of a TLM wall. (a) Figure 8. In-plane shear w (a) (b) ng of TLM masonry walls [72]: (a) in-plane s (b) y wall (b) ure 8 (a) ment and (b) sliding failure of a TLM wall. I additio Ve de Mee et al [74] ha e i e ti ated ee a d h i ka e h Figure 8. In-plane shear wall testing of TLM masonry walls [72]: (a) in-plane shear testing arrange- ment and (b) sliding failure of a TLM wall. Figure 8. In-plane shear wall testing of TLM masonry walls [72]: (a) in-plane shear testing arrangement and (b) sliding failure of a TLM wall. teristics of TLM masonry made of calcium silicate blocks to develop post-tensioned TLM masonry walls. It was reported that the final prestress loss due to creep and shrinkage were relatively less in the range of 16–24% due to reduced mortar thickness in TLM ma- sonry. Later, the same researchers [75] evaluated the in-plane shear behaviour of post– tensioned TLM masonry made of calcium silicate blocks and highlighted that the system behaved quite similar to conventional masonry with improved shear resistance. Overall, it can be stated that the TLM masonry is a prospect to develop a prefabricated masonry walling system. Similar design concepts as those of conventional masonry can be adopted for TLM masonry, with relatively higher bonding strength characteristics according to the used mortar types. on, Ven der Meer et al. [74] have investigated creep and shrinkage charac- M masonry made of calcium silicate blocks to develop post-tensioned TLM s. 2.3. Thin Layered Mortared Masonry [72] revealed that under low pre-compression (<0.5 MPa), which corresponded to less than 5% of the masonry compressive strength, the TLM concrete masonry shear walls would fail by base sliding due to the higher bond strength between unit and mortar, as shown in Figure 8, where the wall behaved similarly to reinforced concrete walls. Moreover, out-of-plane bending tests carried out by Kanyeto and Fried [73] revealed that the resistance is nearly four times higher than that of conventional masonry. 9 of 22 g of-plane l Buildings 2021, 11, 294 slidi whe Figure 7. Lifting of TLM masonry walls made of concrete blocks [72]. (a) (b) Figure 8. In-plane shear wall testing of TLM masonry walls [72]: (a) in-plane shear testing arran ment and (b) sliding failure of a TLM wall. I additio Ve de Mee et al [74] ha e i e ti ated ee a d h i ka e ha Figure 7. Lifting of TLM masonry walls made of concrete blocks [72]. four times higher than that of conventional masonry. Figure 7. Lifting of TLM masonry walls made of concrete blocks [72]. (a) (b) Figure 8. In-plane shear wall testing of TLM masonry walls [72]: (a) in-plane shear testing arrange- ment and (b) sliding failure of a TLM wall. Figure 8. In-plane shear wall testing of TLM masonry walls [72]: (a) in-plane shear testing arrangement and (b) sliding failure of a TLM wall. Figure 7. Lifting of TLM masonry walls made of concrete blocks [72]. Figure 7. Lifting of TLM masonry walls made of concrete blocks [72]. gher than that of conventional masonry. ng of TLM masonry walls made of concrete blocks [72]. Figure 7. Lifting of TLM masonry walls made of concrete blocks [72]. Figure 7. Lifting of TLM masonry walls made of concrete blocks [72]. gher than that of conventional masonry. ng of TLM masonry walls made of concrete blocks [72] Figure 7. Lifting of TLM masonry walls made of concrete blocks [72]. Figure 7. Lifting of TLM masonry walls made of concrete blocks [72]. g of TLM masonry walls made of concrete blocks [72] Figure 7. Lifting of TLM masonry walls made of concrete blocks [72]. Figure 7. Lifting of TLM masonry walls made of concrete blocks [72]. g of TLM masonry walls made of concrete blocks [72] (a) (b) Figure 8. 2.3. Thin Layered Mortared Masonry It was reported that the final prestress loss due to creep and shrinkage y less in the range of 16–24% due to reduced mortar thickness in TLM ma- the same researchers [75] evaluated the in-plane shear behaviour of post– M masonry made of calcium silicate blocks and highlighted that the system e similar to conventional masonry with improved shear resistance. Overall, d that the TLM masonry is a prospect to develop a prefabricated masonry m. Similar design concepts as those of conventional masonry can be adopted nry, with relatively higher bonding strength characteristics according to the ypes. In addition, Ven der Meer et al. [74] have investigated creep and shrinkage charac- teristics of TLM masonry made of calcium silicate blocks to develop post-tensioned TLM masonry walls. It was reported that the ﬁnal prestress loss due to creep and shrinkage were relatively less in the range of 16–24% due to reduced mortar thickness in TLM masonry. Later, the same researchers [75] evaluated the in-plane shear behaviour of post–tensioned TLM masonry made of calcium silicate blocks and highlighted that the system behaved quite similar to conventional masonry with improved shear resistance. Overall, it can be stated that the TLM masonry is a prospect to develop a prefabricated masonry walling system. Similar design concepts as those of conventional masonry can be adopted for TLM masonry, with relatively higher bonding strength characteristics according to the used mortar types. 3. Case Study of an Australian Prefabricated Masonry House of an Australian Prefabricated Masonry House 3. Case Study of an Australian Prefabricated Masonry House The review carried out in Section 2 highlights that the RM, PT and TLM construction methods are the prospects for developing prefabricated masonry walling systems. There- fore, for establishing the concept of a prefabricated masonry house design, these three walling systems along with the conventional masonry were taken into consideration for a case study of a typical house unit in Australia. The details of designing these walling systems for the considered house under various actions such as wind and earthquake as per the Australian standards were veriﬁed and outlined in this section. This case study details were then used to assess the life-cycle energy and a cost analysis later in the paper. Buildings 2021, 11, 294 t w 10 of 22 10 of 22 3.1. Prototype House ical house plan, as sh 3.1. Prototype House ical house plan, as sh A typical house plan, as shown in Figure 9, was considered in this case study to explore the prospects of prefabricated masonry design, erection procedure on site, life-cycle energy and cost analyses. The typical housing plan was selected from a wider study of the existing housing types in Australia [76,77]. The house was designed for a regional area in Australia of medium seismicity (with a zone factor of 0.12 as per Australian Earthquake Standards, AS1170.4 [78]), where prefabricated masonry would solve the issue of shortage of labour and provide resistance against bushﬁre and cyclonic destructions. The considered house is a single-story dwelling, which is more common in housing, with a ﬂoor area of around 238 m2. prospects of prefabricated masonry design, erection procedure on site, life-cycle d cost analyses. The typical housing plan was selected from a wider study of the ousing types in Australia [76,77]. The house was designed for a regional area in of medium seismicity (with a zone factor of 0.12 as per Australian Earthquake , AS1170.4 [78]), where prefabricated masonry would solve the issue of shortage and provide resistance against bushfire and cyclonic destructions. The consid- e is a single-story dwelling, which is more common in housing, with a floor area 238 m2. (a) (b) House layout selected for the design and LCE analyses: (a) Plan view and (b) Elevation Figure 9. House layout selected for the design and LCE analyses: (a) Plan view and (b) Elevation view. (a) (a) (b) (b) use layout selected for the design and LCE analyses: (a) Plan view and (b) Elevation Figure 9. House layout selected for the design and LCE analyses: (a) Plan view and (b) Elevation view. For prefabricated masonry walling systems, several possible scenarios as discussed in Section 2 were assessed, as summarised in Table 1. The conventional brick and TLM masonries were considered unreinforced (Type 1 and Type 2), and their thicknesses were assumed as 110 mm and 190 mm, respectively. Grouting was only considered for the RM wall system (Type 3), while the PT masonry system (Type 4) was designed for unbonded tendons without any grout. The detailed design of the house for the PMS as listed in Table 1 is described in the next sub-section. Buildings 2021, 11, 294 11 of 22 11 of 22 Table 1. Considered masonry walling systems. Table 1. 3.1. Prototype House ical house plan, as sh Considered masonry walling systems. Notation Prefab Walling System Wall Thickness Reinforcement Grouting Type 1 Conventional clay brick masonry 110 mm × × Type 2 TLM Hollow block masonry 190 mm × × Type 3 PT block masonry 190 mm √ × Type 4 RM block masonry 190 mm √ √ 3.2. Design Approaches The selected house for this case study was designed to withstand gravity, earthquake and wind actions. A summary of design data used for the design scenarios is given in Table 2. The assumed parameters for soil type and terrain are also included in Table 2. Based on these assumed parameters, the design loads for gravity, earthquake and wind were calculated as shown under each category in the Table 2. The design standards that were followed to compute the different actions are also outlined. Table 2. Loading scenarios and magnitudes. Load Scenario Magnitude Relevant Code Gravity Loads Roof tiles and roof truss load 1.3 kPa AS1170.1 [79] Imposed load 0.5 kPa AS1170.1 [79] Masonry wall load 2.1 kPa AS1170.1 [79] Earthquake Loads Importance level 2 AS1170.0 [80] Soil class (soft rock) Be AS1170.4 [78] Zone factor (Z) 0.12 AS1170.4 [78] Earthquake design category I AS1170.4 [78] Base shear (V) 90 kN AS1170.4 [78] Wind Loads Wind region A3 AS1170.2 [81] Regional wind speed 45 m/s AS1170.2 [81] Terrain category 3 AS1170.2 [81] Wind pressure on walls 0.8 kPa AS1170.2 [81] Table 2. Loading scenarios and magnitudes. The most critical walls subjected to compression due to gravity loads, in-plane shear due to earthquake and out-of-plane bending due to wind forces were identiﬁed. These individual walls were then designed, and their capacities were checked to resist these loads using Australian Masonry Standards (AS3700 [66]) provisions. The design of the critical walls was carried out for all the proposed conceptual PMS listed in Table 1. For safety against the tensile forces that can be caused due to lifting of prefabricated panels, TLM mortar of 3 mm thickness was used in all types of masonry walls design [82]. The design parameters and determined capacity of each type of masonry system is presented in Table 3 for comparison. The design parameters mentioned in Table 3, such as unit strength, dimensions, ﬂexural and shear bond strengths, were typical values, used for a common masonry design practice in Australia. 12 of 22 Buildings 2021, 11, 294 Table 3. Design details of critical walls for different masonry systems. 3.2. Design Approaches Design Parameter Type 1 Type 2 Type 3 Type 4 Unit strength 15 MPa 15 MPa 15 MPa 15 MPa Unit height 76 mm 190 mm 190 mm 190 mm Face-shell thickness n/a 30 mm 30 mm 30 mm Mortar type M3 M3 M3 M3 Mortar thickness 3 mm 3 mm 3 mm 3 mm compressive strength 7 MPa 8 MPa 8 MPa 8 MPa ﬂexural strength ( f ′ mt) 0.2 MPa 0.2 MPa 0.2 MPa 0.2 MPa shear strength ( f ′ ms) 0.25 MPa 0.25 MPa 0.25 MPa 0.25 MPa Grout strength f ′ cg n/a n/a n/a 25 MPa Vertical bars n/a n/a 12.7 mm strand 1 N16 Horizontal bars n/a n/a n/a 1 N12 Compression Design (Maximum Load = 13.5 kN/m) Compression capacity 190 kN/m (safe) 140 kN/m (safe) 232 kN/m (safe) 242 kN/m (safe) In-plane Shear Design (Maximum Load = 7 kN/m) In-plane shear capacity 17 kN/m (safe) 9 kN/m (safe) 9 kN/m (safe) 70 kN/m (safe) Out-of-plane bending Design (Maximum Load = 0.8 kPa) Out-of-plane bending capacity 0.1 kPa (unsafe) 0.5 kPa(unsafe) 6.2 kPa (safe) 5.7 kPa (safe) Table 3. Design details of critical walls for different masonry systems. As expected, all types of walling systems were found to be very safe in compression as the gravity loading in the selected single-story house was not very signiﬁcant. The most critical wall under in-plane shear caused by the earthquake design loads shown in Table 2 also had sufﬁcient capacity. However, for the out-of-plane wind pressure, both unreinforced systems (brick and block masonry) were found unsafe with less capacity as compared to the design wind pressure. AS 3700 [66] limits the ﬂexural bond strength to be assumed for any masonry is 0.2 MPa; however, for TLM with high bond strength mortars, the bond strength can be up to 1.0 MPa [83]; thus, TLM can be used to resist higher out-of-plane bending if appropriate tensile and shear bond strength values are recommended in the standards. Nevertheless, comparison showed that for a safe design against each type of critical load, RM and PT systems will be more suitable to choose for the prefabricated masonry application. 3.3.1. Design of Wall Lifting VIEW Specially made lifting hook clutches such as Reid Swift Lift or commercially available ferrules such as elephant foot ferrules combined with lifting eyes can be used to lift the prefabricated masonry wall. These lifting systems shall be embedded into the top plinth/lintel beam and can be designed as per AS3850 [84]. These lifting systems should be designed to an appropriate safety factor, which varies from 2.0 to 4.0 based on the type of component. The embedded element shall be veriﬁed according to the design of post-installed and cast-in fastenings in the wall according to AS5216 [85]. Further, the grout depth shall be increased locally where these lifting points are embedded if necessary. Depending on the span of the prefabricated masonry wall, the masonry shall rest on the bottom plinth and shall be suspended from the top plinth through the embedded reinforcement in the grout ﬁlls as shown in Figure 10, which implies that the RM and PT masonries (Type 3 and Type 4) can be effectively used to adopt these kinds of lifting arrangement. The spacing of the vertical reinforcement shall be determined based on the depth of the bottom plinth beam and the depth of the top plinth beam shall be designed based on the width of the panels. It must be mentioned that Type 1 and Type 2 masonry wall systems being unreinforced would be vulnerable to damage during transport and lifting, and therefore they are not preferable prefab masonry options. Otherwise, reinforcement bars/anchorages should be added to lifting positions to transfer the stresses. prefabricated masonry wall. These lifting systems shall be embedded into the top plinth/lintel beam and can be designed as per AS3850 [84]. These lifting systems should be designed to an appropriate safety factor, which varies from 2.0 to 4.0 based on the type of component. The embedded element shall be verified according to the design of post- installed and cast-in fastenings in the wall according to AS5216 [85]. Further, the grout depth shall be increased locally where these lifting points are embedded if necessary. 3.3.1. Design of Wall Lifting VIEW De- pending on the span of the prefabricated masonry wall, the masonry shall rest on the bot- tom plinth and shall be suspended from the top plinth through the embedded reinforce- ment in the grout fills as shown in Figure 10, which implies that the RM and PT masonries (Type 3 and Type 4) can be effectively used to adopt these kinds of lifting arrangement The spacing of the vertical reinforcement shall be determined based on the depth of the bottom plinth beam and the depth of the top plinth beam shall be designed based on the width of the panels. It must be mentioned that Type 1 and Type 2 masonry wall systems being unreinforced would be vulnerable to damage during transport and lifting, and therefore they are not preferable prefab masonry options. Otherwise, reinforcement bars/anchorages should be added to lifting positions to transfer the stresses Figure 10. Lifting method of masonry walls. Figure 10. Lifting method of masonry walls. Figure 10. Lifting method of masonry walls. Figure 10. Lifting method of masonry walls. 3.3.2. Erection Methods of Walls Figure 10. Lifting method of masonry walls. 3.3.2. Erection Methods of Walls The prefabricated masonry wall system can be installed on the footing slab. Once the wall is lifted and placed on the location, it must be supported through temporary props, as illustrated in Figure 11. The temporary props are commonly called push and pull props which can be used to prop the masonry panels temporarily onto the ground floor slab. The props shall be connected to the top plinth beam and the inserts need to be designed based on AS 5216 [85]. The props shall be spaced based on the wind and intended lateral action during the erection of the walls. Figure 10. Lifting method of masonry walls. 3.3.2. Erection Methods of Walls The prefabricated masonry wall system can be installed on the footing slab. Once the wall is lifted and placed on the location, it must be supported through temporary props, as illustrated in Figure 11. The temporary props are commonly called push and pull props which can be used to prop the masonry panels temporarily onto the ground ﬂoor slab. The props shall be connected to the top plinth beam and the inserts need to be designed based on AS 5216 [85]. 3.3. Wall Erection Details In this section, the possible concepts of prefabricated wall transportation and erections are discussed for the considered masonry walling systems in Section 3.1. Primarily, the prefabricated masonry wall sizes and shapes should be design based on the transport and lifting regulations as per the local requirements as well as considering the economy of the construction. Further, the prefabricated masonry wall support system relies on the type of wall to ﬂoor/foundation connection, lifting and transportation methods. Some of these concepts are drawn from the methods of transporting and erecting prefabricated concrete/reinforced concrete walls and are highlighted in the following sub-sections. Buildings 2021, 11, 294 13 of 22 13 of 22 3.3.1. Design of Wall Lifting VIEW 3.3.1. Design of Wall Lifting VIEW The props shall be spaced based on the wind and intended lateral action during the erection of the walls. Figure 10. Lifting method of masonry walls. Figure 10. Lifting method of masonry walls. Figure 10. Lifting method of masonry walls Figure 10. Lifting method of masonry walls. 3 3 2 Erection Methods of Walls 3.3.2. Erection Methods of Walls 3.3.2. Erection Methods of Walls The prefabricated masonry wall system can be installed on the footing slab. Once the wall is lifted and placed on the location, it must be supported through temporary props, as illustrated in Figure 11. The temporary props are commonly called push and pull props which can be used to prop the masonry panels temporarily onto the ground floor slab The props shall be connected to the top plinth beam and the inserts need to be designed based on AS 5216 [85]. The props shall be spaced based on the wind and intended lateral action during the erection of the walls The prefabricated masonry wall system can be installed on the footing slab. Once the wall is lifted and placed on the location, it must be supported through temporary props, as illustrated in Figure 11. The temporary props are commonly called push and pull props which can be used to prop the masonry panels temporarily onto the ground ﬂoor slab. The props shall be connected to the top plinth beam and the inserts need to be designed based on AS 5216 [85]. The props shall be spaced based on the wind and intended lateral action during the erection of the walls. 3.3.2. Erection Methods of Walls The prefabricated masonry wall system can be installed on the footing slab. Once the wall is lifted and placed on the location, it must be supported through temporary props, as illustrated in Figure 11. The temporary props are commonly called push and pull props which can be used to prop the masonry panels temporarily onto the ground floor slab The props shall be connected to the top plinth beam and the inserts need to be designed based on AS 5216 [85]. The props shall be spaced based on the wind and intended lateral action during the erection of the walls The prefabricated masonry wall system can be installed on the footing slab. Once the wall is lifted and placed on the location, it must be supported through temporary props, as illustrated in Figure 11. The temporary props are commonly called push and pull props which can be used to prop the masonry panels temporarily onto the ground ﬂoor slab. The props shall be connected to the top plinth beam and the inserts need to be designed based on AS 5216 [85]. 3 3 2 Erection Methods of Walls 3.3.2. Erection Methods of Walls The props shall be spaced based on the wind and intended lateral action during the erection of the walls. 3.3.2. Erection Methods of Walls The prefabricated masonry wall system can be installed on the footing slab. Once the wall is lifted and placed on the location, it must be supported through temporary props, as illustrated in Figure 11. The temporary props are commonly called push and pull props which can be used to prop the masonry panels temporarily onto the ground floor slab. The props shall be connected to the top plinth beam and the inserts need to be designed based on AS 5216 [85]. The props shall be spaced based on the wind and intended lateral action during the erection of the walls The prefabricated masonry wall system can be installed on the footing slab. Once the wall is lifted and placed on the location, it must be supported through temporary props, as illustrated in Figure 11. The temporary props are commonly called push and pull props which can be used to prop the masonry panels temporarily onto the ground ﬂoor slab. The props shall be connected to the top plinth beam and the inserts need to be designed based on AS 5216 [85]. The props shall be spaced based on the wind and intended lateral action during the erection of the walls. 14 of 22 Buildings 2021, 11, 294 Figure 11. Propping method of masonry walls. REVIEW Push & Pull Props Figure 11. Propping method of masonry walls. REVIEW 14 of 2 Push & Pull Props Figure 11. Propping method of masonry walls. Figure 11. Propping method of masonry walls. Moreover, Figure 12 shows the connection system between the prefabricated masonry wall and the ﬂooring slab/foundation. Dowels shall be used between the ground ﬂoor and the bottom plinth beam to connect the modules to the ground ﬂoor. These dowels can be installed during the casting of the footing slab. The dowels shall be shorter and the same as the depth of the bottom plinth beam as there is minimal or no tension expected in dowels. The corrugated tubes on the bottom plinth shall be grouted using two grouting tubes (top and bottom) to ensure proper contact between the ground ﬂoor and the prefabricated masonry wall. Figure 11. Propping method of masonry walls. 300-600 Figure 12. Connection of masonry walls with the base. 3 3 2 Erection Methods of Walls 3.3.2. Erection Methods of Walls Pump until the grout exit through the top grouting tube Pump grout exit through the bottom grouting tube Seal the edges Figure 12. Connection of masonry walls with the base. Pump until the grout exit through the top grouting tube Seal the edges Pump grout exit through the bottom grouting tube Pump grout exit through the bottom grouting tube Figure 12. Connection of masonry walls with the bas Figure 12. Connection of masonry walls with the base. 4. Life-Cycle Energy/Cost Analysis 4. Life-Cycle Energy/Cost Analysis In order to evaluate the sustainability of PMS with conventional construction, th life-cycle energy and cost analysis were carried out and reported in this section for th four types of masonry systems considered in Section 3. The energy consumptions an related greenhouse gas emissions (GHGE) were estimated for all four types of masonr wall systems. The energy consumptions and related GHGE for all the systems were as sumed to be similar to the construction materials and the location of the house was th same for all systems. Thus, the energy and GHGE estimations were limited only to pro duction and construction phases. The energy consumption and GHGE related to raw ma terials and transportation were accounted for in the production phase. Then, the energ consumption and GHGE from the construction or installation was considered in the con In order to evaluate the sustainability of PMS with conventional construction, the life-cycle energy and cost analysis were carried out and reported in this section for the four types of masonry systems considered in Section 3. The energy consumptions and related greenhouse gas emissions (GHGE) were estimated for all four types of masonry wall systems. The energy consumptions and related GHGE for all the systems were assumed to be similar to the construction materials and the location of the house was the same for all systems. Thus, the energy and GHGE estimations were limited only to production and construction phases. The energy consumption and GHGE related to raw materials and transportation were accounted for in the production phase. Then, the energy consumption and GHGE from the construction or installation was considered in the Buildings 2021, 11, 294 15 of 22 15 of 22 construction phase. The equivalent coefﬁcient for GHGE from Environmental Performance in Construction (EPiC) database [86] was used to derive the GHGE from the materials. The type of equipment and fuel to be used, and travel distance, were accounted for to derive the energy consumptions and related GHGE from transportation to installation. Thereafter, the bottom-up and top-down approach based on the economic data and energy intensity were used to estimate the embodied energy (EE). An average national input–output data [87] with hybrid energy coefﬁcients were used as per Equation (1) to calculate the EE. 4. Life-Cycle Energy/Cost Analysis 4. Life-Cycle Energy/Cost Analysis where Di is the travel distance; fE is the fuel energy factor (0.002275 MJ/kg·km); and fGHGE is the GHGE factor for fuel (0.07 kg CO2-e/MJ) [89]. Further, in order to consider the workmanship in relation to the energy consumption, it was assumed that three skilled masons were employed to construct the wall systems and the average working hours were 6 h/day. The construction of the conventional brick house in the case study consumed 28 days, whilst the other three types of masonry houses consumed 40 days when traditional construction methods [90] are used. The number of days required were selected from the labour catalogue data in the Australian context and conservatively, 28 days for a brick masonry house and 40 days for RM, PT and TLM were ﬁxed. As illustrated from the previous studies [91–93], the offsite construction time for the prefabricated element is less (i.e., about 20–30%). This offsite construction time can be further reduced by 20–40%, when using automation technologies. Thus, the construction duration was assumed to be reduced by 40% for manufacturing at a factory (i.e., prefabrication masonry systems), as the off-site construction reduces the construction complexity. Tam et al. [92] highlighted that the onsite installation time for precast structural wall is 15 min. Thus, the duration of on-site installation of a prefabricated masonry wall system was assumed to be 1.5 days. The ofﬁce equipment (i.e., computers, printer, air conditioner, telephone and lighting) and other construction equipment (i.e., cement mixer and mobile crane) were assumed to be run on temporary power with a diesel engine. The energy content and GHGE factors for this engine were taken as 38.6 GJ/kL and 69.9 kg CO2-e/GJ [88]. The total energy consumption during the production and construction phases (EP.C) was calculated based on Equation (6): EP.C = EE + ET + EP + ES (6) (6) where ES and EP are the energy consumed on-site and off-site construction, respectively. Tables 5 and 6 show the energy consumption and GHGE for the production and construc- tion of the prefabricated and traditional (control) masonry wall construction, respectively. The energy consumption of traditional construction during the onsite construction phase is signiﬁcantly higher than the prefabricated construction. This was due to the construction time, as the prefabricated construction was consumed only 1.5 days onsite, whilst the tradi- tional construction consumed more than 20 days. 4. Life-Cycle Energy/Cost Analysis 4. Life-Cycle Energy/Cost Analysis During these periods, the construction activities require energy for equipment, cement mixture, lights, etc. Table 6 highlights that about 94% of GHGEP.C was contributed by raw materials in the prefabricated masonry wall system, whilst it was about 68% in the traditional masonry wall. Off-site and on-site construction of prefabricated wall system contributed about 16% of GHGEP.C. The con- struction of a traditional masonry wall accounted for about 30% of total GHGE. The GHGE from transportation for prefabricated and traditional masonry wall systems contributed about 3% and 2% of GHGEP.C, respectively. The total GHGEP.C related to the production and construction phases of prefabricated and traditional masonry wall systems is shown in Figure 13. The traditional masonry wall system showed 20% higher GHGE compared with the prefabricated masonry wall for the Type 1 system, and 7%, 14% and 18% in Type 2, Type 3 and Type 4, respectively. This was due to the high amount of energy intensive materials and their high embodied energy and GHGE coefﬁcients as well as the higher construction time of the traditional masonry wall, compared to that of the prefabricated masonry. where ES and EP are the energy consumed on-site and off-site construction, respectively. Tables 5 and 6 show the energy consumption and GHGE for the production and construc- tion of the prefabricated and traditional (control) masonry wall construction, respectively. The energy consumption of traditional construction during the onsite construction phase is signiﬁcantly higher than the prefabricated construction. This was due to the construction time, as the prefabricated construction was consumed only 1.5 days onsite, whilst the tradi- tional construction consumed more than 20 days. During these periods, the construction activities require energy for equipment, cement mixture, lights, etc. Table 6 highlights that about 94% of GHGEP.C was contributed by raw materials in the prefabricated masonry wall system, whilst it was about 68% in the traditional masonry wall. Off-site and on-site construction of prefabricated wall system contributed about 16% of GHGEP.C. The con- struction of a traditional masonry wall accounted for about 30% of total GHGE. The GHGE from transportation for prefabricated and traditional masonry wall systems contributed about 3% and 2% of GHGEP.C, respectively. The total GHGEP.C related to the production and construction phases of prefabricated and traditional masonry wall systems is shown in Figure 13. 4. Life-Cycle Energy/Cost Analysis 4. Life-Cycle Energy/Cost Analysis Table 4 shows the embodied energy intensities of materials used in both prefabricated and traditional masonry wall systems: EE = M ∑ m=1 QmEEm (1) (1) where Qm and EEm are the quantity of material and the embodied energy coefﬁcient (GJ/unit), respectively. Table 4. The EE intensity, embodied greenhouse gas (GHGEE) intensity and density of construction material used in the wall systems [88]. Table 4. The EE intensity, embodied greenhouse gas (GHGEE) intensity and density of construction material used in the wall systems [88]. Material Density (kg/m3) Unit EE Coefﬁcient (MJ/Unit) GHGEE Coefﬁcient (kg CO2-e/Unit) Grout (25 MPa) 2400 m3 2581 361 Steel 7850 kg 38.8 2.9 Mortar 1858 kg 3.9 0.1 Gypsum Plasterboard 885 kg 6.5 0.4 Brick 1920 kg 3.5 0.32 Rockwool 70 kg 57.1 3.8 Block 1400 kg 35.2 3.2 Then, Equation (2) was used to determine the GHGE from the production and construc- tion phases (GHGEP.C). Where GHGEE is the embodied GHGE of construction materials, GHGEP is the GHGE from the production of prefabricated wall systems, which includes consumption of electricity and fuel by equipment. GHGET and GHGES are GHGE from transportation of materials and on-site construction activities (including fuel and electricity consumption by the equipment), respectively. The GHGEE was obtained from Equation (3): GHGEP.C = GHGEE + GHGEP + GHGET + GHGES (2) GHGEE = M ∑ m=1 QmGHGEm (3) GHGEP.C = GHGEE + GHGEP + GHGET + GHGES (2) M GHGEP.C = GHGEE + GHGEP + GHGET + GHGES (2) (2) GHGEE = M ∑ m=1 QmGHGEm (3) (3) where GHGEE is the embodied GHGE coefﬁcient (kg CO2-e/unit) of the construction materials, which was obtained from Table 4. Yan et al. [89] speciﬁed that the GHGE factor for diesel trucks used inland transport, which was used to estimate the GHGET using Equation (4). This study assumed that the construction site was located within an 80 km radius of the material sourced location. Then, the Equation (5) was used to calculate the energy consumption for transportation (ET): GHGET = ∑QmDi fE fGHGE (4) ET = ∑QmDi fE (5) (4) (5) Buildings 2021, 11, 294 16 of 22 16 of 22 where Di is the travel distance; fE is the fuel energy factor (0.002275 MJ/kg·km); and fGHGE is the GHGE factor for fuel (0.07 kg CO2-e/MJ) [89]. 4. Life-Cycle Energy/Cost Analysis 4. Life-Cycle Energy/Cost Analysis Prospects, Challenges and Need for Research It can be stated that the prefabricated masonry can be an alternative to the conven- tional labour intensi e masonry construction From Section 2 it was established that the 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Type 1 Type 2 Type 3 Type 4 GHGE (t CO2-e/m2) Masonry types Prefab Traditional Figure 13. Comparison of GHGE for prefabricated and conventional masonry construction. 5. Prospects, Challenges and Need for Research It can be stated that the prefabricated masonry can be an alternative to the conven- tional labour-intensive masonry construction From Section 2 it was established that the Table 5. Energy consumption (GJ) production to and construction phases. Table 6. Comparison of GHGE between conventional and prefabricated constructions. Table 6. Comparison of GHGE between conventional and prefabricated constructions. Figure 13. Comparison of GHGE for prefabricated and conventional masonry construction. Figure 13. Comparison of GHGE for prefabricated and conventional masonry construction. 5. Prospects, Challenges and Need for Research 5. Prospects, Challenges and Need for Research It can be stated that the prefabricated masonry can be an alternative to the conven- tional labour-intensive masonry construction. From Section 2, it was established that the RM, PT and TLM masonries are the main options for masonry prefabrication, as these systems facilitate the transport and erection of the walls within the regulations allowed for prefabricated construction systems. Nevertheless, systematic research studies are needed to establish fully fledged PMS using these construction methods. It can be men- tioned that the design guidelines for reinforced masonry, post-tensioned masonry and thin layered mortared masonry under various stress-states such as in-plane shear, out of plane bending and axial compression are already available in the standards. Therefore, system level research studies such as for the required connection configurations between the components (e.g., wall to floor/foundation and wall to wall), transportation and erec- It can be stated that the prefabricated masonry can be an alternative to the conven- tional labour-intensive masonry construction. From Section 2, it was established that the RM, PT and TLM masonries are the main options for masonry prefabrication, as these systems facilitate the transport and erection of the walls within the regulations allowed for prefabricated construction systems. Nevertheless, systematic research studies are needed to establish fully ﬂedged PMS using these construction methods. 4. Life-Cycle Energy/Cost Analysis 4. Life-Cycle Energy/Cost Analysis The traditional masonry wall system showed 20% higher GHGE compared with the prefabricated masonry wall for the Type 1 system, and 7%, 14% and 18% in Type 2, Type 3 and Type 4, respectively. This was due to the high amount of energy intensive materials and their high embodied energy and GHGE coefﬁcients as well as the higher construction time of the traditional masonry wall, compared to that of the prefabricated masonry. Buildings 2021, 11, 294 17 of 22 Table 5. Energy consumption (GJ) production to and construction phases. Details Type 1 Type 2 Type 3 Type 4 Prefab Conv Prefab Conv Prefab Conv Prefab Conv Embodied Raw materials (EE) 373 455 1328 1697 1337 1708 1442 1844 Offsite construction (EP) 70 - 75 - 75 - 75 - Onsite Transport (ET) 12 12 7 7 8 8 23 23 Onsite construction (ES) 7 174 7 187 7 187 7 187 Total (EP.C) 462 641 1418 1891 1426 1902 1546 2054 Table 6. Comparison of GHGE between conventional and prefabricated constructions. Details Type 1 Type 2 Type 3 Type 4 Prefab Conv Prefab Conv Prefab Conv Prefab Conv Embodied raw materials (GHGEE) 28 29 116 117 192 216 345 415 Offsite construction (GHGEP) 5.1 - 5.4 - 5.4 - 5.4 - Onsite Transport (GHGET) 0.9 0.9 0.5 0.5 0.5 0.5 1.6 1.6 Onsite construction (GHGES) 0.5 13 0.5 14 0.5 14 0.5 14 Total (GHGEP.C) 34 43 123 132 199 230 352 430 Buildings 2021, 11, x FOR PEER REVIEW 17 of 22 Table 6. Comparison of GHGE between conventional and prefabricated constructions. Details Type 1 Type 2 Type 3 Type 4 Prefab Conv Prefab Conv Prefab Conv Prefab Conv Embodied raw materials (GHGEE) 28 29 116 117 192 216 345 415 Offsite construction (GHGEP) 5.1 - 5.4 - 5.4 - 5.4 - Onsite Transport (GHGET) 0.9 0.9 0.5 0.5 0.5 0.5 1.6 1.6 Onsite construction (GHGES) 0.5 13 0.5 14 0.5 14 0.5 14 Total (GHGEP.C) 34 43 123 132 199 230 352 430 Figure 13. Comparison of GHGE for prefabricated and conventional masonry construction. 5. 4. Life-Cycle Energy/Cost Analysis 4. Life-Cycle Energy/Cost Analysis It can be mentioned that the design guidelines for reinforced masonry, post-tensioned masonry and thin layered mortared masonry under various stress-states such as in-plane shear, out of plane bending and axial compression are already available in the standards. Therefore, system level re- search studies such as for the required connection conﬁgurations between the components (e.g., wall to ﬂoor/foundation and wall to wall), transportation and erection of PMS are Buildings 2021, 11, 294 18 of 22 18 of 22 needed. Primarily, the system level performance against static and dynamic (e.g., seismic) actions should also be evaluated for the PMS. Furthermore, masonry is inherently a better material for ﬁre and sound insulations. However, the performance of prefabricated masonry along with connection components should be assessed for ﬁre and sound resistance. Additionally, sealing methods of connec- tions and components of PMS is another area of concern against different environmental conditions, which needs systematic research studies to address the gap in the knowledge. Further, the choice of a prefabrication masonry system must be based on the economic aspects of the construction I to make it viable with regard to the life cycle cost of the system in addition to an adequate structural performance. Author Contributions: Conceptualization, J.T. and S.N.; Methodology, J.T. and S.N.; Formal analysis, T.Z. and S.N.; Investigation, J.T., T.Z. and S.N.; Data curation, M.A.; Writing—original draft prepara- tion, J.T., T.Z. and S.N. Writing—review and editing, K.P.; Supervision, J.T. and K.P. All authors have read and agreed to the published version of the manuscript. 6. Summary and Conclusions Researchers and practitioners have paid limited attention to understanding and de- signing PMS in the past. The reason for limited research in establishing the PMS differs across different countries due to cost effectiveness, limited awareness and lack of under- standing of such systems’ performance. Therefore, in this study, potentiality of developing PMS in the Australian context has been assessed. Initially, the available studies with regard to masonry prefabricated systems and possible conventional masonry construction systems that can be used as prefabricated systems were appraised. Thereafter, to establish the concept of prefabricated design and construction, a prototype single storey house was selected as a case study to design three types of prefabricated masonry walling systems and their design and construction approaches were highlighted. Further to evaluate the sustainability of the prefabricated masonry construction systems, LCA analysis in terms of energy and carbon emissions were assessed and compared against conventional masonry construction system. Consequently, the following conclusions can be drawn on the prospect of prefabricated masonry construction in the Australian context. • Reinforced, post-tensioned and thin layered mortared masonry systems are better options for establishing prefabricated masonry systems (PMS), as they have been shown to possess adequate structural capacities in different states of actions and their components facilitate providing better solutions for lifting and erection processes. • The design concepts of prefabricated masonry can be drawn from masonry design standards for conventional masonry, while provisions for lifting and erections of the walling systems can be taken from well-established regulations available for prefabricated reinforced concrete walls. However, more systematic studies are needed to verify these provisions for prefabricated masonry walling systems. • In terms of the sustainability perspective, the prefabricated masonry walling systems may perform better than the conventional masonry construction depending on the type of construction method adopted. Additionally, the LCA of the prefabricated masonry walls can be further enhanced by the selection of more sustainable materials and proper executions methods. More systematic research studies are needed to establish fully-ﬂedged PMS, espe- cially structural performance at system level, where the behaviour of wall panels with its connection components against various actions should be assessed. Additionally, stud- ies are needed for prefabricated masonry walls against realistic ﬁre loadings and sound insulations. Author Contributions: Conceptualization, J.T. and S.N.; Methodology, J.T. and S.N.; Formal analysis, T.Z. and S.N.; Investigation, J.T., T.Z. and S.N.; Data curation, M.A.; Writing—original draft prepara- tion, J.T., T.Z. and S.N. References es not limit itself to only one structural material: Interlocked masonry versus cohesive masonry. J. . [CrossRef] 1. Foraboschi, P. Masonry does not limit itself to only one structural material: Interlocked masonry versus cohesive masonry. J. Build. Eng. 2019, 26, 100831. [CrossRef] g 0 9, , 0083 [C oss e ] 2. Thamboo, J.A.; Zahra, T.; Dhanasekar, R. Development of design methodology for mortarless masonry system: Case study—A resettlement housing colony. J. Build. Eng. 2020, 27, 100973. [CrossRef] 2. Thamboo, J.A.; Zahra, T.; Dhanasekar, R. Development of design methodology for mortarless masonr resettlement housing colony. J. Build. Eng. 2020, 27, 100973. [CrossRef] 3. Zahra, T.; Dhanasekar, M. 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https://openalex.org/W4327741449	https://www.mdpi.com/2079-3200/11/3/56/pdf?version=1679044813	English	null	Generalization of Skill for a Working Memory Recognition Procedure in Children: The Benefit of Starting with Easy Materials	Journal of intelligence	2,023	cc-by	12,507	Article Generalization of Skill for a Working Memory Recognition Procedure in Children: The Beneﬁt of Starting with Easy Materials Chenye Bao and Nelson Cowan * Department of Psychological Sciences, Faculty of Arts and Science, University of Missouri, Columbia, MO 65211, USA * Correspondence: cowann@missouri.edu Abstract: When children practice a new task, they need to learn both the task procedure and the materials tested. It is often unclear if improvements with practice reﬂect learning of the task procedure or familiarity with the materials. We sought to examine learning of the task procedure by switching from one set of materials to another in a working memory recognition task. We recruited 70 children (34 female, M = 11.27 years, SD = 0.62, ranging from 10.08 to 12.39) in the United States who were to remember sequences of orientations and of shapes for recognition immediately following the list. Half of the children began with orientation, an easier task, and the other half began with difﬁcult-to-name shapes, a harder task. When children began with the easier task, the acquisition of the recognition task skill in the easy condition transferred to the more difﬁcult task, optimizing the mean performance across tasks. Transfer was less potent when children began with the more difﬁcult task. The results showed that sufﬁcient practice is crucial to avoid poor initial performance, which might be important for the student’s rate of progress and task engagement. Keywords: working memory; practice; transfer effects; generalization Intelligence Journal of Intelligence Journal of Intelligence Journal of Intelligence Journal of https://www.mdpi.com/journal/jintelligence When children are asked to carry out a laboratory task, their performance depends upon both familiarity with the material and practice with the task with which it is typically tested. It is typically difﬁcult to know how much learning consists of familiarity with the materials and how much consists of practice with the task. We investigated this issue in elementary school children by changing the materials but using the same working memory recognition task for two sets of materials in sequence. Any transfer of learning of the task would result in better performance for the ﬁrst part of the second set of materials, compared with the performance for the ﬁrst part of the ﬁrst set of materials. Citation: Bao, Chenye, and Nelson Cowan. 2023. Generalization of Skill for a Working Memory Recognition Procedure in Children: The Benefit of Starting with Easy Materials. Journal of Intelligence 11: 56. https://doi.org/ 10.3390/jintelligence11030056 p p Working memory refers to a system that provides the temporary storage and manipu- lation of information that is necessary for engaging in a wide range of complex activities (Baddeley 2012). It is generally regarded as a limited capacity to maintain only a relatively small number of items, with a capacity that varies among individuals (Baddeley 2012; Cowan 2001). An individual’s capacity, and the manageability of the particular materials to be remembered given that capacity, is important for learning and cognitive performance of many kinds. For example, Cowan (2014) summarized evidence that working memory ca- pacity helps determine how well information can be learned, inasmuch as learning involves holding the combination of various pieces of information in the focus of attention at once so that new associations can be formed. Relevant information must be held in mind while a process is carried out. Presumably, for that reason, working memory is highly correlated with success in a broad range of cognitive activities, including as language comprehension (Daneman and Merikle 1996), reading (Peng et al. 2018), problem-solving and arithmetic (Swanson and Beebe-Frankenberger 2004), and decision-making (Hoffmann et al. 2020). Received: 3 February 2023 Revised: 9 March 2023 Accepted: 15 March 2023 Published: 17 March 2023 2. Working Memory and Task Familiarity It is clear that familiarity with a stimulus increases performance in short-term recogni- tion tasks. (e.g., Curby and Gauthier 2007; Morrison and Chein 2011). For example, Buttle and Raymond (2003) presented a pair of faces followed by a second pair, with one face the same and the other changed to a different individual of the same gender. Recognition was superior when the change involved a famous face as the pre-change face, the post-change face, or both. However, this effect occurred only when the change was on the left side of the screen. This effect of face familiarity was eliminated when the faces were inverted, which suggested that it required facial recognition. Others, however, have not found a beneﬁcial effect of familiarity. Chen et al. (2006) compared short-term recognition of novel and trained polygons. The results show that performance improved during practice, but that this improvement was not limited to polygons used in training. These ﬁndings indicated that familiarity with unnamable shapes played a limited role in inﬂuencing the ability of visual working memory. g y Various studies have been conducted in recent years under the heading of working memory training, but these studies mostly have examined effects of practice, not the introduction of a speciﬁc strategy for the participants to use. A growing number of working memory training studies have explored the effects of working memory training on diverse cognitive abilities in different populations (Shipstead et al. 2012; Sternberg 2008). However, the current conclusions regarding working memory training are inconsistent (Chen et al. 2006; Corbin and Camos 2011; Henry et al. 2014; Holmes et al. 2009; Klingberg et al. 2002; Melby-Lervåg and Hulme 2013; Melby-Lervåg et al. 2016; Stephenson and Halpern 2013). Claims have been made that working memory training can improve intelligence (Klingberg et al. 2002), reading comprehension (Dahlin 2011), mathematical ability (Holmes et al. 2009), and even future scholastic success (Alloway 2009; Cowan et al. 2005). “Transfer of training” to other tasks or situations (for example, training on a working memory task leading to better performance on scholastic tasks) is a critical indicator of working memory training used to evaluate the enhancement and efﬁciency of processing through training (Barnett and Ceci 2002). It is quite common in working memory training literature to equate post-test improvements in a trained task to improvements in cognitive ability (Jaeggi et al. 2008; Klingberg 2010). 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/). https://www.mdpi.com/journal/jintelligence J. Intell. 2023, 11, 56. https://doi.org/10.3390/jintelligence11030056 J. Intell. 2023, 11, 56 2 of 14 Here, in a version of a short-term recognition task, we disentangled two factors that were confounded in past work, both of which are potentially important in inﬂuencing performance, namely familiarity with the materials to be learned, on the one hand, and practice with the task, on the other hand. Either factor could underlie the effects of practice that have been observed previously (e.g., Chen et al. 2006; Morrison and Chein 2011; Rudebeck et al. 2012). We separated the factors by having the participants practice a short-term recognition task, ﬁrst with one set of materials, either line orientations or shapes, and then with another set, then ﬁnally returning to the ﬁrst set. The issue was whether practicing this task with one set of materials would enhance performance when the participant switched to the other set. We ﬁrst reviewed prior research on working memory and task familiarity and explain here how we went beyond this literature. 3. The Present Study Within the near transfer that occurs, it is not clear whether the transferred beneﬁt comes from similarities in the materials (e.g., transferring from a task using digits to a task using letters as the memoranda) or similarities in the task (e.g., transferring from recognizing to recalling digits). We believe that one way to begin to assess the role of task practice with a transfer of the training procedure is to keep the task’s requirements ﬁxed and change only the materials to be remembered from one type to a different type, within that task. We distinguished between these possibilities by using the same short-term recognition procedure with two kinds of materials, line orientations and shapes, with a switch from one type to the other and back again for a single participant. yp g g p p The purpose of the present work was to document the role of task practice within the near-training effects that have been examined. We did this by changing the materials to be remembered while keeping the requirements of the task ﬁxed. The basic procedure is illustrated in Figure 1. Items were presented one at a time in a sequence, each in the middle of the viewing screen, followed by a test of the recognition of one item, which was to be judged to have been included in the most recent sequence or absent from it. After an initial block of trials with one type of item (line orientations or unfamiliar shapes), the same type was used in a second block that began with the open-ended encouragement to remember the items through whatever strategy worked best. This was followed by two blocks of trials in which the second type of items was used instead: shapes if orientations had been presented ﬁrst, or orientations if shapes had been presented ﬁrst. Finally, there was a trial block in which the initial type of items was presented once more. Testing each new type of item introduced began with practice trials that were not counted. 2. Working Memory and Task Familiarity However, better performance on a particular task does not signal an improvement in working memory’s capacity per se (Shipstead et al. 2012). The literature has mostly shown that it is possible to become better with practice and that it is possible to show the transfer of skills from one task to a similar task, called near transfer (Shipstead et al. 2010). For example, improvements on simple-span tasks following training on an adaptive n-back task can be considered a near-transfer effect. There is little evidence of “far transfer,” that is, improvement in tasks very unlike the working memory tasks that were used for training, such as ﬂuid intelligence, reasoning, nonverbal ability, verbal ability, arithmetic, word decoding, or reading comprehension (for reviews, see Shipstead et al. 2012; Melby-Lervåg et al. 2016). What is known about the near transfer of training within working memory studies does not completely distinguish between learning of the task procedures and familiarity J. Intell. 2023, 11, 56 3 of 14 3 of 14 with the materials to be learned. Melby-Lervåg et al. (2016) conducted a meta-analysis study involving 87 working memory training studies and found no far-transfer effects but found reliable near-transfer effects. A few studies explored the potentially domain-speciﬁc nature of working memory training effects, the tendency for training to apply to one particular domain, such as transference from one visual memory task to another, but not another domain, such as transference from visual to verbal memory (e.g., Melby-Lervåg and Hulme 2013; Von Bastian and Oberauer 2014). A recent empirical study also focused on domain-speciﬁc transfer effects (Stephenson and Halpern 2013), reporting transfer effects for matrix reasoning following visual-spatial working memory training but not following auditory n-back training. However, even the extent of within-domain transfer is uncertain. 4.1. Participants 4.1. Participants a i ipa We recruited 70 children (34 female and 36 male, M = 11.27 years, SD = 0.62, ranging from 10.08 to 12.39 years) from primary schools in the United States. They were randomly assigned into two groups, which differed in the order in which two types of stimuli were presented to each group. Group One had 14 females and 21 males, M = 11.18 years, SD = 0.61, ranging from 10.11 to 12.35 years; Group Two had 20 females and 15 males, M = 11.35 years, SD = 0.62, ranging from 10.08 to 12.39 years. Parents were encouraged to complete the electronic consent and demographic information before children participated in the online experiment. The sample was composed of approximately 72.86% White children, 15.71% Asian-American children, 7.14% African-American children, 1.43% children of other races, and 2.86% of participants preferring not to say according to the parents’ self- reported records. The study was approved by the institutional review board of the Uni- it f Mi i We recruited 70 children (34 female and 36 male, M = 11.27 years, SD = 0.62, ranging from 10.08 to 12.39 years) from primary schools in the United States. They were ran- domly assigned into two groups, which differed in the order in which two types of stimuli were presented to each group. Group One had 14 females and 21 males, M = 11.18 years, SD = 0.61, ranging from 10.11 to 12.35 years; Group Two had 20 females and 15 males, M = 11.35 years, SD = 0.62, ranging from 10.08 to 12.39 years. Parents were encouraged to complete the electronic consent and demographic information before children participated in the online experiment. The sample was composed of approximately 72.86% White chil- dren, 15.71% Asian-American children, 7.14% African-American children, 1.43% children of other races, and 2.86% of participants preferring not to say according to the parents’ self-reported records. The study was approved by the institutional review board of the University of Missouri. versity of Missouri. Our analyses of the data are based on Bayesian inferential statistics (e.g., Rouder et al. 2012). These statistics yield evidence that can provide positive support either for or against the existence of an effect. 4.1. Participants 4.1. Participants Increasing the sample size offers more certainty about the correct interpretation of the findings but, unlike frequentist analyses, it does not run the risk of eventually finding a significant effect that is too small to be of practical signifi- cance; instead, the existence versus nonexistence of each effect becomes clearer with an increasing N. As is common using Bayesian methods, therefore, we allowed ourselves to increase the sample size past an initial minimum size of 40, subsequently increasing it to 70 th t th lt l i i d t i t y Our analyses of the data are based on Bayesian inferential statistics (e.g., Rouder et al. 2012). These statistics yield evidence that can provide positive support either for or against the existence of an effect. Increasing the sample size offers more certainty about the correct interpretation of the ﬁndings but, unlike frequentist analyses, it does not run the risk of eventually ﬁnding a signiﬁcant effect that is too small to be of practical signiﬁcance; instead, the existence versus nonexistence of each effect becomes clearer with an increasing N. As is common using Bayesian methods, therefore, we allowed ourselves to increase the sample size past an initial minimum size of 40, subsequently increasing it to 70 so that the results were no longer in an indeterminate range. 3. The Present Study The main predictions were (1) that the performance at the beginning of the materials tested second would be at a higher level than at the beginning of the materials tested ﬁrst, due to learning of the recognition test procedure; and (2) that the performance at the return to the initial test procedure in the ﬁnal block would be at the same practiced level as when it was last tested. In the process of pilot testing, we learned that recognition of unfamiliar shapes was considerably more difﬁcult than recognition of line orientations. This observation led us to the eventual realization that learning the recognition procedure might be facilitated by starting with the easier line-orientation task. Given the relevance of our investigation for classroom learning, we decided to examine performance in 10- and 12-year-old children, who seemed old enough to carry out the recogni- tion procedure according to instructions and benefit from practice, but young enough to have a simple memory span approaching adult-like levels (e.g., Cowan et al. 1987). 4 of 14 4 of 15 J. Intell. 2023, 11, 56 J. Intell. 2023, 11, x F Figure 1. Detailed illustration of an orientation and a shape trial. Note. The upper row was an ori- entation test trial; the lower one was a shape test trial. Figure 1. Detailed illustration of an orientation and a shape trial. Note. The upper row was an orientation test trial; the lower one was a shape test trial. Figure 1. Detailed illustration of an orientation and a shape trial. Note. The upper row was an ori- entation test trial; the lower one was a shape test trial. Figure 1. Detailed illustration of an orientation and a shape trial. Note. The upper row was an orientation test trial; the lower one was a shape test trial. 4.3. Stimuli and Procedure Each participant was tested individually in a quiet room via a popular program allow- ing interactions between the experimenter and the participant over the computer by audio and video, ZOOM. An experimenter was present throughout the whole experiment for the children online. The experimental program was created and run in PsychoPy v2021.2.3. The researcher ran the experimental program on her own computer and shared the screen with one participant at a time. Participants were allowed to use a desktop computer, laptop, or iPad to carry out the tasks. Before starting the experiment, an experimenter explained the purpose of the present study and the instructions for each phase. Then there was an op- portunity to ask questions, and the participant was encouraged to complete the experiment. They were told that they could stop the experiment at any time if they felt uncomfortable. No participant asked to stop the experiment. Lastly, an electronic payment form was to be completed by their parents after the online session. They received $15 for completing the whole experiment, which lasted about 35–50 min. For Group 1, the ﬁrst phase was practicing with the orientation stimuli. Only one orientation item was presented at the center of the screen in the ﬁrst trial. Then the second practice trial presented two orientation stimuli in sequence, then three, and ﬁnally four, summing to four practice trials in total. In each trial, participants were asked whether they had seen a probe stimulus or not within the just-seen sequence. The options “Yes” and “No” were in the lower left part and the lower right part of the screen, respectively. Children spoke their responses, and then the researcher clicked their answers immediately. In the practice phases, the children were required to correctly answer each time before starting the next trial to ensure they understood the experiment. Children received feedback after they selected the answer in the practice phase. Then the test trials were presented, each with a four-object visual sequence followed by a single probe object, to be judged as the same as one of the four sequence objects or different from all four (in either shape or orientation, depending on the current task). They did not receive feedback during the test phases. g p In Group 1, the test phases Stim1Part1 and Stim1Part2 had 20 four-item trials each, with orientation stimuli. 4.2. Design 4.2. Design There were seven phases of the experiment, each of which involved trials with a brief presentation of a four-object sequence followed by a probe to be recognized as present in the sequence or absent from it. The phases were: (1) practicing Task 1 (Practice1), (2) test- ing on Task 1 (Stim1Part1), (3) further testing on Task 1 (Stim1Part2), (4) practicing Task 2 (Practice2), (5) testing on Task 2 (Stim2Part1), (6) further testing on Task 2 (Stim2Part2), and (7) further testing on Task 1 (Stim1Return). For Group 1, Task 1 involved sequences There were seven phases of the experiment, each of which involved trials with a brief presentation of a four-object sequence followed by a probe to be recognized as present in the sequence or absent from it. The phases were: (1) practicing Task 1 (Practice1), (2) testing on Task 1 (Stim1Part1), (3) further testing on Task 1 (Stim1Part2), (4) practicing Task 2 (Practice2), (5) testing on Task 2 (Stim2Part1), (6) further testing on Task 2 (Stim2Part2), and (7) further testing on Task 1 (Stim1Return). For Group 1, Task 1 involved sequences of identical objects with different orientations and Task 2 involved sequences of objects of J. Intell. 2023, 11, 56 5 of 14 5 of 14 different shapes, whereas for Group 2, Task 1 used shapes and Task 2 used orientations. There were 4 practice trials in each practice phase and 20 trials in each test phase, 10 with the probe present in the sequence and 10 with the probe absent from the sequence. Before Stim1Part2 and Stim2Part2, the second phase for each stimulus type, there were instructions for the participants to try to ﬁnd the best way to remember the stimuli. Figure 1 illustrates one orientation trial and one shape trial. 4.3. Stimuli and Procedure Importantly, the instructions to ﬁnd the best way to remember these stimuli were given to participants at the beginning of Stim1Part2. Then the participant switched to the second set of stimuli, shape stimuli. Practice2 included four practice trials, and Stim2Part1 and Stim2Part2 were test trials, with four-item shape stimulus sequences. The instructions to ﬁnd the best way to remember these stimuli were presented at the beginning of Stim2Part2. Finally, the test phase Stim1Return was again the orientation stimulus with four-item sequences. Across the test phases, there were 100 test trials (60 trials for the ﬁrst set of stimuli and 40 trials for the second set of stimuli) and 8 practical trials. p Group 2 followed the same procedure as Group 1, but the stimulus types used were the opposite. Speciﬁcally, Group 2’s participants were presented with the sets of shape stimuli in Practice1, Stim1Part1, Stim1Part2, and Stim1Return, and with orientation stimuli in Practice2, Stim2Part1, and Stim2Part2. As shown in Figure 1, all items were presented on a screen with a uniform medium gray background. A trial began with a 1000 ms ﬁxation cross at the center of the screen, followed by a 1000 ms blank interval. Then came the four different items to be remembered in sequence, each lasting 1000 ms. The items were separated by an interstimulus interval (ISI) of 1000 ms. The test item was presented last and remained on the screen until a response was recorded. Additionally, the practice trials displayed feedback reporting J. Intell. 2023, 11, 56 6 of 14 correct or incorrect responses and instructing the participant to restart the trial or begin the next trial. correct or incorrect responses and instructing the participant to restart the trial or begin the next trial. As Figure 1 (top row) shows, the orientation items presented in each trial were ar- ranged in eight different directions (north, south, east, west, northeast, northwest, southeast, and southwest). The ﬁgure used for the orientation test was an arrow presented in white on a gray background and, when viewed horizontally, took up 21% of the screen’s width (on the experimenter’s screen, 545 of 2560 pixels in width, scaled up or down to equal the same proportion on the participant’s screen). It was rotated around the center of the ﬁgure, and four different orientations were shown in each test sequence to be remembered. 4.4. Statistical Analysis In the present study, the software package R Studio was used for descriptive statistical analyses for the proportion of responses correct in the two groups in different test phases. We relied on Bayesian tests (ANOVAs and t-tests) for inferential purposes but we also provide frequentist results, for descriptive purposes. We conducted a Bayesian analysis, which is a statistical inference method that calculates the ratio of the likelihood of each model including an effect to the likelihood of the identical model excluding that effect. The Bayesian analyses used the Cauchy prior distributed as π µ, σ2 = 1 σ2 (Rouder et al. 2012). The R package we used assumed a Cauchy distribution of effect sizes centered on 0, with a scale of 0.707 (Ly et al. 2018). The resulting Bayes factor for the inclusion of the effect (BFincl) of no less than 3 is usually considered evidence for an effect, while a BF less than 0.33 is considered to be evidence against the effect, with more extreme values indicating stronger evidence. 4.3. Stimuli and Procedure The probe to be identiﬁed as an orientation present in the series or absent from it was shown as a white pin within a black square taking up 21% of the width of the screen (on the experimenter’s screen, 545 of 2560 pixels in width and 545 of 1600 pixels in height), to distinguish the probe from the memoranda. As shown in Figure 1 (bottom row), the shape items included eight different irregular shapes, which were developed by Li et al. (2020). Each shape was centered on the screen and was presented in black (Figure 1), ﬁlling most of a white square that covered 21% of the screen’s width. After four stimuli were presented, the test item was shown with a black border around the white background to distinguish the test item from the other stimuli (Figure 1). For the serial presentation of lists of objects for an immediate recognition task, a previous study showed that preventing articulatory activity upon the presentation of each object did not change the pattern of the results (Cowan et al. 2011), so we did not add that complication to the present study. 5. Results Group N Phase Stimulus Proportion Correct Reaction Time Mean SD Mean SD 1 35 Stim1Part1 Orientation 0.78 0.42 3.47 1.75 Stim1Part2 Orientation 0.82 0.38 3.37 1.73 Stim2Part1 Shape 0.72 0.45 3.57 1.80 Stim2Part2 Shape 0.73 0.45 3.55 1.86 Stim1Return Orientation 0.79 0.41 3.56 2.13 Average 0.77 0.42 3.50 1.86 2 35 Stim1Part1 Shape 0.64 0.48 3.57 2.00 Stim1Part2 Shape 0.70 0.46 3.45 1.76 Stim2Part1 Orientation 0.75 0.43 3.83 1.88 Stim2Part2 Orientation 0.78 0.42 3.79 1.92 Stim1Return Shape 0.72 0.45 3.62 1.93 Average 0.72 0.45 3.65 1.90 Grand average 0.74 0.44 3.58 1.88 5.1. Proportion of Correct Recognition Figure 2 illustrates the mean proportion correct for five test phases for both groups. The patterns for the two groups in these five test phases were different. One can see that the performance for Group 2 was initially low for the shape trials and that the perfor- mance was higher in Group 2 later on for both kinds of stimuli. One can see that the per- formance began higher in Group 1 and that the switch to shape trials in this group in Stim2Part1 and Stim2Part2 did not result in the same poor performance that Group 2 ini- tially showed. That is to say, the kind of stimulus interacted with the order in which the stimuli were presented. This description was confirmed with the statistical analyses of the proportion correct, as reported below. Figure 2. The proportion correct in each phase (x-axis) of each group. Note. Dashed line: Group 1 started with the orientation stimuli. Solid line: Group 2 started with the shape stimuli. Practice 0.5 0.6 0.7 0.8 0.9 1.0 Proportion Correct Experimental Phase Orientation Shape Group 1 Group 2 Figure 2. The proportion correct in each phase (x-axis) of each group. Note. Dashed line: Group 1 started with the orientation stimuli. Solid line: Group 2 started with the shape stimuli. Practice phases for the two stimuli were omitted from the ﬁgure. The error bars are the standard errors of the mean. J. Intell. 2023, 11, 56 7 of 14 Table 1. The descriptive statistics of the proportion correct and reaction time for each phase. 5. Results Group N Phase Stimulus Proportion Correct Reaction Time Mean SD Mean SD 1 35 Stim1Part1 Orientation 0.78 0.42 3.47 1.75 Stim1Part2 Orientation 0.82 0.38 3.37 1.73 Stim2Part1 Shape 0.72 0.45 3.57 1.80 Stim2Part2 Shape 0.73 0.45 3.55 1.86 Stim1Return Orientation 0.79 0.41 3.56 2.13 Average 0.77 0.42 3.50 1.86 2 35 Stim1Part1 Shape 0.64 0.48 3.57 2.00 Stim1Part2 Shape 0.70 0.46 3.45 1.76 Stim2Part1 Orientation 0.75 0.43 3.83 1.88 Stim2Part2 Orientation 0.78 0.42 3.79 1.92 Stim1Return Shape 0.72 0.45 3.62 1.93 Average 0.72 0.45 3.65 1.90 Grand average 0.74 0.44 3.58 1.88 Group N Phase Stimulus Proportion Correct Reaction Time Mean SD Mean SD 1 35 Stim1Part1 Orientation 0.78 0.42 3.47 1.75 Stim1Part2 Orientation 0.82 0.38 3.37 1.73 Stim2Part1 Shape 0.72 0.45 3.57 1.80 Stim2Part2 Shape 0.73 0.45 3.55 1.86 Stim1Return Orientation 0.79 0.41 3.56 2.13 Average 0.77 0.42 3.50 1.86 2 35 Stim1Part1 Shape 0.64 0.48 3.57 2.00 Stim1Part2 Shape 0.70 0.46 3.45 1.76 Stim2Part1 Orientation 0.75 0.43 3.83 1.88 Stim2Part2 Orientation 0.78 0.42 3.79 1.92 Stim1Return Shape 0.72 0.45 3.62 1.93 Average 0.72 0.45 3.65 1.90 Grand average 0.74 0.44 3.58 1.88 Table 1. The descriptive statistics of the proportion correct and reaction time for each phase. Phase Stimulus Proportion Correct Reaction Time Mean SD Mean SD 5.1. Proportion of Correct Recognition 5.1. Proportion of Correct Recognition Figure 2 illustrates the mean pro 5.1. Proportion of Correct Recognition 5.1. Proportion of Correct Recognition Figure 2 illustrates the mean pro Figure 2 illustrates the mean proportion correct for ﬁve test phases for both groups. The patterns for the two groups in these ﬁve test phases were different. One can see that the performance for Group 2 was initially low for the shape trials and that the performance was higher in Group 2 later on for both kinds of stimuli. One can see that the performance began higher in Group 1 and that the switch to shape trials in this group in Stim2Part1 and Stim2Part2 did not result in the same poor performance that Group 2 initially showed. That is to say, the kind of stimulus interacted with the order in which the stimuli were presented. This description was conﬁrmed with the statistical analyses of the proportion correct, as reported below. Figure 2 illustrates the mean proportion correct for five test phases for both groups. The patterns for the two groups in these five test phases were different. 5. Results We examined three dependent variables: the proportion of correct recognition, which indicated how well the stimuli were remembered; the reaction time, as reported in the online supplement; and bias, a measure of an individual’s overall tendency to say that a probe was or was not included in the studied sequence. For all measures, we excluded trials with reaction times exceeding the overall mean plus 3 standard deviations, which were trials with reaction times of 13.81 s or above. Out of 7000 test trials, 78 trials (1.11%) were omitted on the basis of this criterion. The averages of the proportion correct and the reaction time in each test phase for the two groups are shown in Table 1. Intell. 2023, 11, 56 7 of 14 Table 1. The descriptive statistics of the proportion correct and reaction time for each phase. Group N Phase Stimulus Proportion Correct Reaction Time Mean SD Mean SD 1 35 Stim1Part1 Orientation 0.78 0.42 3.47 1.75 Stim1Part2 Orientation 0.82 0.38 3.37 1.73 Stim2Part1 Shape 0.72 0.45 3.57 1.80 Stim2Part2 Shape 0.73 0.45 3.55 1.86 Stim1Return Orientation 0.79 0.41 3.56 2.13 Average 0.77 0.42 3.50 1.86 2 35 Stim1Part1 Shape 0.64 0.48 3.57 2.00 Stim1Part2 Shape 0.70 0.46 3.45 1.76 Stim2Part1 Orientation 0.75 0.43 3.83 1.88 Stim2Part2 Orientation 0.78 0.42 3.79 1.92 Stim1Return Shape 0.72 0.45 3.62 1.93 Average 0.72 0.45 3.65 1.90 Grand average 0.74 0.44 3.58 1.88 5.1. Proportion of Correct Recognition Figure 2 illustrates the mean proportion correct for ﬁve test phases for both groups. The patterns for the two groups in these ﬁve test phases were different. One can see that the performance for Group 2 was initially low for the shape trials and that the performance was higher in Group 2 later on for both kinds of stimuli. One can see that the performance began higher in Group 1 and that the switch to shape trials in this group in Stim2Part1 and Stim2Part2 did not result in the same poor performance that Group 2 initially showed. That is to say, the kind of stimulus interacted with the order in which the stimuli were presented. This description was conﬁrmed with the statistical analyses of the proportion correct, as reported below. Table 1. The descriptive statistics of the proportion correct and reaction time for each phase. 5. Results 2023, 11, 56 8 of 14 8 of 14 The ANOVA of the proportion of correct recognition was conducted with one within- participant factor (phase, with ﬁve phases) and one between-participants factor (Group, 1 or 2). We examined seven models by comparing them with a null hypothesis model that did not include group, phase, or their interaction. The models, summarized in Table 2, included those with all combinations of the group and phase variables. On the basis of the table, we used three different methods to calculate BFincl (the Bayes factor for including an effect) for each effect: phase, group, and their interaction (Table 3). In the ﬁrst method, we compared the factor with the null model. For example, for the interaction effect, the Bayes factor of Model 4, which only included the interaction effect, indicated BFincl = 268,376,003. Then the second method was to divide the effects that appeared to exist alone plus the tested effect by the effects that appeared to exist, without the tested effect. For the effect of the interaction, we divided the BF of the group model plus the interaction by the BF of the group model, that is, BFincl = 238,534,312,357/796.57 = 299,453,294. Given that the Bayes factor for phase was small, we did not use it in a similar calculation to assess the interaction. Third, we compared the full model to a model omitting only the effect in question. For the interaction, we divided the full model by the model with both main effects but no interaction, so BFincl = BFModel 7/BFModel 3 = 2,686,584,969/8.67 = 310,050,198. Overall, these three methods of calculating BFincl produced quite similar results. We also used these three methods for calculating the BFincl for the main effects of phase and group. When all three methods were used, the BFincl of the effect of group was close to 800. However, the BFincl of the effect of phase was about 0.01, as Table 3 shows. In summary, the best model in the present study was Model 6, which included the group and interaction variables but not the phase variable. Table 2. Statistical results from frequentist and Bayesian ANOVA for the proportion correct. 5. Results One can see that the performance for Group 2 was initially low for the shape trials and that the perfor- mance was higher in Group 2 later on for both kinds of stimuli. One can see that the per- formance began higher in Group 1 and that the switch to shape trials in this group in Stim2Part1 and Stim2Part2 did not result in the same poor performance that Group 2 ini- tially showed. That is to say, the kind of stimulus interacted with the order in which the stimuli were presented. This description was confirmed with the statistical analyses of the proportion correct, as reported below. Figure 2. The proportion correct in each phase (x-axis) of each group. Note. Dashed line: Group 1 started with the orientation stimuli. Solid line: Group 2 started with the shape stimuli. Practice 0.5 0.6 0.7 0.8 0.9 1.0 Proportion Correct Experimental Phase Orientation Shape Group 1 Group 2 Figure 2. The proportion correct in each phase (x-axis) of each group. Note. Dashed line: Group 1 started with the orientation stimuli. Solid line: Group 2 started with the shape stimuli. Practice phases for the two stimuli were omitted from the ﬁgure. The error bars are the standard errors of the mean. 0.5 0.6 0.7 0.8 0.9 1.0 Proportion Correct Experimental Phase Orientation Shape Group 1 Group 2 Experimental Phase Figure 2. The proportion correct in each phase (x-axis) of each group. Note. Dashed line: Group 1 started with the orientation stimuli. Solid line: Group 2 started with the shape stimuli. Practice Figure 2. The proportion correct in each phase (x-axis) of each group. Note. Dashed line: Group 1 started with the orientation stimuli. Solid line: Group 2 started with the shape stimuli. Practice phases for the two stimuli were omitted from the ﬁgure. The error bars are the standard errors of the mean. Figure 2. The proportion correct in each phase (x-axis) of each group. Note. Dashed line: Group 1 started with the orientation stimuli. Solid line: Group 2 started with the shape stimuli. Practice Figure 2. The proportion correct in each phase (x-axis) of each group. Note. Dashed line: Group 1 started with the orientation stimuli. Solid line: Group 2 started with the shape stimuli. Practice phases for the two stimuli were omitted from the ﬁgure. The error bars are the standard errors of the mean. J. Intell. 5. Results Bolded numbers are the critical cross-group comparisons, which are reliable only for the ﬁrst phase for the more difﬁcult shape stimuli, BFincl = 3.44. 5.2. Reaction Times As shown in Table 1 and in the online supplement, starting with the easier orientati task in Group 1 resulted in a faster response throughout the entire experiment (M = 3.50 than starting with the more difﬁcult shape task in Group 2 (M = 3.65 s). This ﬁndi was supported by the ANOVA analysis of reaction times (see the online supplemen BFincl = 6.00 to 6.33 depending on the method of analysis. This difference in speed betwe the groups persisted even when the tasks were switched, so there was no interaction A three-way Bayesian ANOVA analysis that included not only the phase and group but also the participants’ gender did not show any reliable effects of gender (main effect, BFincl = 0.48; interaction with group, BFincl = 0.51; interaction with phase, BFincl = 0.08; three-way interaction, BFincl = 0.09). The ﬁrst two effects were indeterminate, whereas the last two effects were small enough (<0.33) to provide positive evidence of a null effect, indicating that there was no gender difference in the learning process across phases. g g g p p To learn more about the patterns of the results, Bayesian t-tests were conducted for pairwise comparisons of the ﬁve test phases in each group (Table 4). Because each t- test yielded a Bayes factor that expresses a ratio that can either support the alternative hypothesis or support the null (or can be indeterminate), there was no bias toward ﬁnding an effect with more tests, and we believed that no correction for multiple testing would be appropriate. Table 4 shows the results of these comparisons. Within a group, most of the reliable differences occurred when the stimulus has changed from orientations to shapes or vice versa, indicating that orientations were generally easier than shapes. One exception was that, in Group 2, the performance on shapes improved from Phase 2 (Stim1Part1) to Phase 7 (Stim1Return), BFincl = 12.39, a practice effect. We also carried out pairwise comparisons of the test phases that used the same stimulus across two groups (Table 4, bolded numbers). First, we compared the mean proportion correct for orientation as the ﬁrst and the second stimulus (Stim1Part1 in Group 1 vs. Stim2Part1 in Group 2; Stim1Part2 in Group 1 vs. 5. Results Conditions selected for comparison included all those within the same group as well as cross-group comparisons of the ﬁrst two phases for each type of stimulus. G1P2 = Group 1, Phase 2, and so on. Group 1 began with orientation stimuli and Group 2 began with shape stimuli. Phase 1 was practice, Phase 2 was Stim1Part1, Phase 3 was Stim1Part 2, Phase 4 was Practice, Phase 5 was Stim2Part1, Phase 6 was Stim2Part2, and Phase 7 was Stim1Return. Bolded numbers are the critical cross-group comparisons, which are reliable only for the ﬁrst test phase for the more difﬁcult shape stimuli, BFincl = 3.44. Note. Conditions selected for comparison included all those within the same group as well as cross-group comparisons of the ﬁrst two phases for each type of stimulus. G1P2 = Group 1, Phase 2, and so on. Group 1 began with orientation stimuli and Group 2 began with shape stimuli. Phase 1 was practice, Phase 2 was Stim1Part1, Phase 3 was Stim1Part 2, Phase 4 was Practice, Phase 5 was Stim2Part1, Phase 6 was Stim2Part2, and Phase 7 was Stim1Return. Bolded numbers are the critical cross-group comparisons, which are reliable only for the ﬁrst test phase for the more difﬁcult shape stimuli, BFincl = 3.44. 5. Results G1P2 G1P3 G1P5 G1P6 G1P7 G2P2 G2P3 G2P5 G2P6 G1P2 - G1P3 0.59 - G1P5 1.33 8263.23 - G1P6 0.34 1201.70 0.05 - G1P7 0.05 0.21 6.18 1.79 - G2P2 - - 3.44 - - - G2P3 - - - 0.14 - 0.44 - G2P5 0.09 - - - - 2103.25 0.84 - G2P6 - 0.50 - - - 771,403.70 31.13 0.08 - G2P7 - - - - - 12.39 0.09 0.09 0.88 Note. Conditions selected for comparison included all those within the same group as well as cross-group comparisons of the ﬁrst two phases for each type of stimulus. G1P2 = Group 1, Phase 2, and so on. Group 1 began with orientation stimuli and Group 2 began with shape stimuli. Phase 1 was practice, Phase 2 was Stim1Part1, Phase 3 was Stim1Part 2, Phase 4 was Practice, Phase 5 was Stim2Part1, Phase 6 was Stim2Part2, and Phase 7 was Stim1Return. Bolded numbers are the critical cross-group comparisons, which are reliable only for the ﬁrst test phase for the more difﬁcult shape stimuli, BFincl = 3.44. 5.2. Reaction Times As shown in Table 1 and in the online supplement, starting with the easier orientation task in Group 1 resulted in a faster response throughout the entire experiment (M = 3.50 s) than starting with the more difﬁcult shape task in Group 2 (M = 3.65 s). This ﬁnding was supported by the ANOVA analysis of reaction times (see the online supplement), BFincl = 6.00 to 6.33 depending on the method of analysis. This difference in speed between the groups persisted even when the tasks were switched, so there was no interaction of Note. Conditions selected for comparison included all those within the same group as well as cross-group comparisons of the ﬁrst two phases for each type of stimulus. G1P2 = Group 1, Phase 2, and so on. Group 1 began with orientation stimuli and Group 2 began with shape stimuli. Phase 1 was practice, Phase 2 was Stim1Part1, Phase 3 was Stim1Part 2, Phase 4 was Practice, Phase 5 was Stim2Part1, Phase 6 was Stim2Part2, and Phase 7 was Stim1Return. Bolded numbers are the critical cross-group comparisons, which are reliable only for the ﬁrst test phase for the more difﬁcult shape stimuli, BFincl = 3.44. Note. 5. Results Stim2Part2 in Group 2). The results did not show reliable differences, indicating that the order in which the orientation stimuli were presented did not affect the children’s proportion of correct recognition. Second, we carried out similar comparisons for the mean proportion correct for shape (Stim1Part1 in Group 2 vs. Stim2Part1 in Group 1; Stim1Part2 in Group 2 vs. Stim2Part2 in Group 1). The results indicated only one reliable difference: the mean proportion correct for shape in Stim1Part1 in Group 2 was reliably lower than the one for shape in Stim2Part1 in Group 1 (BFincl = 3.44), indicating that practice with the easier orientation stimuli transferred, to some extent, to the more difﬁcult shape stimuli (see Figure 2). Table 4. Bayes factors for t-test comparisons of the proportion of correct recognition for comparable conditions. Table 4. Bayes factors for t-test comparisons of the proportion of correct recognition for comparable conditions. G1P2 G1P3 G1P5 G1P6 G1P7 G2P2 G2P3 G2P5 G2P6 G1P2 - G1P3 0.59 - G1P5 1.33 8263.23 - G1P6 0.34 1201.70 0.05 - G1P7 0.05 0.21 6.18 1.79 - G2P2 - - 3.44 - - - G2P3 - - - 0.14 - 0.44 - G2P5 0.09 - - - - 2103.25 0.84 - G2P6 - 0.50 - - - 771,403.70 31.13 0.08 - G2P7 - - - - - 12.39 0.09 0.09 0.88 Note. Conditions selected for comparison included all those within the same group as well as cross-group comparisons of the ﬁrst two phases for each type of stimulus. G1P2 = Group 1, Phase 2, and so on. Group 1 began with orientation stimuli and Group 2 began with shape stimuli. Phase 1 was practice, Phase 2 was Stim1Part1, Phase 3 was Stim1Part 2, Phase 4 was Practice, Phase 5 was Stim2Part1, Phase 6 was Stim2Part2, and Phase 7 was Stim1Return. Bolded numbers are the critical cross-group comparisons, which are reliable only for the ﬁrst test phase for the more difﬁcult shape stimuli, BFincl = 3.44. 5.2. Reaction Times Table 4. Bayes factors for t-test comparisons of the proportion of correct recognition for comparable conditions. 5. Results Model Factors Predictor df F Cohen’s f p BFincl of Model 1 Phase Phase 4 276 1.97 0.046 0.10 0.01 2 Group Group 1 6920 20.65 0.055 <0.001 796.57 3 Phase + Group Phase 4 276 1.97 0.046 0.10 8.67 Group 1 64 3.55 0.071 0.06 4 Interaction Interaction 8 272 6.38 0.110 <0.001 268,376,003 5 Phase + interaction Phase 4 272 2.24 0.046 0.07 3,036,658 Interaction 4 272 10.51 0.100 <0.001 6 Group + interaction Group 1 60 2.39 0.059 0.13 238,534,312,357 Interaction 8 272 6.38 0.110 <0.001 7 Phase + Group + interaction Phase 4 272 2.24 0.046 0.07 2,686,584,969 Group 1 60 3.49 0.071 0.07 Interaction 4 272 10.51 0.100 <0.001 Note. The BFincl of each model was in comparison with the null model. Table 2. Statistical results from frequentist and Bayesian ANOVA for the proportion correct. Table 3. Three methods for calculating the Bayes factor for the proportion correct. Factor Method Formula BFincl Phase 1 Phase/null model 0.01 2 (Phase + Group)/Group 0.01 3 All effects/(Group + interaction) 0.01 Group 1 Group/null model 796.57 2 (Group + interaction)/interaction 888.81 3 All effects/(Phase + interaction) 884.72 Interaction 1 Interaction/null model 268,376,003 2 (Group + interaction)/Group 299,453,294 3 All effects/(Group + Phase) 310,050,198 J. Intell. 2023, 11, 56 9 of 14 9 of 14 ll. 2023, 11, 56 9 o A three-way Bayesian ANOVA analysis that included not only the phase and gro but also the participants’ gender did not show any reliable effects of gender (main effe BFincl = 0.48; interaction with group, BFincl = 0.51; interaction with phase, BFincl = 0. three-way interaction, BFincl = 0.09). The ﬁrst two effects were indeterminate, whereas t last two effects were small enough (<0.33) to provide positive evidence of a null effe indicating that there was no gender difference in the learning process across phases. To learn more about the patterns of the results, Bayesian t-tests were conducted pairwise comparisons of the ﬁve test phases in each group (Table 4). Because each test yielded a Bayes factor that expresses a ratio that can either support the alternati hypothesis or support the null (or can be indeterminate), there was no bias toward ﬁndi an effect with more tests, and we believed that no correction for multiple testing would appropriate. Table 4 shows the results of these comparisons. 5. Results Within a group, most of t reliable differences occurred when the stimulus has changed from orientations to shapes vice versa, indicating that orientations were generally easier than shapes. One excepti was that, in Group 2, the performance on shapes improved from Phase 2 (Stim1Par to Phase 7 (Stim1Return), BFincl = 12.39, a practice effect. We also carried out pairw comparisons of the test phases that used the same stimulus across two groups (Table bolded numbers). First, we compared the mean proportion correct for orientation as t ﬁrst and the second stimulus (Stim1Part1 in Group 1 vs. Stim2Part1 in Group 2; Stim1Pa in Group 1 vs. Stim2Part2 in Group 2). The results did not show reliable differenc indicating that the order in which the orientation stimuli were presented did not affect t children’s proportion of correct recognition. Second, we carried out similar compariso for the mean proportion correct for shape (Stim1Part1 in Group 2 vs. Stim2Part1 in Group Stim1Part2 in Group 2 vs. Stim2Part2 in Group 1). The results indicated only one reliab difference: the mean proportion correct for shape in Stim1Part1 in Group 2 was reliab lower than the one for shape in Stim2Part1 in Group 1 (BFincl = 3.44), indicating that pract with the easier orientation stimuli transferred, to some extent, to the more difﬁcult sha stimuli (see Figure 2). Table 4. Bayes factors for t-test comparisons of the proportion of correct recognition for compara conditions. G1P2 G1P3 G1P5 G1P6 G1P7 G2P2 G2P3 G2P5 G2P6 G1P2 - G1P3 0.59 - G1P5 1.33 8263.23 - G1P6 0.34 1201.70 0.05 - G1P7 0.05 0.21 6.18 1.79 - G2P2 - - 3.44 - - - G2P3 - - - 0.14 - 0.44 - G2P5 0.09 - - - - 2103.25 0.84 - G2P6 - 0.50 - - - 771,403.70 31.13 0.08 - G2P7 - - - - - 12.39 0.09 0.09 0.88 Note. Conditions selected for comparison included all those within the same group as well as cross-gro comparisons of the ﬁrst two phases for each type of stimulus. G1P2 = Group 1, Phase 2, and so on. Group 1 be with orientation stimuli and Group 2 began with shape stimuli. Phase 1 was practice, Phase 2 was Stim1Pa Phase 3 was Stim1Part 2, Phase 4 was Practice, Phase 5 was Stim2Part1, Phase 6 was Stim2Part2, and Phase 7 w Stim1Return. 6. Discussion The main ﬁnding of this study was that children showed near transfer from one task to another in a working memory training session. Children performed better on the more difﬁcult task when they started to learn the easier materials ﬁrst. Transfer effects of this sort are crucial for demonstrating the practical or clinical beneﬁts of working memory training (Melby-Lervåg and Hulme 2013). Below, we summarize the results and then discuss the aspects of task difﬁculty, the equivalence of the initial ability, practice, and bias before examining the limitations of the study and prospects for future research. By comparing the training performance of the two groups of participants, the ﬁndings of the Bayesian t-test comparison imply that if the training starts at a different level of difﬁculty, then it may affect the participants’ subsequent proportion correct during the training. Speciﬁcally, Group 1 began with the easier stimuli (orientation) before tackling the more difﬁcult shape stimuli, whereas Group 2 began with the opposite, that is, they began with the more difﬁcult stimuli (shapes) ﬁrst and then switched to orientations. We found better performance for the more difﬁcult materials in the short-term shape recognition task for the group that received these materials only after having practiced the short-term orientation recognition task. Speciﬁcally, Group 1′s ﬁrst test performance levels with the difﬁcult task in Phase 5 were much higher than Group 2′s ﬁrst performance levels with the difﬁcult task in Phase 2 (see Figure 2), which was a reliable difference (BFincl = 3.44). There was no comparable transfer for initial training on the more difﬁcult task; the switch from shape to orientation recognition in Group 2 produced a performance in Phase 5 that was no better than the performance of Group 1 in Phase 2 regarding orientation. The short- term orientation recognition task, which involved easier materials, yielded approximately equivalent performance in both groups; for the initial phase in each group, BFincl = 0.09 or 11.11 to 1 (1/.09) in favor of the null hypothesis (see Table 4, ﬁrst column). On the basis of these ﬁndings, we suggest that the order of presentation of the stimuli may inﬂuence children’s proportion of correct recognition at the beginning of training. The advantage of starting with the easier stimuli is that the recognition procedure is already familiar by the time the more difﬁcult shape stimuli are introduced. 5.2. Reaction Times As shown in Table 1 and in the online supplement, starting with the easier orientation task in Group 1 resulted in a faster response throughout the entire experiment (M = 3.50 s) than starting with the more difﬁcult shape task in Group 2 (M = 3.65 s). This ﬁnding was supported by the ANOVA analysis of reaction times (see the online supplement), BFincl = 6.00 to 6.33 depending on the method of analysis. This difference in speed between the groups persisted even when the tasks were switched, so there was no interaction of J. Intell. 2023, 11, 56 10 of 14 10 of 14 group and the phase of the experiment (BFincl = 0.0005, or 2000 to 1 in favor of the null hypothesis). This may be related to the performance advantage of Group 1 conferred by starting with an easier stimulus (orientation) ﬁrst. More details of the analysis and the results for the reaction time and bias are provided in the Supplementary Materials. 6. Discussion In summary, we found asymmetrical effects of working memory training on the transfer of training in a relatively short working memory training period for the ﬁrst time in the literature, to our knowledge. g y g p g The above conclusions depend on the fact that the initial ability levels of the partici- pants in both groups were equivalent prior to working memory training. In the current study, we did not perform an initial measurement of each participant’s ability, but when the participants had practiced, the scores of the groups were almost identical across the last two test phases (one orientation and one shape trial phase in each participant, in the opposite order (see Figure 2, Stim2Part2 and Stim1Return data). This equivalence suggests that the ability levels of the two groups were comparable and that the initial differences in performance within the groups were related to the material presented when it was unpracticed. Thus, we can conclude that the order of presentation inﬂuenced performance and that the learning procedure with more success along the way was the one starting with the easier materials. The reason that starting with easier materials is helpful could be that children’s work- ing memory is improved by the application of metamemory or insight into the capabilities of one’s own working memory (Forsberg et al. 2021), but this metamemory consumes some of the same resources as working memory storage. Metamemory, or knowledge of one’s own memory, can be used to ﬁne-tune performance. For example, if you know that you can only hold three items of a certain kind, it is counterproductive to try to hold four, because that attempt will make you lose more of them (similar to dropping a too-tall stack of dishes). J. Intell. 2023, 11, 56 11 of 14 11 of 14 A more difﬁcult set of materials (shapes compared with line orientations) leaves less of working memory’s capacity to be devoted to using metamemory to maximize performance. A more difﬁcult set of materials (shapes compared with line orientations) leaves less of working memory’s capacity to be devoted to using metamemory to maximize performance. This pattern of results underscores the potentially critical role of the amount of practice when comparing different experimental conditions in children. 6. Discussion Moreover, starting with the easier orientation task in Group 1 resulted in faster responses throughout the entire experiment than starting with the more difﬁcult shape task in Group 2. These ﬁndings suggest that by starting with simple tasks, children learn to respond more efﬁciently than when starting with more difﬁcult tasks. In our procedure, the order of presentation ultimately did not matter for the ﬁnal performance when there had been sufﬁcient practice. However, in educational settings, it can be helpful to set up an experience of success rather than an initial experience of failure, which is an argument for starting with easier materials. Moreover, some learning situations do not afford enough practice to result in the ﬁnal equivalent levels that we observed. This type of working memory experience we have examined might be applicable to children’s development in other cognitive abilities and academic achievement, such as arithmetic and writing. Given that, throughout childhood, the contents of working memory are related to how much information children will transfer to their long-term memory (Forsberg et al. 2022), we expect that our ﬁndings are relevant to learning. For example, on the basis of the ﬁndings of this study, we hypothesize that children may learn to memorize their arithmetic multiplication tables more efﬁciently if they start by memorizing the lower part of the table soundly before problems with higher numbers are presented. Our ﬁnding can also be of use in ﬁne-tuning studies of near transfer so they can be conducted in a way that distinguishes whether it is the materials or the task skill that is trained, or some of both. 7. Limitations and Prospects for Research 2023, 11, 56 12 of 14 12 of 14 models: domain-general versus domain-speciﬁc (Shah and Miyake 1996). There is an ongoing debate on whether working memory’s capacity is a domain-general construct or a separable, domain-speciﬁc construct (Melby-Lervåg and Hulme 2013The domain-general perspective of working memory involves processes that are not related to speciﬁc types of information or sensory modalities but which still help to encode, maintain, and retrieve information from working memory. It is meant to apply to any area (Morrison and Chein 2011). Morrison and Chein thought that domain-general processes of working memory involved mechanisms that control attention, gate information ﬂowing into and out of the working memory buffer in our brain, avoid interference from unnecessary information sources, and manage engagement with domain-speciﬁc strategies. Therefore, the type of stimulus, such as verbal or visual-spatial, used for working memory training should not affect the effect of training (Peng and Fuchs 2017). Others take a different view, namely the domain-speciﬁc perspective that working memory involves strategies that are speciﬁc to the maintenance and operation of speciﬁc types of information. Children’s studies tend to favor this point of view. For instance, children who participate in visual-spatial working memory training tasks would perform better in visual-spatial working memory and visual-spatial related tasks (e.g., arithmetic calculation) than verbal working memory tasks (Peng and Fuchs 2017). In clinical research, Swanson et al. (2009) thought that children with serious learning difﬁculties exhibited working memory deﬁcits in both the verbal and visual-spatial domains. However, verbal working memory deﬁcits are more related to reading difﬁculties in children, whereas visual-spatial deﬁcits tend to be more important for difﬁculties with mathematics (Swanson and Jerman 2006). To expand the applicability of our ﬁndings, it would therefore be useful to examine whether domain-general skills are learned that would apply when there is a switch from visual to verbal working memory recognition tasks or vice versa, and from sequence item recognition tasks to list item recognition tasks or vice versa. 7. Limitations and Prospects for Research To our knowledge, our study is the ﬁrst to ﬁnd a near-transfer effect for working memory training (or practice) in a single session lasting less than an hour. Our study also had some limitations that need to be considered. First, because our training period was relatively short, between approximately 30–40 min, this may have result in training effects that did not last long. Therefore, future studies could conduct longer training periods to further explore the transfer effects (e.g., in longitudinal research). In addition, some past working memory training studies have found that working memory training improves individuals’ cognitive abilities (e.g., Jaeggi et al. 2008; Klingberg 2010), whereas other studies have not found this (e.g., Melby-Lervåg et al. 2016). Our training studies did not measure the children’s cognitive abilities before and after training. Although it seems unlikely that any such transfer would emerge in a short period, it might be helpful to check this, inasmuch as a positive result could reveal aspects of apparent far transfer that might be due to the transient situation, rather than due to true long-term improvement. Importantly, to reiterate the purpose and meaning of our working memory training, the goal was not to make individuals expert performers of speciﬁc tasks, but to explore the positive effects of rapid working memory training (or practice) that may indicate previously overlooked capabilities that are relevant to children’s development and, possibly, more generally to people’s daily lives (Chase and Ericsson 1982). p p y In our study, we trained children on one visual working memory task and transferred them to another visual memory task, but it is possible that there were skills needed in these tasks that would apply to other tasks, such as verbal working memory recognition tasks. The domain-generality of the ﬁnding has not been explored yet. Plenty of existing studies have demonstrated that working memory’s capability affects a variety of individuals’ cognitive abilities such as general ﬂuid intelligence (Kane et al. 2005; Oberauer et al. 2005), reading comprehension (Daneman and Carpenter 1980; Turner and Engle 1989), reasoning (Kyllonen and Christal 1990; Von Bastian and Oberauer 2013), learning computer languages (Shute 1991), and academic achievement (Alloway 2009). One factor that might lead to the inconsistent results of working memory training studies is the nature of the training task. This argument reﬂects the intense debate over two competing working memory J. Intell. 8. Conclusions Data Availability Statement: Experimental materials, data, and analysis codes can be viewed at an anonymized OSF link: https://osf.io/g2s5h/?view_only=e5dffc06f2cf442081ed97945abf500a. Acknowledgments: The authors would like to thank all participants and parents for their cooperation. We thank Bret Glass for assistance. The work was supported by NIH grant R01-HD021338. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Alloway, Tracy Packiam. 2009. Working memory, but not IQ, predicts subsequent learning in children with learning difﬁculties. European Journal of Psychological Assessment 25: 92–98. [CrossRef] Baddeley, Alan. 2012. Working memory: Theories, models, and controversies. Annual review of psychology 63: 1–29. [CrossRef] [PubMed] Barnett, Susan. M., and Stephen J. Ceci. 2002. When and where do we apply what we learn?: A taxonomy Bulletin 128: 612. [CrossRef] Buttle, Heather, and Jane E. Raymond. 2003. High familiarity enhances visual change detection for face stimuli. Perception and Psychophysics 65: 1296–306. [CrossRef] y p y [ ] Chase, William G., and K Anders Ericsson. 1982. Skill and working memory. In Psychology of Learning and Motivation. Cambridge: Academic Press, vol. 16, pp. 1–58. pp Chen, Diyu, Hing Yee Eng, and Yuhong Jiang. 2006. 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Conclusions The ﬁndings of the current working memory training (or practice) study showed that children were able to show near transfer from one task to another after a working memory training session, with an advantage of starting with the easier task. The difﬁculty of tasks affected the children’s performance at the beginning of training. Moreover, starting with the easier task resulted in faster responses throughout the entire experiment than starting with the more difﬁcult task. After some training (experience with the task), children’s average proportion correct tended to be similar across groups, which underscores the potentially critical role of the amount of practice when comparing different experimental conditions in children. Children may develop strategies on their own with practice during the training process. These ﬁndings may shed some light on children’s cognitive development. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jintelligence11030056/s1, Figure S1: The mean reaction time in each phase (x-axis) of each group; Figure S2: The bias in each test phase (x-axis) for both groups; Table S1: Statistical results of the frequentist and Bayesian ANOVA for reaction time; Table S2: Three methods for calculating the Bayes factor for reaction time; Table S3: Statistical results of the frequentist and Bayesian ANOVA for bias; Table S4: Three methods of calculating the Bayes factor for bias. Author Contributions: Conceptualization, C.B. and N.C.; Methodology, C.B. and N.C.; Software, C.B.; Validation, N.C.; Investigation, C.B.; Data curation, C.B.; Writing—original draft, C.B.; Writing— review & editing, N.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Eunice Kennedy Shriver National Institute of Child Health and Human Development, grant number R01-HD021338. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review of the University of Missouri (protocol IRB Project Number 99-04-095; IRB Review Number 356686; approved 11 January 2022). J. Intell. 2023, 11, 56 13 of 14 13 of 14 Informed Consent Statement: Online informed consent was obtained from one parent of each child and additionally, oral assent was obtained from the child. Informed Consent Statement: Online informed consent was obtained from one parent of each child and additionally, oral assent was obtained from the child. Data Availability Statement: Experimental materials, data, and analysis codes can be viewed at an anonymized OSF link: https://osf.io/g2s5h/?view_only=e5dffc06f2cf442081ed97945abf500a. 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https://openalex.org/W2152833463	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0016492&type=printable	English	null	Analysis of Normal-Tumour Tissue Interaction in Tumours: Prediction of Prostate Cancer Features from the Molecular Profile of Adjacent Normal Cells	PloS one	2,011	cc-by	12,773	Abstract Statistical modelling, in combination with genome-wide expression profiling techniques, has demonstrated that the molecular state of the tumour is sufficient to infer its pathological state. These studies have been extremely important in diagnostics and have contributed to improving our understanding of tumour biology. However, their importance in in- depth understanding of cancer patho-physiology may be limited since they do not explicitly take into consideration the fundamental role of the tissue microenvironment in specifying tumour physiology. Because of the importance of normal cells in shaping the tissue microenvironment we formulate the hypothesis that molecular components of the profile of normal epithelial cells adjacent the tumour are predictive of tumour physiology. We addressed this hypothesis by developing statistical models that link gene expression profiles representing the molecular state of adjacent normal epithelial cells to tumour features in prostate cancer. Furthermore, network analysis showed that predictive genes are linked to the activity of important secreted factors, which have the potential to influence tumor biology, such as IL1, IGF1, PDGF BB, AGT, and TGFb. Citation: Trevino V, Tadesse MG, Vannucci M, Al-Shahrour F, Antczak P, et al. (2011) Analysis of Normal-Tumour Tissue Interaction in Tumours: Prediction of Prostate Cancer Features from the Molecular Profile of Adjacent Normal Cells. PLoS ONE 6(3): e16492. doi:10.1371/journal.pone.0016492 Editor: Diego Di Bernardo, Fondazione Telethon, Italy Received August 4, 2010; Accepted January 3, 2011; Published March 30, 2011 Received August 4, 2010; Accepted January 3, 2011; Published March 30, 2011 Copyright: 2011 Trevino 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 Trevino et al. This is an open-access article distributed under the terms of t unrestricted use, distribution, and reproduction in any medium, provided the original author and so Copyright: 2011 Trevino et al. This is an open-access article distributed under the terms of the Creative Commons unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Victor Trevino was a recipient of a Darwin Trust Fellowship and CONACyT. This work is in part funded by grants from the CRUK (C8504/A9488), Spanish Ministry of Science and Innovation (BIO2008-04212), GVA-FEDER (PROMETEO/2010/001). Abstract The authors also thank the support of the National Institute of Bioinformatics (www.inab.org) and the RTICC (grant RD06/0020/1019) both initiatives of the Instituto de Salud Carlos III (MICINN). Marina Vannucci is partially supported by NIH-NHGRI R01-HG003319. 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: ffalciani@googlemail.com [3]. These cells secrete most of the enzymes involved in ECM breakdown, for example they produce growth factors that have a role in controlling tumour cell proliferation, apoptosis, and migration. They also secrete pro-inflammatory cytokines involved in chemoattraction and activation of specific leucocytes and therefore play a role in determining inflammatory responses [4]. Growth factors and cytokines are also involved in the neoplastic transformation of cells, angiogenesis, tumour clonal expansion and growth, passage through the ECM, intravasation into blood or lymphatic vessels and the non-random homing of tumor metastasis to specific sites. Many of these factors are also secreted by normal epithelial cells, immune cells and endothelial cells in proximity of the tumour mass. It has also been shown that the stroma may impact on the response to anti-tumour therapy. Indeed, the presence of CD11b+ leucocytes confers resistance to anti- angiogenesis therapy [5]. [3]. These cells secrete most of the enzymes involved in ECM breakdown, for example they produce growth factors that have a role in controlling tumour cell proliferation, apoptosis, and migration. They also secrete pro-inflammatory cytokines involved in chemoattraction and activation of specific leucocytes and therefore play a role in determining inflammatory responses [4]. Growth factors and cytokines are also involved in the neoplastic transformation of cells, angiogenesis, tumour clonal expansion and growth, passage through the ECM, intravasation into blood or lymphatic vessels and the non-random homing of tumor metastasis to specific sites. Many of these factors are also secreted by normal epithelial cells, immune cells and endothelial cells in proximity of the tumour mass. It has also been shown that the stroma may impact on the response to anti-tumour therapy. Indeed, the presence of CD11b+ leucocytes confers resistance to anti- angiogenesis therapy [5]. Victor Trevino1,6, Mahlet G. Tadesse2, Marina Vannucci3, Fatima Al-Shahrour4, Philipp Antczak1, Sarah Durant1, Andreas Bikfalvi8,9, Joaquin Dopazo4, Moray J. Campbell5,7, Francesco Falciani1* Victor Trevino1,6, Mahlet G. Tadesse2, Marina Vannucci3, Fatima Al-Shahrour4, Philipp Antczak1, Sarah Durant1, Andreas Bikfalvi8,9, Joaquin Dopazo4, Moray J. Campbell5,7, Francesco Falciani1* 1 School of Biosciences and IBR, University of Birmingham, Edgbaston, United Kingdom, 2 Department of Epidemiology & Biostatistics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America, 3 Rice University, Houston, Texas, United States of America, 4 Bioinformatics Department, Centro de Investigacio´n Prı´ncipe Felipe, Valencia, Spain, 5 Institute of Biomedical Research, School of Medicine, The Birmingham University, Birmingham, United Kingdom, 6 Computer Science Department & Biomedical Engineering Program, Instituto Tecnolo´gico y de Estudios Superiores de Monterrey, Nuevo Leon, Mexico, 7 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York, United States of America, 8 INSERM U920, Talence, France, 9 University Bordeaux I, Talence, France PLoS ONE \| www.plosone.org Specificity of gene signatures predictive of cancer histo- pathological features The initial objective of our analysis was to test whether the molecular profile of normal cells is predictive of cancer features. We initially considered two important aspects of prostate tumour physiology: the degree of organization of tumour cells defined by a histo-pathological scoring system called Gleason score, and the ability of tumour cells to penetrate the organ capsule summarized by a binary histo-pathological score called capsular penetration. The level of differentiation of tumour cells measures the tendency of cells to aggregate in glandular-like structures that are reminiscent of the organization of the normal tissue. The Gleason score can be used to define two main classes. The first is characterized by low-grade tumours that display a highly organised structure (correspondent to a score below or equal to 6) whereas a second class is characterized by high-grade tumours cells that are dispersed in the matrix and do not show a tendency to form glandular-like structures (correspondent to a score above or equal to 7). By contrast capsular penetration describe the extent to which cells have evaded the capsule that surrounds the prostate. Adjacent normal and tumour tissues are morphologically distinct. However, they show a degree of molecularly similarity which is in part a consequence of sharing the same micro- environment [10]. We therefore wondered whether the predictive models we have developed from normal epithelial cells represent a molecular signature that is specific to normal tissue or whether the expression of the predictive genes in tumour cells may also be predictive of tumour features. In order to address this hypothesis, we took genes selected by our modelling strategies developed from the normal tissue datasets and tested whether their expression in the tumour issue was predictive of cancer features. of cells to aggregate in glandular-like structures that are reminiscent of the organization of the normal tissue. The Gleason score can be used to define two main classes. The first is characterized by low-grade tumours that display a highly organised structure (correspondent to a score below or equal to 6) whereas a second class is characterized by high-grade tumours cells that are dispersed in the matrix and do not show a tendency to form glandular-like structures (correspondent to a score above or equal to 7). By contrast capsular penetration describe the extent to which cells have evaded the capsule that surrounds the prostate. Results The link between normal and tumour shown in this analysis is also supported by a univariate analysis which we have performed using a broad spectrum of available methodologies (Figure S4). Normal-Tumour Cell Interaction in Prostate Cancer This illustrates that the quality of the tumour stroma may significantly influence tumour development. represent optimal predictive subsets that are based on a very similar number of genes and have a high degree of overlap at the gene level, suggesting that our results are independent of the methodology used (Figure 1 and Figures S1, S2, and S3). Consistent with the relatively small degree of overlap between the microarray platforms (, = 8%, see the Data Processing section in the Text S1 for details), the representative models developed from the two independent datasets have no genes in common. The importance of the micro-environment in determining the onset and progression of cancer arises the question whether it may be possible to predict the patho-physiology and clinical outcome of the tumour from specific components of the molecular state of normal cells. If possible, we would expect these molecular signatures to represent important components of cell-cell cross- talk involved in specifying the development of cancer. p g Further analysis of the relative contribution of the individual genes to the sample separation was performed using a principal component analysis (Figure 2). This approach has revealed that genes involved in cell communication pathways are predictive of capsular penetration. Within the gene set selected by the GA in the normal tissue dataset, a combination of higher expression of the gene PRELP and a lower expression of the genes UBE4A, ZNF146 in the normal cells was predictive of tumour capsular penetration. In the gene set developed by applying the BVS procedure on the normal tissue dataset, a high expression of PPP2R4, PRELP, CALLA, ISG20L2 was predictive of tumour capsular penetration. The models developed from the Lapointe dataset revealed that lower expression of OAT and higher expression of PCGF5 and MYCN in the GA model and lower expression of IGF1 and PRAC and higher expression of PCGF5 and CPSF7 are predictive of capsular penetration. We addressed this question by developing statistical models based on a genome wide profiling of normal tissue adjacent the tumour and identifying aspects that are predictive of cancer features. We have analyzed two different prostate cancer microarray datasets available in the public domain [8,9]. We show that in both datasets the molecular state of cells adjacent to the tumour is predictive of clinically relevant cancer features. These pathways are informative molecular signatures and represent pathways involved in the production and response to secreted factors. Specificity of gene signatures predictive of cancer histo- pathological features We also challenged the prediction accuracy of models deve- loped from the tumour data by performing the corresponding comparison in the normal dataset. In both cases, the prediction accuracy of the models is close to 50% (which correspond to the expected accuracy of a random guess) (Figure 3). This analysis therefore shows that the molecular signatures we have identified are specific for the tissues (normal or tumour) they have been selected to represent. Our analysis aimed to link the molecular profile of normal cells to differentiation level (low versus high differentiation) and capsular penetration (positive versus negative). This was achieved through the development of statistical models that were based on the molecular profile of normal cells and predictive of the sample classes, specifically Gleason score and capsular penetration. Normal-Tumour Cell Interaction in Prostate Cancer These findings support the potential relevance of normal tissue biopsies in the diagnosis and prognosis of prostate cancer. This approach also provides a generally applicable analysis strategy to identify key pathways involved in cell to cell communication. PLoS ONE \| www.plosone.org Introduction The application of functional genomics technologies, particu- larly gene expression profiling, has provided the scientific community with the tools to characterize the molecular state of cells and tissues at a genome level. These technologies coupled with the ability to dissect specific cell types from a complex tissue have created an unprecedented opportunity to characterise the molecular identity of specific cell types in the context of a complex tissue [1]. Following this approach, gene expression profiling have been applied to generate the transcriptional profile of tumour cells that are predictive of both tumour features and clinical outcome in a variety of human cancers [2]. Many genome-wide studies however are often analyzed not taking explicitly into consideration that components of the extra-cellular matrix (ECM) (matrix proteins, soluble grow factors and chemokines) secreted by normal cells, adjacent to the tumour site, heavily influence the biology of the tumour. Recently, stromal cells have emerged as primary candidates for playing a role into normal-tumour cell interaction Furthermore, pre-treatment of the stroma with anti-angiogen- esis molecules prior to tumour implantation in mouse tumour models may paradoxically increase tumour development [6,7]. PLoS ONE \| www.plosone.org March 2011 \| Volume 6 \| Issue 3 \| e16492 1 Normal-Tumour Cell Interaction in Prostate Cancer Functional networks linked to predictive signatures representing normal epithelial cells expression profiles include important cytokine and growth factor signals For this purpose we applied two different multivariate modelling approaches (GA-MLHD and BVS methods) to two independent datasets developed by Singh et al. [9] and by Lapointe et al. [8]. The two statistical modelling approaches are designed to search for multi-gene markers that maximise the distinction between sample classes. Using these methods, we have developed represen- tative models that were predictive of tumour features by means of the gene expression profile of normal cells. Classification accuracy and size of these models were comparable to the ones developed using the molecular state of tumour cells (Table 1). Representa- tive models developed with the BVS and GA-MLHD methods In order to facilitate the biological interpretation of the genes represented in our statistical models we used the IPA analysis software to perform an in depth analysis at the network level. To ensure our analysis covered the full spectrum of possible solutions, we used as input to the IPA software the list of genes represented in the collection of predictive models identified from the normal tissue by the GA procedure. These covers a wider spectrum of the solution space respect to the representative models described above (Figure 1 and 2) and represent 239 and 259 genes for Singh and Lapointe datasets respectively. In this analysis we PLoS ONE \| www.plosone.org March 2011 \| Volume 6 \| Issue 3 \| e16492 2 Normal-Tumour Cell Interaction in Prostate Cancer Table 1. Accuracy, size, and gene content of representative models developed from normal and tumour data. Table 1. Accuracy, size, and gene content of representative models developed from normal and tumo Table 1. Accuracy, size, and gene content of representative models developed from normal and tumour data. Class+Tissue Dataset GA-MLHD Acc BVS Acc CP+N Singh et al. WDR18, ZNF146, MBD3, UBE4A, PRELP, MXRA7, MME/CALLA* 86.1 (7) RPS2, CCL13, VCP, PRELP, PPP2R4, ISG20L2, MXRA7, ARAF, MME/CALLA* 78.0 (9) CP+T Singh et al. LUZP1, SORL1, TMSL8, HYOU1, ST14, TALDO1, DGCR6L 97.4 (7) TMSL8, RPL35, HIST1H2BK, KRT8, RAB1A, TSPAN1, TALDO1, PDLIM5, GADD45G, GDF15 92.0 (10) GS+N Singh et al. BZRPL1, TEGT, IDH3B, MID1, D83779, PTGDS, PFDN5, PTGDS* 89.7 (8) HBA1/2, PABPC1/3, TEGT, PRSS22, DNAJC4, PTGDS, PNPLA2, USP9X, PTGDS* 92.5 (9) GS+T Singh et al. TMSB4X/L3, SLC6A7, AA524802, ABCC10, INHBB, SULT2B1, PHYHIP, SLC1A5, ACPP, C7, ACPP, NR4A1* 91.6 (12) VIM, R42599, ARF1, RBM3, EIF4G2, ACPP, VEGF, SPARCL1, COL4A2, HLA-DPB1, DSTN, UBB, ACPP, NR4A1* 90.0 (14) CP+N Lapointe et al. Functional networks linked to predictive signatures representing normal epithelial cells expression profiles include important cytokine and growth factor signals H27617, PCGF5, IGF1, OAT, EPHB3, BEX1, C12orf56, H08136, IDH3G, CYR61, TNRC6B, CX3CL1, MYCN 89.2 (13) H27617, FLJ12529, PCGF5, PRAC, IGF1, H08136, TNRC6B, CX3CL1 97.4 (8) CP+T Lapointe et al. MT1X, R20199, NEBL, ACSL3, CXCL14, AI018472 96.5 (6) AA420602, H19, MT1X, CIP29, R20199, PLGLB2, ZNF533, CXCL14, NAT1 100.0 (9) GS+N Lapointe et al. FOLR1, APOD, NALP2, CLSPN, N39101, ISL1, KITLG, N46872, APOD, KBTBD10, ZNF185, AA699363, FUT8, KLK2* 93.8 (14) NALP2, ISL1, KIAA1244, KBTBD10, ZNF185, AI018026, KLK2, RERG 97.1 (8) GS+T Lapointe et al. MOCOS, ITGBL1, PLEKHH2, WDR72, DUSP8, RBM12B, MCOLN3 94.4 (7) ITGBL1, S100A1, KBTBD10, AA699944, WDR72, MTMR9, DUSP8, PUNC 97.1 (8) Accuracy (Acc) are expressed in percentage and model size are shown in brackets. Marked genes in bold and asterisk appear in both methods (GA-MLHD and BVS). Dataset is indicated. CP+N – Capsular Penetration class from Normal data, CP+T – Capsular Penetration Tumour, GS+N – Gleason Score Normal, GS+T – Gleason Score Tumour. doi:10.1371/journal.pone.0016492.t001 Accuracy (Acc) are expressed in percentage and model size are shown in brackets. Marked genes in bold and asterisk appear in both methods (GA-MLHD and BVS). Dataset is indicated. CP+N – Capsular Penetration class from Normal data, CP+T – Capsular Penetration Tumour, GS+N – Gleason Score Normal, GS+T – Gleason Score Tumour. doi:10 1371/journal pone 0016492 t001 Accuracy (Acc) are expressed in percentage and model size are shown in brackets. Marked genes in bold and asterisk appear in both methods (GA-MLHD and BVS). Dataset is indicated. CP+N – Capsular Penetration class from Normal data, CP+T – Capsular Penetration Tumour, GS+N – Gleason Score Normal, GS+T – Gleason Score T focused on Capsular penetration because of its clinical and prognostic relevance. The network analysis was performed independently in the two datasets and the most significant networks (statistically significant and with .50% target genes represented in the network) were selected for further analysis. (Table S3). With the purpose of limiting the interference of stromal cell contaminants, we selected a dataset representing a microarray analysis of seven types of normal and tumour epithelial cells populations, purified by laser-capture micro-dissection (LCM) reported by Tomlins et al. [11]. These included, normal prostate cells purified from healthy prostates (Nor), normal cells from benign prostate hyperplasia (BPH), normal cells adjacent the tumour (adj), tumour cells from prostatic intraepithelial neoplasia (PIN), tumour cells from low grade prostate carcinoma (L-PCA), tumour cells from high grade prostate carcinoma (H-PCA) and tumour cells from prostate cancer metastases (Meta). Figure 4 describes the most significant networks identified by the IPA application representative of the models based on the molecular state of normal cells and predictive of capsular penetration in the Lapointe et al. dataset (see Table S2 for the full list of significant networks identified by IPA). Figure 4A shows a network represented by the interaction between the pro- inflammatory cytokine IL1b and the transcription factor NFkB. y y p Figure 4B–D represent three interconnected sub-networks which involve the interaction between several growth factors genes and the transcription factors P53 (TP53) and C-MYC (MYC). More specifically, Figure 4C represent a network including the growth factors IGF1, its receptor IGF1R and PDGF BB. Figure 4B represents the interaction between the extracellular factors Angiotensin (AGT), the growth factor TGFb and the Notch receptor ligand Jagged (JAG). Figure 4D on the other hand represents genes that are either directly or indirectly connected to the transcription factor c-myc (MYC). Figure 4B–D represent three interconnected sub-networks which involve the interaction between several growth factors genes and the transcription factors P53 (TP53) and C-MYC (MYC). More specifically, Figure 4C represent a network including the growth factors IGF1, its receptor IGF1R and PDGF BB. Figure 4B represents the interaction between the extracellular factors Angiotensin (AGT), the growth factor TGFb and the Notch receptor ligand Jagged (JAG). Figure 4D on the other hand represents genes that are either directly or indirectly connected to the transcription factor c-myc (MYC). We hypothesize that since the 20 genes we selected were included in models highly predictive of tumour capsular penetration, they may also be differentially expressed during prostate cancer progression. We tested this hypothesis by comparing the seven LCM cell populations. Expression of predictive cytokines, growth factors and their receptors in Prostate Cancer progression In order to improve understanding of the biological significance of the IPA networks we analysed the expression of genes in different stages of prostate cancer progression. We focused the investigation on a small subset of 20 genes representing the secreted factors included in the IPA networks and their receptors (Table S3). In both datasets, predictive genes were part of networks linking extracellular molecules such as the pro-inflammatory cytokine IL1b, the pro-metastatic chemokines CX3CL1 and CCL20 and the growth factors IGF1, TGFb and PDGF BB with the activity of the nuclear transcription factors NFKb, HF4A, TP53, and MYC. (Table S3). Each quadrant in the figure represents a combination of a modelling approach and a specific dataset. Genes present in GA-MLHD and BVS for the same dataset are highlighted in red. Accuracy is reported below each heatmap. GeneBank accession number and gene symbol are shown on the left side of the heatmap. Brighter green or red colours in heatmaps represent lower or higher relative expression respectively. t-test p-value is shown for comparison with the differential expression criteria commonly used in univariate variable selection approaches. doi:10.1371/journal.pone.0016492.g001 not in Meta cells (Figure 5E). The expression of the LOX gene was found higher in all tumour cell populations relative to adjacent and normal cells (Figure 5F) consistent with the fact that higher expression of LOX has been associated to hypoxia-induced metastasis in breast, head, neck cancers [12,13]. not in Meta cells (Figure 5E). The expression of the LOX gene was found higher in all tumour cell populations relative to adjacent and normal cells (Figure 5F) consistent with the fact that higher expression of LOX has been associated to hypoxia-induced metastasis in breast, head, neck cancers [12,13]. Of relevance for understanding the biological basis of the predictive power of normal cells signature is the observation that normal cells adjacent the tumour showed significant differences in respect to Normal cells and BPH (Figure 5B). Five genes (IL1R, LOX and TGFBR, CX3CL1 and CYR61) were differentially expressed between the three populations of normal cells. More specifically, normal cells adjacent to the tumour (Norm) were characterized by a lower expression of the tumour suppressor gene LOX, the receptors for interleukin 1 (IL1R) and TGFb (TGFBR) and by a higher expression of the pro-tumour genes CYR61 and CX3CL1. The expression of AGT, TGFB and JAG1 were linked in a different IPA network (Figure 4B). The expression of Angioten- sinogen (AGT) is higher in adjacent cells compared to PIN, L-PCA and H-PCA whereas JAG1 follows an opposite trend (down regulated in adjacent cells respect to L-PCA, H-PCA and Meta). If angiotensinogen is produced at higher levels in adjacent cells one of the activating enzymes which convert the product of the AGT gene in angiotensin II (ACE) is instead higher in PIN and L-PCA, suggesting the potential for utilization in tumour cells at lower stages of prostate cancer development. (Table S3). We discovered that a surprising large proportion of these genes were differentially expressed (75% at p,0.001 and 95% at p,0.05) (Table S3, Figure 5, S7 and S8). Further support to the relevance of the gene expression signature we had identified came from the observation that the two dimensional cluster analyses performed using the matrix of differential gene expression profiles (average expression for each group), recapitulated the expected relationship between the different stages in the development of prostate cancer (Figure 5A). More precisely, normal cell populations clustered together followed by PIN and a cluster of L- PCA and H-PCA. The Metastatic cell group clustered aside. The top four most significant networks identified from the Singh dataset (Figure S5) represent genes connected to the same cytokines and growth factors identified in the Lapointe dataset. This interesting observation suggests that, despite the limited amount of overlap at the gene level, models derived from the two dataset may represent functionally similar molecular networks. The top four most significant networks identified from the Singh dataset (Figure S5) represent genes connected to the same cytokines and growth factors identified in the Lapointe dataset. This interesting observation suggests that, despite the limited amount of overlap at the gene level, models derived from the two dataset may represent functionally similar molecular networks. PLoS ONE \| www.plosone.org March 2011 \| Volume 6 \| Issue 3 \| e16492 3 Normal-Tumour Cell Interaction in Prostate Cancer Figure 1. Multivariate Models for Capsular Penetration using Normal data. The figure shows the heat maps representing the expression profile of genes selected by the GA and BVS models in both Lapointe and Singh datasets from the normal tissue data. Each quadrant in the figure represents a combination of a modelling approach and a specific dataset. Genes present in GA-MLHD and BVS for the same dataset are highlighted in red. Accuracy is reported below each heatmap. GeneBank accession number and gene symbol are shown on the left side of the heatmap. Brighter green or red colours in heatmaps represent lower or higher relative expression respectively. t-test p-value is shown for comparison with the differential expression criteria commonly used in univariate variable selection approaches. doi:10.1371/journal.pone.0016492.g001 Figure 1. Multivariate Models for Capsular Penetration using Normal data. The figure shows the heat maps representing the expression profile of genes selected by the GA and BVS models in both Lapointe and Singh datasets from the normal tissue data. PLoS ONE \| www.plosone.org (Table S3). The finding that AGT and JAG1 have opposite trends supports the hypothesis that AGT may repress the expression of JAG1 (Figure S8 panels E and F). This connection was reported by the IPA software (Figure 4B) but was supported by an endothelial cell culture experimental model [14]. These results are consistent with the hypothesis that this mechanism may also be relevant in prostate cancer. We then examined the expression of individual genes across the different stages of tumour progression in relation to the networks identified by the IPA software (Figure 4). The cytokine IL1b, identified by the IPA analysis as linked to the activation of the pro-metastatic chemokines CX3CL1 and CCL20 (Figure 4A), was up-regulated in the tumour cell populations PIN and H-PCA (Figure 5A, and 5C), whereas the expression of IL1R1, which mediated the activity of IL1b, follows an opposite trend (Figure 5A, B and C). The pro- metastatic chemokine CX3CL1 was expressed at higher levels in adjacent cell population respect to PIN, L-PCA and H-PCA but PLoS ONE \| www.plosone.org March 2011 \| Volume 6 \| Issue 3 \| e16492 4 Normal-Tumour Cell Interaction in Prostate Cancer Figure 2. Principal component representation for Capsular Penetration using Normal Data. The figure shows the result of a PCA representing sample separation on the basis of the expression in normal tissue of genes selected by the modelling procedures. Each quadrant in the figure represents a combination of a modelling approach and a specific dataset. Each quadrant contains a 2D plot representing the separation of capsular penetration negative (black close circles) and positive (red close circles) samples (plots B, D, F and H) and a bar chart (plots A, C, E and G) representing the PC loadings (x axis) for each gene component (y axis). Note that PC loadings represent the contribution of every gene to class separation. Dashed lines delimitated genes with larger contribution that are discussed in the manuscript. Genes present in GA-MLHD and BVS for the same dataset are highlighted in red. doi:10.1371/journal.pone.0016492.g002 Figure 2. Principal component representation for Capsular Penetration using Normal Data. The figure shows the result of a PCA representing sample separation on the basis of the expression in normal tissue of genes selected by the modelling procedures. Each quadrant in the figure represents a combination of a modelling approach and a specific dataset. Discussion We have demonstrated that normal epithelial cell signatures are predictive of important features of prostate cancer. This finding has potential clinical implications as it may suggests that the molecular state of normal cells has prognostic value. At the molecular level, network analysis has revealed that our approach has the potential to identify genes involved in the disease pathogenesis. These include key genes encoding cytokines and growth factors expressed by normal epithelial cells and known to influence the biology of the tumour. The analysis of the LCM dataset showed that IL1b is expressed at higher levels in PIN and H-PCA than in adjacent cell populations whereas the latter expressed higher levels of the receptor (ILR1) (Figure 5). (Table S3). Each quadrant contains a 2D plot representing the separation of capsular penetration negative (black close circles) and positive (red close circles) samples (plots B, D, F and H) and a bar chart (plots A, C, E and G) representing the PC loadings (x axis) for each gene component (y axis). Note that PC loadings represent the contribution of every gene to class separation. Dashed lines delimitated genes with larger contribution that are discussed in the manuscript. Genes present in GA-MLHD and BVS for the same dataset are highlighted in red. doi:10.1371/journal.pone.0016492.g002 A third IPA network represents the interaction between the tumour-promoting factors IGF1, PDGF BB and CYR61 (Figure 4C). Although the expression of PDGF is constant in all cell populations, its receptor (PDGFR) is higher in H-PCA and Meta cell populations compared to adjacent cells. The expression of CYR61 is higher in adjacent cells respect to PIN and Meta cell populations (Figure S8). through the activation of the NFkB complex. The IPA software linked IL1b to the expression of the known pro-metastatic chemokines CX3CL1 [15] and CCL20 [16] in an endothelial cell culture model [14]. Although induction of these chemokines by IL1b has not been demonstrated to date, several pieces of evidence support the relevance of this mechanism in prostate cancer progression. Voronov et al. [17] have shown that IL1b is required for tumour invasiveness and angiogenesis in a mouse breast cancer model and provided evidence for the same mechanism in prostate cancer. More recently, an Interleukin-1 receptor antagonist haplotype have been found to be associated with prostate cancer risk [18] suggesting that the results of the animal model may be relevant in a clinical setting. PLoS ONE \| www.plosone.org Normal-Tumour Cell Interaction in Prostate Cancer Normal-Tumour Cell Interaction in Prostate Cancer number of cancers [20] including prostate [21]. The production of this chemokine by epithelial cells adjacent the tumour has th f th t ti l t i d t ll i ti Si il l prostate cancer cell line DU 145 via a mechanis interference with FGF2 binding and signalling c H LOX h l b d h Figure 3. Accuracy and Tissue specificity of representative models. The predictive accuracy of the models developed using (panel A, filled circles) is comparable to those models developed using tumour tissue (panel B, filled diamonds). When models dev normal tissue are trained and tested using data from tumour tissue, the prediction power is decreased considerably (empty circles). Lik models trained and tested with data from normal tissue are also non predictive (empty diamonds). doi:10.1371/journal.pone.0016492.g003 Figure 3. Accuracy and Tissue specificity of representative models. The predictive accuracy of the models developed using normal tissue (panel A, filled circles) is comparable to those models developed using tumour tissue (panel B, filled diamonds). When models developed using normal tissue are trained and tested using data from tumour tissue, the prediction power is decreased considerably (empty circles). Likewise, tumour models trained and tested with data from normal tissue are also non predictive (empty diamonds). doi:10.1371/journal.pone.0016492.g003 Figure 3. Accuracy and Tissue specificity of representative models. The predictive accuracy of the models developed using normal tissue (panel A, filled circles) is comparable to those models developed using tumour tissue (panel B, filled diamonds). When models developed using normal tissue are trained and tested using data from tumour tissue, the prediction power is decreased considerably (empty circles). Likewise, tumour models trained and tested with data from normal tissue are also non predictive (empty diamonds). doi:10.1371/journal.pone.0016492.g003 prostate cancer cell line DU 145 via a mechanism involving interference with FGF2 binding and signalling cascade [23]. However, LOX has also been reported to have an important tumour promoting activity by favouring metastasis in breast, head, and neck cancers [12] [13]. The analysis of the LCM dataset has shown that the expression of the LOX is higher in all tumour cell populations (PIN, L-PCA, H-PCA and Meta) respect to adjacent cells. This may be consistent with the tumour-promoting role of LOX but it raises the question whether the amount of LOX produced by epithelial cells would be able to significantly affect tumour cells. PLoS ONE \| www.plosone.org March 2011 \| Volume 6 \| Issue 3 \| e16492 Cytokine induced production of pro-metastatic chemokines Furthermore, normal epithelial cells express higher levels of CX3CL1 respect to their tumour counterpart while its receptor (CX3CR1) is expressed in tumour cells [19]. This chemokine promotes migration of cancer cells and metastases formation in a The network shown in Figure 4A and Figure S5 represents signalling of the pro-inflammatory cytokine interleukin 1 (IL1b) PLoS ONE \| www.plosone.org March 2011 \| Volume 6 \| Issue 3 \| e16492 March 2011 \| Volume 6 \| Issue 3 \| e16492 5 Normal-Tumour Cell Interaction in Prostate Cancer Normal-Tumour Cell Interaction in Prostate Cancer Normal-Tumour Cell Interaction in Prostate Cancer Figure 4. Functional networks representing known interaction between genes expressed in normal tissue and selected in the models predictive of capsular penetration. The figure represents the four most significant networks selected by the IPA software. Genes represented by blue shapes are present in the collection of models collected by the GA-MLHD procedure. Genes represented with red shapes represent genes in the collection of models but also included in the representative most predictive models. Genes in the networks are arranged by cellular localization (extracellular, membrane, cytoplasm and nucleus). Note that the IPA software search for statistically significant sub-networks of a given maximum size to simplify their visualization. Nevertheless, in this case these are linked as indicated by red dashed arrows connecting specific network components. doi:10.1371/journal.pone.0016492.g004 Figure 4. Functional networks representing known interaction between genes expressed in normal tissue and selected in the Figure 4. Functional networks representing known interaction between genes expressed in normal tissue and selected in the models predictive of capsular penetration. The figure represents the four most significant networks selected by the IPA software. Genes represented by blue shapes are present in the collection of models collected by the GA-MLHD procedure. Genes represented with red shapes represent genes in the collection of models but also included in the representative most predictive models. Genes in the networks are arranged by cellular localization (extracellular, membrane, cytoplasm and nucleus). Note that the IPA software search for statistically significant sub-networks of a given maximum size to simplify their visualization. Nevertheless, in this case these are linked as indicated by red dashed arrows connecting specific network components. doi:10.1371/journal.pone.0016492.g004 The hypothesis that IL1b may trigger the activation of pro- metastatic signals in normal epithelial cells is an interesting one. We have initially tested this hypothesis by treating the normal prostate cell line RWPE1 with recombinant IL1b and discovered that both CCL20 and CX3CL1 are significantly up regulated 6 and 24 hours after stimulation. LOX is instead only transiently up- regulated six hours after IL1b stimulation (Figure S6). This observation suggests that our hypothesis may be correct. PDGF BB has a dual role on prostate cancer development. It directly promotes tumour cell proliferation and invasion [27]. Platelet-derived growth factor induces proliferation of hyperplastic human prostatic stromal cells [27]. Normal-Tumour Cell Interaction in Prostate Cancer In addition, PDGF BB has been described as a potent inductor of angiogenesis and promotes pericyte recruitment [28]. Its activity is synergistic to IGF1 in promoting migration of human arterial smooth muscle cells [29]. The IPA network shows that PDGF BB and IGF1 can transcriptionally activate CYR61 [30] [31]. CYR61 is an extracel- lular matrix-associated protein that promotes adhesion, migration, proliferation, and angiogenesis. CYR61 is required for breast tumorigenesis and cancer progression [32] [33] and promote prostatic cell adhesion and proliferation [34] [35]. CYR61 also promotes invasion when tumor stroma is irradiated before tumor implantation in a model of skin cancer [36]. Relative to LCM normal (norm) cells, CYR61 is up regulated in LCM BPH, normal Role of IGF1 and PDGF BB in Prostate Cancer development PLoS ONE \| www.plosone.org Normal-Tumour Cell Interaction in Prostate Cancer number of cancers [20] including prostate [21]. The production of this chemokine by epithelial cells adjacent the tumour has therefore the potential to induce tumour cell migration. Similarly, prostate normal epithelial cells produce the chemokine CCL20 and the expression of its receptor (CCR6) in prostate cancer cells has been recently found to be a predictor of tumour aggressiveness [22]. The network also includes LOX, which is represented as an indirect repressor of the NFKb complex [23] [24]. The biological role of LOX in cancer is complex. LOX has been reported to have tumour suppressor activity [25] and can inhibit proliferation of the March 2011 \| Volume 6 \| Issue 3 \| e16492 6 Role of IGF1 and PDGF BB in Prostate Cancer development The IPA software identified a network representing interactions with the growth factors PDGF BB and IGF1 (Figure 4C and S5B-C). The role of IGF1R in malignant transformation is well documented [26]. IGF1R is over-expressed by many tumour cell lines and targeted disruption of the IGF1R gene can abolish cell transformation. PLoS ONE \| www.plosone.org March 2011 \| Volume 6 \| Issue 3 \| e16492 7 Normal-Tumour Cell Interaction in Prostate Cancer Figure 5. Analysis of LCM cell populations representative of prostate cancer progression. The figure represents the results of the analysis performed on the dataset developed by Tomlins et al. [11]. Different cell populations are labelled as follows. Normal cells (norm), normal cells adjacent the tumour (adj), benign prostate hyperplasia (MPH), low grade prostate carcinoma (L-PCA), high-grade prostate carcinoma (H-PCA) and metastatic cells (meta). Panel A shows a two-dimensional cluster analysis performed on the genes differentially expressed (p,0.01) across the seven LCM purified normal and tumour epithelial cell populations. Panel B represents the expression level (y axis) of genes differentially expressed between norm, adjacent and BPH (represented on the y axis). Levels of individual genes across all stages are presented in panels C-F and in Figure S8. doi:10.1371/journal.pone.0016492.g005 Figure 5. Analysis of LCM cell populations representative of prostate cancer progression. The figure represents the results of the analysis performed on the dataset developed by Tomlins et al. [11]. Different cell populations are labelled as follows. Normal cells (norm), normal cells adjacent the tumour (adj), benign prostate hyperplasia (MPH), low grade prostate carcinoma (L-PCA), high-grade prostate carcinoma (H-PCA) and metastatic cells (meta). Panel A shows a two-dimensional cluster analysis performed on the genes differentially expressed (p,0.01) across the seven LCM purified normal and tumour epithelial cell populations. Panel B represents the expression level (y axis) of genes differentially expressed between norm, adjacent and BPH (represented on the y axis). Levels of individual genes across all stages are presented in panels C-F and in Figure S8. doi:10.1371/journal.pone.0016492.g005 Figure 5. Analysis of LCM cell populations representative of prostate cancer progression. The figure represents the results of the analysis performed on the dataset developed by Tomlins et al. [11]. Different cell populations are labelled as follows. Normal cells (norm), normal cells adjacent the tumour (adj), benign prostate hyperplasia (MPH), low grade prostate carcinoma (L-PCA), high-grade prostate carcinoma (H-PCA) and metastatic cells (meta). Role of IGF1 and PDGF BB in Prostate Cancer development Panel A shows a two-dimensional cluster analysis performed on the genes differentially expressed (p,0.01) across the seven LCM purified normal and tumour epithelial cell populations. Panel B represents the expression level (y axis) of genes differentially expressed between norm, adjacent and BPH (represented on the y axis). Levels of individual genes across all stages are presented in panels C-F and in Figure S8. doi:10.1371/journal.pone.0016492.g005 doi:10.1371/journal.pone.0016492.g005 cells adjacent to the tumor and both low and high-grade prostate carcinoma (Figure 5B and S8). The receptor of PDGF BB, PDGFBR, has a similar trend, which is consistent with its potential activator role. development of a pro-tumour activity of TGFb in tumour progression is often associated to mutations, which eliminate the tumour suppressor activities of TGFb and promote growth and invasion. Another pro-tumour effect of TGFb is linked to induce immune system tumour tolerance [37]. Consistent with these findings, recent reports suggest that in prostate cancer TGFb may be relevant therapeutic target [40] [39]. Although protein measurements may be necessary to support this analysis, it is not unreasonable to hypothesize that adjacent normal epithelial cells may produce sufficient CYR61 to influence tumor cells. We found that in the LCM cell populations TGFb is expressed at high levels in normal cells adjacent the tumour (Figure 5 and S7). The predictive power of TGFb response signatures in normal epithelial cells may therefore be the reflection of the amount of active TGFB present in the microenvironment that, at least in part may be produced by normal epithelial cells adjacent to the tumour. PLoS ONE \| www.plosone.org The expression of targets of TGFb in the normal tissue predict tumour capsular penetration The networks represented in Figure S5D and 4B represent the connection between genes including models predictive of tumour capsular penetrations and TGF. TGFb has a complex role in tumour development. It can either promote or inhibit tumour development in a context dependent manner [37]. In normal epithelia and early stages of tumour development, TGFb has role in regulating tissue homeostasis and is considered an anti-tumour factor preventing incipient tumours from progressing towards malignancy [5]. Furthermore, TGFbin- hibits recruitment of pericytes to the vasculature, thus decreasing vessel maturation and flow which may also negatively impact on tumour development [38]. gy Figure S4. Classification methods with multivariate variable selection. In order to consider the effect of combinations of genes in the prediction of the histo-pathological variables we have used a statistical modeling approach in combination with mul- tivariate variable selection procedures. In order to demon- strate that our results are independent of a particular method- ology we developed and compared multivariate classification models obtained using two independent procedures. These methods differ for both the variable selection strategy and for the classification algorithms used. The first approach is a modification of the Genetic Algorithm –maximum likelihood discriminant analysis (GA-MLHD) method originally developed by Ooi and Tan [54]. This method uses a genetic algorithm approach for variable selection coupled to a MLHD functions classifier. The GA-MLHD methodology uses an initial random population of models (called chromosomes) and evolves from them highly accurate classifiers using a process that mimics natural selection. Accuracy was estimated as the proportion of guesses in test samples in a cross-validated manner. In our implementation [55] we have improved the error estimation strategy by using two- levels of cross-validations. The first level is used in the evolutionary step of the GA to evaluate the error in a subset of the dataset using a k-fold-cross-validation procedure (k = 5). The second level is used at the end of the evolutionary process, when all chromosomes are selected, to estimate the classification error as an average of the test error in 40 random splits (2/3 for training and 1/3 for testing) using the entire dataset. Model sizes of 5 were used, which showed a higher accuracy than 10 and 20 in average for 10,000 models. In addition, we have compared the results with models obtained using a Bayesian variable selection (BVS) approach that we have developed [56]. This method uses a multinomial probit model as classifier and Markov Chain Monte Carlo (MCMC) methods to search multivariate space for informative subsets of the variables. Classification methods with multivariate variable selection. In order to consider the effect of combinations of genes in the prediction of the histo-pathological variables we have used a statistical modeling approach in combination with mul- tivariate variable selection procedures. In order to demon- strate that our results are independent of a particular method- ology we developed and compared multivariate classification models obtained using two independent procedures. These methods differ for both the variable selection strategy and for the classification algorithms used. gy Figure S4. The first approach is a modification of the Genetic Algorithm –maximum likelihood discriminant analysis (GA-MLHD) method originally developed by Ooi and Tan [54]. This method uses a genetic algorithm approach for variable selection coupled to a MLHD functions classifier. The GA-MLHD methodology uses an initial random population of models (called chromosomes) and evolves from them highly accurate classifiers using a process that mimics natural selection. Accuracy was estimated as the proportion of guesses in test samples in a cross-validated manner. In our implementation [55] we have improved the error estimation strategy by using two- levels of cross-validations. The first level is used in the evolutionary step of the GA to evaluate the error in a subset of the dataset using a k-fold-cross-validation procedure (k = 5). The second level is used at the end of the evolutionary process, when all chromosomes are selected, to estimate the classification error as an average of the test error in 40 random splits (2/3 for training and 1/3 for testing) using the entire dataset. Model sizes of 5 were used, which showed a higher accuracy than 10 and 20 in average for 10,000 models. In addition, we have compared the results with models obtained using a Bayesian variable selection (BVS) approach that we have developed [56]. This method uses a multinomial probit model as classifier and Markov Chain Monte Carlo (MCMC) methods to search multivariate space for informative subsets of the variables. An important question is whether the statistical relationships we have discovered with our analysis reflect a key aspect of tumour microenvironment in which normal epithelial cells influence tumour biology. Although it is hard to provide a conclusive answer, the information available in the literature and our experimental validation in a normal epithelia prostate cell line (Figure S6) indicates that this may be a plausible hypothesis. Despite the limited overlap at the gene level, mainly caused by our stringent pre-processing criteria (see methods section for details), the analyses we have performed on the two independent datasets provided similar results at the network level. This finding reinforces the validity of the overall analysis strategy. From a methodological standpoint our approach therefore has potential for formulating hypothesis on genes playing a role in controlling the development of cancer. The approach is general and likely to be applicable to other datasets for which tumour and adjacent normal samples are available. Conclusions Ultimately, our approach provides a way to identify molecular networks whose activity in normal epithelial cells is predictive of tumour features. Prostate cancer progression rates among so-called ‘‘favourable prognosis’’ localized tumours (e.g. Gleason score ,6) are not precisely predicted by grade and stage at diagnosis. This lack of diagnostic accuracy has contributed to the conundrum of CaP over-screening and possibly over-treatment [48,49]. A similar lack of prognostic accuracy is apparent when tumours recur during androgen depravation therapy. Greater diagnostic accura- cy is thus imperative to distinguish at early stages indolent disease from aggressive phenotypes that can progress rapidly, and at late stage disease the lethal phenotypes. Normal-Tumour Cell Interaction in Prostate Cancer observation is consistent with a biological role of AGT production by normal epithelial cells. microarrays for expression profiling. The first dataset used in our analysis is derived from a study performed by Singh et al. [9] where 52 samples of prostate tumours and adjacent normal tissues were collected from patients undergoing radial prostatectomy; then profiled using Affymetrix Genechip technology. The second dataset used was collected by Lapointe et al. [52] using cDNA arrays. In this study 41 paired normal and tumor specimens were removed from radical prostatectomy. Information about the histo- pathology of the tumor specimens (Gleason score and Tumor stage) was available for both datasets. Details of the data processing for both datasets are available in the supplementary material. After processing, the two datasets show relatively limited overlap at the gene level (up to 8%, Table S1). Consequently, we have opted for the two datasets to be analyzed separately. The IPA network (Figure 4B) links AGT to the transcription of JAG, another factor known to have context-dependent effects on tumour development. In vascular cells, inhibition of Jagged promotes angiogenesis [46] favouring tumour growth. On the other hand Jagged 1 favours proliferation and expansion of prostate tumours [47]. In LMC cell populations tumour cells express higher levels of JAG1 than adjacent normal epithelial cells (Figure 5 and S7). Hence, the effective contribution of normal cell expressed JAG1 on tumour development is unclear. Statistical Modeling Classification methods with univariate variable selection. Our analysis aims to identify molecular signatures predictive of two binary variables representing relevant features of tumor biology. These are the degree of differentiation of the tumor and the ability of the tumor to penetrate the organ capsule. To develop such signatures we have initially tested a univariate variable selection strategy based on an F test in combination with several classification methods (SVM, DLDA, PAMR, KNN, SOM) as implemented in the software application Prophet available in the Web based microarray analysis suite GEPAS [53]. This application uses a step-wise variable inclusion strategy to construct increasingly large models from a list of genes ranked by the value of the F statistics and implement a cross-validation strategy for error estimation. Results of this analysis are shown in Lessons from breast cancer studies have taught us that significant strides in diagnosis (and thus treatment) can be made by applying multiple genetic parameters to define disease with greater clinical resolution (reviewed in [50]). Such approaches have progressed less quickly in prostate cancer [51]. The current study suggests this need and gap in understanding can be met by utilizing gene expression signatures in the normal prostate tissue adjacent to the tumour as novel functional molecular biomarkers. In early stage disease especially identification and sampling of the tumour within the prostate gland can be highly challenging. Therefore it is highly advantageous and attractive to utilize gene expression signatures in the readily sampled normal tissue to make robust prognostic inferences concerning the tumour. Angiotensinogen and Notch in Prostate Cancer Angiotensinogen and Notch in Prostate Cancer The network shown in Figure 4B involve the interaction between the Angiotensin precursor Angiotensinogen (AGT) and the Notch ligand JAG1. A functional Renin-Angiotensin system has been demonstrated in prostate cancer [41] [42]. In addition its canonical role in regulating blood pressure it is now recognized that Angiotensin can influence several growth factor pathways [41], including oncogene activation [43]. It has been recently shown to be a clinically relevant factor in the progression of prostate cancer and a potential avenue for treatment [41] [44] [45]. AGT is up regulated in normal epithelial cells adjacent the tumour compared to PIN and PCA (Figure S8E). This At later stages of tumour development, TGFb has been shown to promote tumour development and metastases formation. Of particular relevance, TGFb1 reverses inhibition of COX-2 with NS398 and increases invasion in prostate cancer cells [39]. The March 2011 \| Volume 6 \| Issue 3 \| e16492 8 PLoS ONE \| www.plosone.org Analysis of LCM cell populations The dataset developed by Tomlins et al. [11] was downloaded from the GEO database and raw data normalized using print tip normalization. The expression profiles of a subset of 20 genes (representative of secreted factors and their receptors from the IPA networks) across samples representing normal and tumour epithelial cells were then selected to create a secondary dataset. Differentially expressed genes were then identified by one factor ANOVA using the software application TMEV [57]. Normal-Tumour Cell Interaction in Prostate Cancer Error estimation and parameters settings have been described in [56]. Two runs were made for model sizes 10 and 20. The model with higher average accuracy was then chosen. sample classification and to estimate the contribution of each gene for class distinction, we used principal component analysis (PCA). PCA reduce the original variable space in a handful of principal components (PC). A PC is defined as a weighted sum of variables (genes). The weight or loading given to a variable is interpreted as its importance. For discussion, we focused in genes having absolute loadings values larger than 0.3 (Figure 2, S1, S2 and S3). In all cases, the chosen PC (first two) show evident class separation providing further support for the association of the selected genes and the sample classes. Selecting representative models. Both GA-MLHD and BVS modeling approaches provide a number of alternative models with comparable predictive value. These models tend to have a degree of overlap in their gene composition. It is therefore meaningful to select a single summary model that represents the most frequent solutions. In order to do so, for the GA-MLHD approach, we have used a forward selection procedure applied to the top 1% most predictive models selected using the GA procedure. In the case of BVS, we have tested models developed with the genes that were included in the subsets of variables most frequently visited by the MCMC search. The final list of models was generated by the union of the two chains with minimum average miss-classification error [56]. Interestingly, we have discovered that representative models developed with the GA- MLHD procedure largely overlaps with the pooled models from the BVS approach. We tested the overlap between models selected by the GA-MLHD procedure in the two datasets at different processing thresholds. The overlap between the top 50 raking genes (by frequency of inclusion in the model populations) in the model populations was always significant (see Tables S4 and S5). Tissue specificity of representative models An important component of our strategy is to demonstrate that molecular signatures are tissue specific hence they are not representing a mere reflection of the overall similarity between normal and tumour tissues. The strategy to demonstrate the specificity of the gene signatures obtained with the multivariate variable selection strategy implemented in the GA-MLHD procedure is described below in two steps. Step 1: development of representative models. Expression data from the normal tissue samples are split between training and test sets (respectively 2/3 and 1/3 of the original dataset). The training set is used to develop a classification model to predict cancer features with a cross-validation strategy. Once the represen- tative models have been developed their classification accuracy is estimated on the test set. Score~{log10 1{ X f {1 i~0 C(G,i)C(N{G,s{i) C(N,s) ! Score~{log10 1{ X f {1 i~0 C(G,i)C(N{G,s{i) C(N,s) ! Step 2: Specificity test. Expression data from the tumour tissues samples are split between training and test sets (respectively 2/3 and 1/3 of the original dataset). The expression profile of genes selected in Step 1 (in the samples selected in the training set) is used to train a classification model to predict Cancer features. The classification accuracy of the trained model is then estimated on the test set. The classification accuracy estimated in step 2 is then compared to the classification accuracy estimated in step 1 to establish the tissue specificity of the gene signatures (Figure 3). In order to demonstrate the tissue specificity of models based on the molecular profile of tumour tissues we have also performed the reverse test. Where N is the number of genes in the genomic network, of which G are focus genes, for a pathway of s genes, f of which are focus genes. C(n,k) is the binomial coefficient. Pathways whose Score were greater than 5 (p,0.0001) were selected for biological interpretation. Canonical pathway analysis was performed using the IPA tools and significance for the enrichment of the genes with a particular Canonical Pathway was determined by right-tailed Fisher’s exact test with a = 0.01 and a whole database as a reference set. The assessment of the tissue specificity of the molecular signatures obtained with the BVS procedure has been performed using a cross-validation procedure for the error estimation as described in [56]. Interaction networks and functional analysis of multivariate signatures: The Ingenuity Pathway Analysis (IPA) software The gene sets represented in the populations of models selected using the GA-MLHD procedure have been analyzed using the Ingenuity Pathway Analysis (IPA) application (Palo Alto, http:// www.ingenuity.com), a web based application that enables discovery, visualization, and exploration of biologically interaction networks. Gene lists represented in the model populations developed with normal or tumor expression data to predict capsular penetration or Gleason score were uploaded into in the application. Each gene identifier was mapped to its correspond- ing gene object in the Ingenuity Pathways Knowledge Base. These genes, called focus genes, were overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. Networks of these focus genes were then algorithmically generated based on their connectivity according to the following procedure implemented in the IPA software application. The specificity of connection for each focus gene was calculated by the percentage of its connection to other focus genes. The initiation and the growth of pathways proceed from the gene with the highest specificity of connections. Each network had a maximum of 35 genes for easier interpretation and visual inspection. Pathways of highly interconnected genes were identified by statistical likelihood using the following equation: Datasets Our analysis is based on two independent large prostate cancer studies performed using different array technologies. In both studies, cells from tumour and adjacent normal tissues have been isolated and the extracted RNA has been hybridized on human PLoS ONE \| www.plosone.org March 2011 \| Volume 6 \| Issue 3 \| e16492 9 Normal-Tumour Cell Interaction in Prostate Cancer PLoS ONE \| www.plosone.org Supporting Information Figure S1 Multivariate Models for Capsular Penetra- tion using Tumour data. Genes present in GA-MLHD and BVS for the same dataset are highlighted in red. Accuracy is estimated as described in the Material and Methods section. GeneBank accession number and gene symbol is shown. Brighter green or red colours in heatmaps represent lower or higher relative expression respectively. t-test p-value is shown for comparison with the differential expression criteria commonly used in univariate variable selection approaches. PCA plots and loadings are used to show the putative contribution of every gene to class separation. For example, TALDO1 gene in top heatmap seems to contribute strongly to positive Capsular Penetration whereas ST14 contribute weakly to negative Capsular Penetration. PCs were selected by visual inspection. (EPS) Figure S5 Functional networks representing known interaction between genes expressed in normal tissue and selected in the models predictive of capsular penetration. The figure represents the four most significant networks selected by the IPA software for the Singh et al. dataset [4]. Genes represented in the predictive models are represented by blue shapes. Genes in the networks are arranged by cellular localization (extracellular, membrane, cytoplasm and nucleus). (TIFF) Figure S5 Functional networks representing known interaction between genes expressed in normal tissue and selected in the models predictive of capsular penetration. The figure represents the four most significant networks selected by the IPA software for the Singh et al. dataset [4]. Genes represented in the predictive models are represented by blue shapes. Genes in the networks are arranged by cellular localization (extracellular, membrane, cytoplasm and nucleus). (TIFF) Figure S6 Induction of pro-metastatic cytokines in RWPE1 cells by Interleukin 1b. The transcriptional response of normal prostate epithelial cells (RWPE1) was measured with human Agilent microarrays 6 hours and 24 hours after addition of 100 ng/ml of recombinant human Interleukin 1b (eBioscience, USA). The experiments were performed three times in different days. Genes represented in Figure 4A were then tested for differential expression using a t-test. Only the pro-metastatic chemokines CCL20 (Panel B) and CX3CL1 (Panel C) were differentially expressed (*, FDR,1%) at both time points. The gene LOX was only transiently activated by Interleukin 1b six hours post exposure (Panel D). Panel A shows the portion of the network in Figure 4A where genes are differentially expressed in RWPE1 in response to Interleukin 1b exposure. Normal-Tumour Cell Interaction in Prostate Cancer Machines. See GEPAS [3] for details in F-ratio, error estimation, and classification methods. Dataset, normal or tumour data, and class is specified in each plot. (EPS) Machines. See GEPAS [3] for details in F-ratio, error estimation, and classification methods. Dataset, normal or tumour data, and class is specified in each plot. (EPS) Supporting Information In this experiment RWPE1 cells were grown in 0.4% gelatin coated plates, complete KSFM media supplemented with L-Glutamine, p/s, BPE and EGF. (TIFF) Figure S2 Multivariate Models for Gleason Score using Normal data. Genes present in GA-MLHD and BVS for the same dataset are highlighted in red. Accuracy is estimated as described in the Material and Methods section. GeneBank accession number and gene symbol is shown. Brighter green or red colours in heatmaps represent lower or higher relative expression respectively. t-test p-value is shown for comparison with the differential expression criteria commonly used in univariate variable selection approaches. PCA plots and loadings are used to show the putative contribution of every gene to class separation. For example, TEGT gene in top heatmap seems to contribute strongly to high Gleason grades whereas D89667 contribute to low Gleason Grades. PCs were selected by visual inspection. (EPS) Figure S3 Multivariate Models for Gleason Score using Tumour data. Genes present in GA-MLHD and BVS for the same dataset are highlighted in red. Accuracy is estimated as described in the Material and Methods section. GeneBank accession number and gene symbol is shown. Brighter green or red colours in heatmaps represent lower or higher relative expression respectively. t-test p-value is shown for comparison with the differential expression criteria commonly used in univariate variable selection approaches. PCA plots and loadings are used to show the putative contribution of every gene to class separation. For example, ACPP gene in top heatmap seems to contribute strongly to low Gleason grades whereas TM8B4X contribute to low Gleason Grades. PCs were selected by visual inspection. (EPS) Figure S7 Expression of selected secreted factors and receptors in Tomlins et al. dataset. Nor, Adj, BPH, PIN, PCA-Low, PCA-High and Meta samples are described in main paper. (TIFF) Figure S8 Comparison of the expression of selected secreted factors and receptors in Tomlins et al. dataset. Panels A-L represents the expression profile (y axis) of a selection of the genes differentially expressed between all LCM cell populations (shown as a heat map in figure 5 in main paper). The different cell populations are arranged along the x axis. Red close circles represent gene expression levels significantly different (P,0.01) respect to adj cells whereas blue close circles inside red circles represent gene expression levels significantly different (p,0.05) respect to adj cells. Nor, Adj, BPH, PIN, PCA-Low, PCA-High and Meta samples are described in main paper. Analyzing the specific contribution of genes in the predictive models Our approach, which is based on multivariate predictive models selects combination of genes to perform predict tumour features. Therefore, differential expression between sample classes may not be always indicative of the relative contribution of a gene to sample separation. Therefore, in order to graphically represent PLoS ONE \| www.plosone.org March 2011 \| Volume 6 \| Issue 3 \| e16492 10 Normal-Tumour Cell Interaction in Prostate Cancer PLoS ONE \| www.plosone.org Author Contributions Conceived and designed the experiments: FF. Performed the experiments: SD PA. Analyzed the data: VT MGT MV FA JD FF PA. Contributed reagents/materials/analysis tools: JD. Wrote the paper: VT AB FF MJC. Conceived and designed the experiments: FF. Performed the experiments: SD PA. Analyzed the data: VT MGT MV FA JD FF PA. Contributed reagents/materials/analysis tools: JD. Wrote the paper: VT AB FF MJC. References Lapointe J, Li C, Higgins JP, van de Rijn M, Bair E, et al. (2004) Gene expression profiling identifies clinically relevant subtypes of prostate cancer. Proc Natl Acad Sci U S A 101: 811–816. 25. 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(2006) CXCL1 induced by prostaglandin E2 promotes angiogenesis in colorectal cancer. J Exp Med 203: 941–951. 3. Alberti C (2006) Prostate cancer progression and surrounding microenviron- ment. Int J Biol Markers 21: 88–95. 3. Alberti C (2006) Prostate cancer progression and surrounding microenviron- ment. Int J Biol Markers 21: 88–95. 21. Wang W, Li Y, Hong A, Wang J, Lin B, et al. (2009) NDRG3 is an androgen regulated and prostate enriched gene that promotes in vitro and in vivo prostate cancer cell growth. Int J Cancer 124: 521–530. 4. Rollins BJ (2006) Inflammatory chemokines in cancer growth and progression. Eur J Cancer 42: 760–767. 5. Joshi A, Cao D (2010) TGF-beta signaling, tumor microenvironment and tumor progression: the butterfly effect. Front Biosci 15: 180–194. 22. Ghadjar P, Loddenkemper C, Coupland SE, Stroux A, Noutsias M, et al. (2008) Chemokine receptor CCR6 expression level and aggressiveness of prostate cancer. J Cancer Res Clin Oncol 134: 1181–1189. 6. 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Underlined numbers in lower triangular matrix correspond to the p-value testing the corresponding overlap number using a hypergeometric test. All comparisons were significant at the 0.05 level. (DOCX) models developed using the GA-MLHD procedure. The table lists the networks identified from the Lapointe et al. [2] dataset associated to models predictive of tumour capsular penetration from the molecular profile of normal cells. HCG Column highlights the network highest connected gene(s) or complex. Genes in bold were part of the multivariate models used as input for IPA analysis. (DOCX) Table S5 Overlap of the top 50 selected genes in models using larger datasets for Lapointe et al. dataset. Numbers in upper triangular matrix correspond to the number of genes overlapped. Underlined numbers in lower triangular matrix correspond to the p-value testing the corresponding overlap number using a hypergeometric test. All comparisons were significant at the 0.05 level. (DOCX) Table S3 Selected secreted factors and receptors. Genes obtained in IPA networks and present in Tomlins et al. dataset were selected. P-Values were estimated using f-test comparing Nor, Adj, BPH, PIN, PCA-Low, PCA-High and Meta samples as shown in Figure 5 and Supplementary Figure S6. Some genes are represented by different probes in the microarray platform used. Only probes with p-Value ,0.001 were included in Figure 5. (DOCX) Supporting Information (TIFF) Figure S8 Comparison of the expression of selected secreted factors and receptors in Tomlins et al. dataset. Panels A-L represents the expression profile (y axis) of a selection of the genes differentially expressed between all LCM cell populations (shown as a heat map in figure 5 in main paper). The different cell populations are arranged along the x axis. Red close circles represent gene expression levels significantly different (P,0.01) respect to adj cells whereas blue close circles inside red circles represent gene expression levels significantly different (p,0.05) respect to adj cells. Nor, Adj, BPH, PIN, PCA-Low, PCA-High and Meta samples are described in main paper. (TIFF) Figure S4 Univariate gene selection models. Models were generated using a forward selection procedure that includes, progressively, genes ranked by a univariate statistic (F-ratio, horizontal axis). The accuracy is assessed by leave-one-out-cross- validation for a number of classification methods (vertical axis, see legends, and the Prophet tool within www.gepas.org [3]). Maximum accuracy is marked by a dotted horizontal line. Overall, this univariate gene selection generates comparable predictive models irrespective of the classification method. More accurate multivariate models generated by GA-MLHD and BVS used in this chapter are shown for comparison in red and black dots. Legends: DLDA - Diagonal Linear Discriminant Analysis, KNN - K-Nearest-Neighbours, PAMR - Shrunken Centroids, SOM - Self Organized Maps, and SVM - Support Vector Table S1 Datasets annotation. As stated, we used approx- imately the 25% of the database (marked in bold). Overlaps were estimated by Unigene annotation. Similar results are obtained using entrez id or gene symbol as shown in columns. 50% Top genes were estimated relaxing the filter range in both datasets to 25 and 50%. (DOCX) Table S2 Significant Networks identified by the Inge- nuity Pathway Analysis (IPA) software associated to PLoS ONE \| www.plosone.org March 2011 \| Volume 6 \| Issue 3 \| e16492 11 Normal-Tumour Cell Interaction in Prostate Cancer PLoS ONE \| www.plosone.org Normal-Tumour Cell Interaction in Prostate Cancer 35. Sun ZJ, Wang Y, Cai Z, Chen PP, Tong XJ, et al. 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J Exp Med 196: 605–617. 31. Tullai JW, Schaffer ME, Mullenbrock S, Kasif S, Cooper GM (2004) Identification of transcription factor binding sites upstream of human genes regulated by the phosphatidylinositol 3-kinase and MEK/ERK signaling pathways. J Biol Chem 279: 20167–20177. 16. Hinata K, Gervin AM, Jennifer Zhang Y, Khavari PA (2003) Divergent gene regulation and growth effects by NF-kappa B in epithelial and mesenchymal cells of human skin. Oncogene 22: 1955–1964. 32. Tsai MS, Bogart DF, Castaneda JM, Li P, Lupu R (2002) Cyr61 promotes breast tumorigenesis and cancer progression. Oncogene 21: 8178–8185. 17. Voronov E, Shouval DS, Krelin Y, Cagnano E, Benharroch D, et al. (2003) IL-1 is required for tumor invasiveness and angiogenesis. Proc Natl Acad Sci U S A 100: 2645–2650. 33. Menendez JA, Mehmi I, Griggs DW, Lupu R (2003) The angiogenic factor CYR61 in breast cancer: molecular pathology and therapeutic perspectives. Endocr Relat Cancer 10: 141–152. 18. 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W4311812628.txt	http://neonatology.bsmu.edu.ua/article/download/267808/263612	en		EMBRYOLOGICAL INDICATORS AND INCIDENCE OF PREGNANCY IN WOMEN WITH INFERTILITY UNDERGOING ASSISTED REPRODUCTIVE TECNOLOGY PROGRAMS WITH THE USE OF MELATONIN	Neonatologìâ, hìrurgìâ ta perinatalʹna medicina	2,022	cc-by	4,661	РЕЗУЛЬТАТИ ДИСЕРТАЦІЙНИХ ТА НАУКОВО-ДОСЛІДНИХ РОБІТ / RESULTS THESIS AND SCIENTIFIC - RESEARCH РЕЗУЛЬТАТИ ДИСЕРТАЦІЙНИХ ТА НАУКОВО-ДОСЛІДНИХ РОБІТ RESULTS THESIS AND SCIENTIFIC-RESEARCH УДК : 618.177-089.888.11:618.179:615.357 DOI: 10.24061/2413-4260.XIІ.3.45.2022.2 В.О. Юзько 1,2, O. M. Юзько 1,2, T.A. Юзько, I.В. Чемьоркіна 2, О.А. Андрієць 1 ЕМБРІОЛОГІЧНІ ПОКАЗНИКИ ТА ЧАСТОТА НАСТАННЯ ВАГІТНОСТІ У ЖІНОК ІЗ БЕЗПЛІДДЯМ В ПРОГРАМАХ ДОПОМІЖНИХ РЕПРОДУКТИВНИХ ТЕХНОЛОГІЙ ПРИ ЗАСТОСУВАННІ МЕЛАТОНІНУ Буковинський державний медичний університет 1 МОЗ України, КЗОЗ «Медичний центр лікування безпліддя» 2 (м. Чернівці, Україна) Резюме Вступ. Технологія інтрацитоплазматичного введення сперматозоїда в яйцеклітину (ICSI) посіла основне місце серед допоміжних репродуктивних технологій (ДРТ) при лікуванні безпліддя. Підвищення ефективності ДРТ залишається складною медичною проблемою. У великій мірі успіх ICSI залежить від якості яйцеклітин, яка, в свою чергу, залежить від кількості фолікулів, що обумовлено овуляторним резервом (ОВ) та ефективною підготовкою пацієнток до ДРТ. Маркери ОВ добре відомі, а на мелатонін як можливий прогностичний чинник ефективності ДРТ та ОВ, увагу звернули в останні десять років. Не вивчені показники вмісту мелатоніну в фолікулярній рідині стимульованих яєчників. У той же час оцінка ембріологічних показників запліднення при застосуванні препарату мелатоніну для підготовки пацієнток та результативність програм ДРТ в цілому може бути корисною для подальшої розробки алгоритмів лікування пацієнток із безпліддям. Мета дослідження. Оцінка ембріологічних показників, результативності програм ICSI та частоти настання вагітності у жінок із безпліддям при підготовці їх препаратом мелатоніну. Матеріал та методи. У проспективному дослідженні брало участь 67 жінок із різними факторами безпліддя. Всі пацієнтки готувались до проведення контрольованої стимуляції яєчників (КСЯ), а в подальшому до пункції фолікулів, забору яйцеклітин та їх запліднення шляхом ICSI. Всі отримані бластоцисти були кріоконсервовані, зберігались в рідкому азоті та розморожувались за необхідності проведення ембріотрансферу. Оцінювались ембріологічні показники, кількість трансферів та ефективність настання вагітності. Згідно мети та завдання пацієнтки були розподілені на дві групи методом парних-непарних чисел: група 1 – 29 пацієнток із безпліддям, які впродовж місяця до пункції фолікулів отримували препарат «Віта-мелатонін» по 3 мг всередину на ніч, група 2 – 38 пацієнток із безпліддям, які не отримували даний препарат. Статистичну обробку отриманих результатів проводили з використанням ліцензійних програм «Microsoft Excel» і «Statistica». Дизайн дослідження та всі методики, які були нами використані в даному проспективному дослідженні, розглянуті та схвалені комісією з біоетики закладу вищої освіти «Буковинський державний медичний університет» (протокол № 7 від 21.04.2022). НДР «Збереження та відновлення репродуктивного здоров’я жінок та дівчат при акушерській та гінекологічній патології» (державний реєстраційний номер 0121U110020. Термін виконання –01.2021-12.2025 рр.). Результати дослідження. Настання вагітності після розморожування бластоцист та ембріотрансферів в цілому у жінок, які отримували мелатонін, було вірогідно (р ˂ 0,05) вищим в порівнянні з жінками, які не отримували мелатонін, відповідно, 86,2 ± 6,41% та 76,3 ± 6,95%. На всі проведені ембріотрансфери частота настання вагітності склала, відповідно, 56,8 ± 8,24% та 52,8 ± 7,62% (р > 0,05), на перший ембріотрансфер 58,1 ± 8,85% та 55,3 ± 8,15% (р > 0,05), на другий – 43,5 ± 16,53% та 33,3 ± 21,14% (р > 0,05), на третій – 100,0 ± 7,00 % та 80,0 ± 17,95% (р > 0,05). При оцінці ембріологічних показників констатували, що у жінок. які отримували мелатонін, зрілих яйцеклітин було 83,4 ± 6,94%, а в жінок, які не отримували мелатонін, 81,5 ± 6,36%, але вірогідної (р > 0,05) різниці не було. Із зрілих яйцеклітин в процесі інкубації після технології ICSI утворилось, відповідно, 49,5 ± 9,54% та 47,2 ± 8,72 (р > 0,05) бластоцист, а з них бластоцист класу І – 48,6 ± 9,36% та 46,4 ± 8,17 % (р > 0,05), класу ІІ – 36,8 ± 8,94% та 44,4 ± 8,13 % (р > 0,05), класу ІІІ – 14,6 ± 6,67% та 9,3 ± 4,78 % (р > 0,05). Висновок. Використання мелатоніну при підготовці жінок із безпліддям до застосування допоміжних репродуктивних технологій показало свою ефективність щодо настання вагітності. Ключові слова: безпліддя; допоміжні репродуктивні технології; мелатонін; вагітність. Вступ В Україні щорічно реєструється понад 50 тис. випадків безплідних пар [1]. Основним методом лікування у них є допоміжні репродуктивні технології (ДРТ), зокрема запліднення шляхом ін- трацитоплазматичного введення сперматозоїдів (ICSI) [2]. Для підвищення ефективності ДРТ актуальним залишається питання підготовки безплідних пар [3]. У той же час, успіх запліднення яйцеклітин залежить від їх якості, яка оцінюва15 НЕОНАТОЛОГІЯ, ХІРУРГІЯ ТА ПЕРИНАТАЛЬНА МЕДИЦИНА NEONATOLOGY, SURGERY AND PERINATAL MEDICINE Т. ХIІ, № 3(45), 2022 Vol. ХIІ, № 3(45), 2022 ISSN 2226-1230 (Print) ISSN 2413-4260 (Online) лась по ефективності додаткової терапії [4]. Від кількості та якості отриманих яйцеклітин напряму залежить ефективність запліднення при процедурі ICSI, а культивування бластоцист від задовільних умов та оптимального складу хімічного середовища. Слід враховувати максимальне зменшення дії непередбачуваних негативних чинників [2]. Надмірна активація антиоксидатної системи організму, фолікулів та яйцеклітин може негативно впливати на ефективність запліднення та розвиток ембріонів. Дані літератури свідчать про позитивну роль збалансованої антиоксидантної дії в середовищах для культивування яйцеклітин та бластоцист [5]. Науковий пошук позитивної дії тих чи інших гормонів організму, зокрема мелатоніну, на процес запліднення яйцеклітин in vitro та розвиток бластоцист спочатку проводився на тваринах. У той же час слід зазначити, що рівень мелатоніну в цих середовищах суттєво відрізняється серед тих чи інших видів тварин [6, 7, 8, 9]. Як результат наукових пошуків було доведено, що мелатонін виказує суттєвий вплив на дозрівання фолікулів, стан яйцеклітини та, власне, процес овуляції. В деяких роботах підкреслювали, що вміст цього гормону в фолікулярній рідині перевищує його рівень в крові. Тобто, був зроблений висновок, що сам фолікул яєчника синтезує його для себе або поглинає з крові господаря в більшій кількості [16] та відіграє важливу фізіологічну роль у дозріванні фолікулів та яйцеклітин, процесі овуляції та запліднення, розвитку бластоцист та ембріонів на ранніх стадіях [10, 11, 12, 13]. Результати застосування препаратів мелатоніну були опубліковані після проведеного подвійного сліпого рандомізованого плацебо-контрольованого дослідження. Жінки з безпліддям отримували мелатонін в дозі від 7 до 16 мг на добу, а в подальшому було проаналізовано кількість отриманих яйцеклітин, їх якість та частота настання вагітності. Паралельно досліджували концентрацію мелатоніну в сироватці крові та показники оксидантного стресу. Настання вагітності в групах з використанням мелатоніну була вищою. Інші дослідники показали, що рівень мелатоніну в сироватці крові в жінок із безпліддям, які в процесі підготовки до пункції яєчників після їх контрольованої стимуляції був вірогідно вищим в порівнянні з контрольною групою, а в фолікулярній рідині був вдвічі меншим. Зроблений висновок, що фолікул та яйцеклітини активно поглинають екзогенний мелатонін. Мелатонін синтезується з амінокислоти триптофану та є похідним біологічного аміну серотоніну в шишкоподібній залозі. Синтез відбувається переважно ввечері та вночі. У той же час відомо, що мелатонін може синтезуватись і в інших органах людини: яєчниках та яєчках, шлунково-кишковому тракті, тимусі та сполучній тканині. Мелатонін є антиоксидантом та блокатором вільних радикалів, іномодулятором [14]. Даний гормон має суттєвий вплив на становлення репродуктивної системи та менструальної функції [15, 16]. Механізм дії гормону мелатоніну на молекулярному рівні недостатньо вивчений. Вважають, що саме через гіпофізарні рецептори він регулює 16 Key title: Neonatologìâ, hìrurgìâ ta perinatalʹna medicina (Online) Abbreviated key title: Neonatol. hìr. perinat. med. (Online) синтез гормонів гіпофіза та репродуктивну систему [17, 18, 19]. Враховуючи велику зацікавленість науковців у вивченні ролі мелатоніну в репродуктивній медицині, достатньо багато питань недостатньо висвітлені та досліджені, зокрема застосування препаратів мелатоніну для підвищення ефективності лікування безпліддя в жінок в програмах ДРТ [3]. Метою даної роботи була оцінка ембріологічних показників, результативності програм інтрацитоплазматичного введення сперматозоїдів у яйцеклітини та частоти настання вагітності у жінок із безпліддям при підготовці їх мелатоніном. Матеріал і методи дослідження У проспективному дослідженні, яке виконувалось на базі КЗОЗ «Медичний центр лікування безпліддя» (м. Чернівці), брало участь 67 жінок із різними формами безпліддя. Згідно мети і завдання пацієнтки були розподілені на дві групи методом парних-непарних чисел: група 1 – 29 пацієнток із безпліддям, які впродовж одного місяця до пункції фолікулів отримували препарат «Вітамелатонін» по 3 мг всередину на ніч, група 2 – 38 пацієнток із безпліддям, які не отримували даний препарат. Середній вік пацієнток в групі 1 був 31,4 ± 1,3 року, а в групі 2 – 32,3 ± 1,1 року. Серед пацієнток не було жінок, які працювали вночі. Стимуляцію яєчників проводили в протоколі з використанням антагоністів гонадотропін-рилізинг-гормону з 2-3-го дня менструального циклу з використанням рекомбінантних та/або сечових гонадотропінів у добовій дозі 150-300 МО. Овуляцію ініціювали введенням рекомбінантного хоріонічного гонадотропіну в дозі 6500 МО. Ооцити отримували в результаті трансвагінальної пункції фолікулів під контролем ультразвукового дослідження через 34-36 годин після введення овуляторної дози тригера овуляції. Пункцію фолікулів виконували під внутрішньовенним наркозом. Рухливі сперматозоїди відбирали шляхом обробки в 2-ступінчастому градієнті щільності в середовищі Sil-Selectpeus («Ferti Pro», Бельгія), після чого для відбору сперматозоїдів використовували метод «swim up». Для запліднення ооцитів застосовували процедуру ICSI. Клітини кумулюса видаляли м'яким піпетуванням (Flexipet Cook) через 3-4 год. після забору ооцитів, використовуючи розчин гіалуронідази (Hyaluronidase in Ferticult Fluhing Medium, Бельгія). Процедура ICSI виконувалась через 1-2 години після денудації на інвертованому мікроскопі Nicon Eclipse Ti («Wild Leitz GmbH», Німеччина) з використанням системи хоффманівського модуляційного контрасту та комплекту мікроманіпуляторів Narishige («Narishige», Японія). Культивування ембріонів здійснювали в СО 2інкубаторі при температурі 37 0С у зволоженій атмосфері з 5,8% СО 2. Ембріони-сибси культивували в середовищі Global («LifeGlobal Group», Бельгія). Ембріони культивували індивідуально у мікрокраплях під шаром мінеральної олії. На 3 добу після запліднення проводили заміну середовища на аналогічне свіже. Оцінювали частоту розвитку ембріонів до 4-8 клітинної стадії, ком- РЕЗУЛЬТАТИ ДИСЕРТАЦІЙНИХ ТА НАУКОВО-ДОСЛІДНИХ РОБІТ / RESULTS THESIS AND SCIENTIFIC - RESEARCH пактизації та формування бластоцист у період з 2-го по 6-й день ембріонального розвитку. Зиготи та ембріони індивідуально оцінювали під мікроскопом через 18, 45, 72 та 96 год. після запліднення для оцінки їх розвитку та якості. На 5-6-ту добу розвитку проводили оцінку якості бластоцист, що сформувалися, за D. Gardner [20]. Кріоконсервування ембріонів у всіх порівнюваних групах проводили з використанням набору для вітрифікації Vitrification Media (Kitazato Corporation, Японія), а розморожування з використанням набору Thawing Media (Kitazato Corporation, Японія). Процедури виконували згідно з протоколами фірми-виробника. Розморожування кріоконсервованих ембріонів проводили безпосередньо в день перенесення ембріона (ПЕ). Клінічну вагітність визначали при ультразвуковому дослідженні на 5-му тижні після ПЕ за наявності плідного яйця в порожнині матки, куприкотіменному розміру плода 2-4 мм та реєстрації серцебиття. Статистичну обробку отриманих результатів проводили з використанням ліцензійних програм «Microsoft Excel» і «Statistica». Дизайн дослідження та всі методики, які були нами використані в даному проспективному дослідженні, розглянуті та схвалені комісією з біоетики закладу вищої освіти «Буковинський державний медичний університет» (протокол № 7 від 21.04.2022). Дослідження відповідає вимогам Хельсінкської декларації. Результати дослідження та їх обговорення Відомо, що підвищення результативності циклів запліднення при ДРТ в лікуванні безпліддя є складним медичним завданням та забезпечується цілим комплексом складових: чинниками безпліддя [2], оваріальним резервом, якістю та кількістю яйцеклітин, ембріонів [21]. Продовжується пошук схем для підвищення ефективності програм ДРТ [3]. Увагу вчених привернув мелатонін. Вважають, що він є медіатором, який передає екологічні стимули ооцитів і забезпечує взаємодії між факторами навколишнього середовища і епігенетичною системою спадковості [9, 12]. Рис.1. Зрілість отриманих яйцеклітин Рис. 2. Кількість та якість отриманих бластоцист Рис. 3. Частота настання вагітності на перенесення ембріонів (ПЕ) по їх послідовності та в цілому, а також вагітність в загальному Примітка: * - вірогідна різниця, р ˂ 0,05. 17 НЕОНАТОЛОГІЯ, ХІРУРГІЯ ТА ПЕРИНАТАЛЬНА МЕДИЦИНА NEONATOLOGY, SURGERY AND PERINATAL MEDICINE Т. ХIІ, № 3(45), 2022 Vol. ХIІ, № 3(45), 2022 ISSN 2226-1230 (Print) ISSN 2413-4260 (Online) Після пункції фолікулів у пацієнток першої групи ми отримали 451 яйцеклітину, а в другій групі – 616 яйцеклітин. Результати дослідження їх зрілості наведені на рис.1. Як свідчать отримані дані, у пацієнток першої групи зрілих яйцеклітин було 83,4 ± 6,9% від усіх отриманих, а в пацієнток другої групи – 81,5 ± 6,3%, що дещо менше, але вірогідної різниці ми не знайшли (р > 0,05). На рис. 2 представлені результати дослідження отриманих зрілих яйцеклітин. Так, в процесі інкубації у пацієнток першої групи всього утворилось 185 бластоцист, що склало 49,2 ± 9,5%, а в пацієнток другої групи – 237 (47,2 ± 8,7%), що дещо менше в порівнянні з першою групою, але достовірної різниці нами не виявлено (р > 0,05). Якщо розглянути якість отриманих бластоцист від всіх зрілих яйцеклітин по класам, картина наступна: клас І в пацієнток першої групи склав 48,6 ± 9,3%, а в пацієнток другої групи – 46,4 ± 8,1%, тобто дещо менше, але вірогідної різниці не було (р > 0,05), відповідно, клас ІІ – 36,8 ± 8,9% та 44,4 ± 8,1%, клас ІІІ – 14,6 ± 6,6% та 9,3 ±4,7%. Тобто у пацієнток першої групи утворилось дещо менше бластоцист класу ІІ, але більше класу ІІІ у порівнянні з пацієнтками другої групи, але різниця була не достовірною (р > 0,05). Результати дослідження по частоті настання вагітності наведені на рис. 3. Так, частота настання вагітності на перше перенесення ембріонів у пацієнток першої групи була 56,1 ± 8,85%, що дещо більше порівняно з даним показником у пацієнток другої групи (55,3 ± 8,15%), але різниця була не достовірною (р > 0,05). Аналогічна ситуація спостерігалась з частотою настання вагітності на друге перенесення ембріонів (відповідно, 45,5 ± 16,53% та 33,3 ± 21,14%) та третє – 100,0 ± 7,00% та 80,0 ± 17,95%, хоча при всіх трьох перенесеннях ембріонів ми не змогли відмітити вірогідність різниці по групам (р > 0,05). Частота настання вагітності на всі перенесення ембріонів у цілому в першій групі була 56,8 ± 8,24%, а в другій – 52,8 ± 7,62%, що дещо менше, але без вірогідної різниці (р > 0,05). Що стосується загальної частоти настання вагітності, то в першій групі вона склала 86,2 ± 6,41%, що вірогідно (р ˂ 0,05) більше в порівнянні з даним показником у другій групі ( 76,3 ± 6,95%). Таким чином, за результатами проведеного нами дослідження по кількості зрілих яйцеклітин та отриманих з них якісних бластоцист слід визнати, що застосування мелатоніну може бути корисним для покращення ембріологічних показників в програмах ДРТ при лікуванні безпліддя. Дозрівання яйцеклітин у фолікулярній рідині супроводжується активацією окислювальних процесів, а при індукції суперовуляції перекисне окислення ліпідів і білків збільшується в десятки разів. Сам окислювальний стрес (ОС) має руйнівну цитоток- Key title: Neonatologìâ, hìrurgìâ ta perinatalʹna medicina (Online) Abbreviated key title: Neonatol. hìr. perinat. med. (Online) сичну дію та активує апоптоз [22]. Природні антиоксиданти традиційно з успіхом застосовуються в медицині [23]. Мелатонін (від лат. melas – чорний) – локальний антиоксидант, який синтезується в мітохондріях. По суті, це гормон фотоперіодичності, що виробляється переважно вночі. Мелатонін є потужним ендогенним адаптогеном, що має імуномодулюючу та мембраностабілізуючу дію, нормалізує проникність стінки судин, покращує мікроциркуляцію ендотелію, нормалізує гемодинамічні процеси [24, 25]. В останні десятиліття активно вивчається роль мелатоніну як прямого інгібітору вільних радикалів у репродуктивній фізіології. Повідомлялось, що під час проведення стимуляції суперовуляції вміст мелатоніну у фолікулярній рідині в декілька разів перевищує його рівень у сироватці крові. Тобто, мова йде про протекторний вплив на зріючу яйцеклітину за рахунок зменшення ОС напевно, за рахунок нейтралізації вільних радикалів [11], оскільки антиоксидантні властивості гормону перевершують інші антиокислювачі, такі як глютатіон, манітол і вітамін Е. Мелатонін є ефективним нейтралізатором вільних радикалів токсичних реагентів на основі кисню, гідроксильних радикалів та азотистих сполук, збільшує ефективність переходу електронів між мітохондріальними дихальними комплексами, які є основними продуцентами вільних радикалів. Мелатонін має непряму дію за рахунок стимуляції утворення антиоксидантних ферментів і глутатіону як важливого інтрацелюлярного антиокислювача. Кілька метаболітів мелатоніну, які утворюються в процесі нейтралізації ушкоджуючих агентів, самі є акцепторами вільних радикалів [10, 26]. І хоча доказова база ефективності застосування мелатоніну тільки формується, позитивні результати захисту яйцеклітин від вільних радикалів для збереження їх якості відомі [27, 28, 29]. Аналіз частоти настання вагітності у пацієнток, які отримували мелатонін, також засвідчив, що вона була вищою. Це корелює з результатами досліджень інших авторів [3, 30]. Висновок Використання мелатоніну при підготовці жінок із безпліддям до застосування допоміжних репродуктивних технологій показало свою ефективність щодо настання вагітності. Перспектива подальших досліджень У подальшому планується продовжувати з’ясовувати місце і роль мелатоніну в сучасній репродукції людини. Конфлікт інтересів: відсутній Джерела фінансування: самофінансування. Література: 1. Юзько ОМ, Юзько ТА, Руденко НГ. Допоміжні репродуктивні технології в Україні. Жіночий лікар. 2021;3(95):22-26. 2. Li MC, Mínguez-Alarcón L, Arvizu M, Chiu YH, Ford JB, Williams PL, et al. Waist circumference in relation to outcomes of infertility treatment with assisted reproductive technologies. Am J Obstet Gynecol [Internet]. 2019[cited 2021 Dec 17];220(6):578. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545132/pdf/nihms-1521268.pdf doi: 10.1016/j.ajog.2019.02.013 3. Tamura H, Jozaki M, Tanabe M, Shirafuta Y, Mihara Y, Shinagawa M, et al. Importance of Melatonin in Assisted Reproductive Technology and Ovarian Aging. Int J Mol Sci [Internet]. 2020[cited 2021 Dec 12];21(3):1135. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036809/pdf/ijms-21-01135.pdf doi: 10.3390/ijms21031135 18 РЕЗУЛЬТАТИ ДИСЕРТАЦІЙНИХ ТА НАУКОВО-ДОСЛІДНИХ РОБІТ / RESULTS THESIS AND SCIENTIFIC - RESEARCH 4. Wei D, Zhang C, Xie J, Song X, Yin B, Liu Q, Hu L, Hao H, Geng J, Wang P. Supplementation with low concentrations of melatonin improves nuclear maturation of human oocytes in vitro. 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An overview on the relationship between endometriosis and infertility: the impact on sexuality and psychological well-being. J Psychosom Obstet Gynaecol. 2020;41(2):937. doi: 10.1080/0167482X.2019.1659775 29. Levi-Setti PE, Cirillo F, Scolaro V, Morenghi E, Heilbron F, Girardello D, et al. Appraisal of clinical complications after 23,827 oocyte retrievals in a large assisted reproductive technology program. Fertil Steril. 2018;109(6):1038-43. doi: 10.1016/j. fertnstert.2018.02.002 30. Suslikova LV, Kaminskyi VV, Chayka KV, Kaminskyi AV, Serbeniuk AV, Zhykharskyi RV, et al. Effect of endometrial injection scratching in cycles of treatment by assisted reproductive technology methods. Репродуктивна ендокринологія. 2020;3:49-54. doi: 10.18370/2309-4117.2020.53.49-54 19 НЕОНАТОЛОГІЯ, ХІРУРГІЯ ТА ПЕРИНАТАЛЬНА МЕДИЦИНА NEONATOLOGY, SURGERY AND PERINATAL MEDICINE ISSN 2226-1230 (Print) ISSN 2413-4260 (Online) Т. ХIІ, № 3(45), 2022 Vol. ХIІ, № 3(45), 2022 Key title: Neonatologìâ, hìrurgìâ ta perinatalʹna medicina (Online) Abbreviated key title: Neonatol. hìr. perinat. med. (Online) EMBRYOLOGICAL INDICATORS AND INCIDENCE OF PREGNANCY IN WOMEN WITH INFERTILITY U NDERGOING ASSISTED REPRODUCTIVE TECNOLOGY PROGRAMS WITH THE USE OF MELATONIN V.O. Yuzko 1,2, O. M. Yuzko 1,2, T.A. Yuzko 2, I.V. Chemiorkina 2, О.А. Andriets 1 Bukovinian State Medical University1, Medical Center of Infertility Treatment 2 (Chernivtsi, Ukraine) Summary Introduction. The technology of intracytoplasmic sperm injection into the egg cell (ICSI) has taken the main place among assisted reproductive technologies (ART) in the treatment of infertility. Improving the effectiveness of ART remains a complex medical problem. To a large extent, the success of ICSI depends on the quality of the oocytes, which, in its turn, depends on the number of follicles, which is determined by the ovulatory = ovarian reserve (OV) and effective preparation of patients for ART. The markers of OV are well known, and attention has been drawn to melatonin as a possible prognostic factor for the effectiveness of ART and OV in the last ten years. The indicators of melatonin content in the follicular fluid of stimulated ovaries have not been studied. At the same time, the assessment of embryological indicators of fertilization when using the medication melatonin for the preparation of patients and the effectiveness of ART programs in general can be useful for the further development of treatment algorithms for patients with infertility. The objective of the study. Evaluation of embryological indicators, the effectiveness of ICSI programs and the incidence of pregnancy in women with infertility during their preparation with melatonin. Materials and methods. 67 women with various infertility factors participated in the prospective study. All patients were prepared for controlled ovarian stimulation (COS), and subsequently for follicular puncture, oocyte retrieval and fertilization by ICSI. All obtained blastocysts were cryopreserved, stored in liquid Nitrogen and thawed when embryo transfer was necessary. Embryological indicators, the number of transfers and the effectiveness of pregnancy incidence were evaluated. According to the purpose of the task, the patients were divided into two groups by the method of even-odd numbers: group 1 – 29 patients with infertility, who received the preparation "Vita-melatonin" 3 mg orally before bedtime, during one month before the follicles puncture, group 2 – 38 patients with infertility, who did not receive this medicine. Statistical processing of the obtained results was carried out using the licensed programs "Microsoft Excel" and "Statistica". The research design and all the methods we used in this prospective study were reviewed and approved by the bioethics commission of the higher education institution "Bukovinian State Medical University" (protocol No. 7 dated 04/21/2022). SRW "Preservation and restoration of reproductive health of women and girls with obstetric and gynecological pathology" (state registration number 0121U110020. Implementation period – 01.2021-12.2025). Results of the research. The incidence of pregnancy after thawing of blastocysts and embryo transfers in general in women who received melatonin was significantly (p ˂ 0.05) higher compared to women who did not receive melatonin, respectively, 86.2 ± 6.41% and 76.3 ± 6.95%. For all performed embryo transfers, the incidence of pregnancy was 56.8 ± 8.24% and 52.8 ± 7.62% (p > 0.05), respectively, for the first embryo transfer 58.1 ± 8 .85% and 55.3 ± 8.15% (р > 0.05), for the second - 43.5 ± 16.53% and 33.3 ± 21.14% (р > 0.05), for the third – 100.0 ± 7.00% and 80.0 ± 17.95% (р > 0.05). When assessing embryological indicators, it was found that in women who received melatonin, the number of mature oocytes was 83.4 ± 6.94%, and in women who did not receive melatonin, 81.5 ± 6.36%, but there was no significant (p > 0.05) difference. Out of mature oocytes in the process of incubation after the ICSI technology, 49.5 ± 9.54% and 47.2 ± 8.72 (р > 0.05) blastocysts were formed, respectively, and from them blastocysts of class I – 48.6 ± 9 .36% and 46.4 ± 8.17% (р > 0.05), class II – 36.8 ± 8.94% and 44.4 ± 8.13% (р > 0.05), class III – 14.6 ± 6.67% and 9.3 ± 4.78% (р > 0.05). Conclusion. The use of melatonin in the preparation of women with infertility for the use of assisted reproductive technologies has shown its effectiveness in terms of pregnancy incidence. Key words: Infertility; Assisted Reproductive Technologies; Melatonin; Pregnancy. 20 РЕЗУЛЬТАТИ ДИСЕРТАЦІЙНИХ ТА НАУКОВО-ДОСЛІДНИХ РОБІТ / RESULTS THESIS AND SCIENTIFIC - RESEARCH Contact Information: Юзько Вікторія Олександрівна – аспірант Буковинського державного медичного університету, лікар акушер-гінеколог КЗОЗ «Медичний центр лікування безпліддя», м. Чернівці, Україна e-mail: yuzkoviktoriia@gmail.com ORCID ID: https://orcid.org/0000-0003-2793-8851 Researcher ID: GLN-7855-2022 Scopus Authors ID: 57219529058 Юзько Олександр Михайлович – д.мед.н., професор, завідувач кафедри акушерства та гінекології Буковинського державного медичного університету, медичний директор КЗОЗ «Медичний центр лікування безпліддя», м. Чернівці, Україна e-mail: prof.yuzko@gmail.com ORCID ID: https://orcid.org/0000-0003-1270-9095 Researcher ID: D-8126-2017 Scopus Authors ID: 42962929800 Юзько Тамара Анатоліївна – к.мед.н., доцент, лікар акушер-гінеколог, директор КЗОЗ «Медичний центр лікування безпліддя», м. Чернівці, Україна e-mail: reprod.cv@gmail.com Researcher ID: GPX-5721-2022 Чемьоркіна Ірина Василівна – ембріолог КЗОЗ «Медичний центр лікування безпліддя», м. Чернівці, Україна e-mail: chemerkina.irina@gmail.com Researcher ID: GPX-6560-2022 Андрієць Оксана Анатоліївна – д.мед.н., професор кафедри акушерства та гінекології, в.о. ректора закладу вищої освіти «Буковинський державний медичний університет», м. Чернівці, Україна e-mail: oandriiets@bsmu.edu.ua ORCID ID: https://orcid.org/0000-0001-9103-8546 Researcher ID: AAP-9746-2021 Scopus Author ID: 57221797595 © В.О. Юзько, O. M. Юзько, T.A.Юзько, I.В. Чемьоркіна, О.А. Андрієць, 2022 Контактна інформація: Victoria Yuzko – postgraduate student of Bukovinian State Medical University, obstetrician-gynecologist of the Medical Center of Infertility Treatment, Chernivtsi, Ukraine yuzkoviktoriia@gmail.com, +38951299075 ORCID ID: https://orcid.org/0000-0003-2793-8851 Researcher ID: GLN-7855-2022 Scopus Authors ID: 57219529058 Olexandr Yuzko – Doctor of Medical Sciences, Professor, Head of the Department of Obstetrics and Gynecology of Bukovinian State Medical University, Medical Director of the Medical Center of Infertility Treatment, Chernivtsi, Ukraine, prof.yuzko@gmail.com, +380506752334 ORCID ID: https://orcid.org/0000-0003-1270-9095 Researcher ID: D-8126-2017 Scopus Authors ID: 42962929800 Tamara Yuzko – Candidate of Medical Sciences, associate professor, obstetrician-gynecologist, director of the Medical Center for the Treatment of Infertility, Chernivtsi, Ukraine reprod.cv@gmail.com, +380505613722 Researcher ID: GPX-5721-2022 Iryna Chemiorkina – embryologist of the Medical Center for Infertility Treatment, Chernivtsi, Ukraine chemerkina.irina@gmail.com, +380503071325 Researcher ID: GPX-6560-2022 Oksana Аndriiets - Doctor of Medical Sciences, Professor of the Department of Obstetrics and Gynecology, acting rector of Bukovinian State Medical University, Chernivtsi, Ukraine oandriiets@bsmu.edu.ua, +38505116333 ORCID ID: https://orcid.org/0000-0001-9103-8546 Researcher ID: AAP-9746-2021 Scopus Author ID: 57221797595 © V.O. Yuzko, O. M. Yuzko, T.A. Yuzko, I.V. Chemiorkina, О.А. Andriets, 2022 Надійшло до редакції 10.06.2022 р. Підписано до друку 20.08.2022 р. 21
https://openalex.org/W2004469763	https://europepmc.org/articles/pmc4281077?pdf=render	English	null	Section Curve Reconstruction and Mean-Camber Curve Extraction of a Point-Sampled Blade Surface	PloS one	2,014	cc-by	10,245	Section Curve Reconstruction and Mean- Camber Curve Extraction of a Point- Sampled Blade Surface Wen-long Li, He Xie, Qi-dong Li, Li-ping Zhou, Zhou-ping Yin* Wen-long Li, He Xie, Qi-dong Li, Li-ping Zhou, Zhou-ping Yin* State Key Laboratory of Digital Manufacturing Equipment and Technology, School of Mechanical Science & Engineering, Huazhong University of Science and Technology, Wuhan, P. R. China Wen-long Li, He Xie, Qi-dong Li, Li-ping Zhou, Zhou-ping Yin* State Key Laboratory of Digital Manufacturing Equipment and Technology, School of Mechanical Science & Engineering, Huazhong University of Science and Technology, Wuhan, P. R. China g , , g , p g , p g State Key Laboratory of Digital Manufacturing Equipment and Technology, School of Mechanical Science & Engineering, Huazhong University of Science and Technology, Wuhan, P. R. China *yinzhp@mail.hust.edu.cn OPEN ACCESS Citation: Li W-l, Xie H, Li Q-d, Zhou L-p, Yin Z- p (2014) Section Curve Reconstruction and Mean- Camber Curve Extraction of a Point-Sampled Blade Surface. PLoS ONE 9(12): e115471. doi:10. 1371/journal.pone.0115471 Citation: Li W-l, Xie H, Li Q-d, Zhou L-p, Yin Z- p (2014) Section Curve Reconstruction and Mean- Camber Curve Extraction of a Point-Sampled Blade Surface. PLoS ONE 9(12): e115471. doi:10. 1371/journal.pone.0115471 Copyright: 2014 Li et al. This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and repro- duction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This work was supported by the National Natural Science Foundation of China (Grant Nos. 51475187, 51275192 and 51421062) and the National Key Projects (Grant No. 2014ZX04001051-05). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manu- script. Funding: This work was supported by the National Natural Science Foundation of China (Grant Nos. 51475187, 51275192 and 51421062) and the National Key Projects (Grant RESEARCH ARTICLE Abstract The blade is one of the most critical parts of an aviation engine, and a small change in the blade geometry may significantly affect the dynamics performance of the aviation engine. Rapid advancements in 3D scanning techniques have enabled the inspection of the blade shape using a dense and accurate point cloud. This paper proposes a new method to achieving two common tasks in blade inspection: section curve reconstruction and mean-camber curve extraction with the representation of a point cloud. The mathematical morphology is expanded and applied to restrain the effect of the measuring defects and generate an ordered sequence of 2D measured points in the section plane. Then, the energy and distance are minimized to iteratively smoothen the measured points, approximate the section curve and extract the mean-camber curve. In addition, a turbine blade is machined and scanned to observe the curvature variation, energy variation and approximation error, which demonstrates the availability of the proposed method. The proposed method is simple to implement and can be applied in aviation casting-blade finish inspection, large forging-blade allowance inspection and visual- guided robot grinding localization. Curve Reconstruction and Mean-Camber Curve Extraction of Blade aviation blades. These techniques can be categorized into two main groups: contact measurement (coordinate measurement machine) and non-contact measurement (laser/optical scanners, X-ray and CT). A coordinate measurement machine (CMM) is equipped with a contact probe while scanning a freeform surface. The CMM is the most popular measuring method in industrial settings and has a high accuracy (1–3 mm). The low measuring speed and potential collision/interference with parts are the main concerns. With the advent of high- resolution sensors, non-contact measuring techniques, such as Breuckmann stereoSCAN 3D-HE, achieve scanning accuracies as high as 10 mm and can be used to inspect a blade during its manufacturing process (casting, forming and robot polishing). Many efforts have been made in blade inspection and repairing using both contact and non-contact measuring techniques to obtain the optimum operation performance and reliability for aviation engines. In Fig. 1, 1) Inspection: a blade is designed with a thin wall, a crankle surface and a difficult-to-cut material. Geometric deformation is a common problem during the manufacturing process, and it must evaluate the dimensional error of the part. The inspection and analysis of the blade section serve as an important link in blade manufacturing; 2) Repairing: an aviation engine is extremely expensive (costing approximately $9 million), and blade manufacturing accounts for a large proportion of this cost. Because of the high temperature and impact load, used blades may have various defects, such as wear, impact dents and cracks. The manufacturing cost associated with repairing a defected blade is as low as one third of the cost of replacing a blade. Some blade manufacturers (MTU, BCT) have begun to perform this business. The main tasks are to locate the damaged area, calculate the breakage volume and evaluate the repaired quality. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Introduction Blades, which include turbine blades, compressor blades, and propeller blades, are the most critical parts of an aviation engine. They work under high temperatures and pressures, and a small change in the blade geometry can affect the operation performance of the aviation engine. For quality assurance purposes, high- precision measuring techniques are used to evaluate the dimensional error of No. 2014ZX04001051-05). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manu- script. Competing Interests: The authors have declared that no competing interests exist. 1 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Blade inspection Hsu et al. [1] proposed an iterative localization algorithm for airfoil blade inspection. A CMM was used to measure the blade object, and the traditional 3-2- 1 rule was applied to establish a coordinate system. A typical feature of this method is its iterative process incorporating the CMM measurement and coordinate upgrading procedure. Shortly thereafter, the same research group systematically introduced a section inspection and analysis technique for aviation blades [2]. A two-step measuring procedure was used to adapt sharp regions, such as the leading/trailing edges. The design parameters of the blade section were evaluated after an efficient localization between the measured data and their nominal curve. Chang and Lin [3] proposed an automatic blade inspection technique using a 3-axis CMM probe with a 2-axis driving head. Interference from the undercut surfaces and traveling paths were particularly focused. Makem et al. [4] presented a virtual inspection system to localize the data and evaluate the manufacturing accuracy of the aviation blade, where the errors of the root/mid/tip section thickness were inspected and analyzed. Unlike the 3-2-1 rule, the Iterative Closest Point (ICP) algorithm [5] was used to provide significantly better PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 2 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 1. Industrial application of blade measuring. (a) Inspection (GE), (b) Repairing (MTU). doi:10.1371/journal.pone.0115471.g001 Fig. 1. Industrial application of blade measuring. (a) Inspection (GE), (b) Repairing (MTU). Fig. 1. Industrial application of blade measuring. (a) Inspection (GE), (b) Repairing (MTU). doi:10.1371/journal.pone.0115471.g001 doi:10.1371/journal.pone.0115471.g001 registration results between the point cloud and its nominal model. Savio et al. [6] presented a state of metrology of freeform shapes, which focused on the introduction of measuring techniques and related metrological issues. A blade, which is regarded as a typical freeform surface, was cited to analyze the challenging tasks in sampling, alignment and error evaluation. Heo et al. [7] presented a computer-aided measuring technique for an impeller using a CMM based on the ruled line of the CAD model, which could partition the blade surface into several unit measuring regions. To recover the surface shape of the manufacturing part from the nominal curve and measured points, Li and Ni [8] proposed an iterative method of non-rigid registration and section profile reconstruction. Recently, Breuckmann and GE companies [9–10] also developed a blade inspection system based on the non-contact measuring technique and successfully applied it to blade inspection. Blade inspection The Breuckmann 3D inspection system operates based on an adapted structured light projector, and customized software was developed for the inspection requirement. By using the latest state-of-the-art inspection technology of Breuckmann, GE replaced the conventional CMM measuring approach with a highly time-saving and cost-saving control procedure, which significantly reduced the inspection time and generated more conclusive and easily transferrable measurement results. The non-contact measurement points are large-scale, unordered and noisy. In this situation, performing surface alignment and parameter extraction of the blade surface is not easy. Over the past few years, some iterative alignment methods for the blade surface have been developed, such as the improved ICP algorithm [11], SDM algorithm [12], ADF algorithm [13]; however, avoiding the local optimal alignment problem and accurately extracting the blade section parameters from the discrete point cloud remain a challenging task. Curve Reconstruction and Mean-Camber Curve Extraction of Blade the blade repairing processes are manually performed, but they are labor intensive and time consuming, and the quality is inconsistent because curved blades are overly complex for manual treatment. Recently, many practical studies of blade repair have been conducted by researchers at the University of Nottingham. Yilmaz et al. [14] presented the state of the research on machining and repairing turbo-machinery components. Two important repair steps were introduced and discussed: milling tool path generation and robot belt grinding/polishing. Gao et al. [15] and Yilmaz and Grindy [16] also published important studies on blade repairing based on the reverse-engineering technique. In their repair process, a CAD model was used to generate motion paths of laser welding and NC machining. Because used blades typically suffer from wear, cracks and other defects, non-contact measuring sensors were applied to locate the defecting regions. Similar to Gao et al., Zheng et al. [17] used the reverse-engineering technique to digitize the blade surface, perform point-to-surface alignment, identify the worn area and undamaged area, and generate a laser welding procedure. Berger et al. [18] proposed an integrated process for the multi-axis milling of aviation hard-cutting materials, such as titanium and nickel alloys, which is applied to the intelligent equipment and control of the ESPRIT project. Rong et al. [19] proposed a deformable-template-based method to recover the blade surface from section profiles. This method can automatically deform the nominal curve to best fit the measured points of a blade section. the blade repairing processes are manually performed, but they are labor intensive and time consuming, and the quality is inconsistent because curved blades are overly complex for manual treatment. Recently, many practical studies of blade repair have been conducted by researchers at the University of Nottingham. Yilmaz et al. [14] presented the state of the research on machining and repairing turbo-machinery components. Two important repair steps were introduced and discussed: milling tool path generation and robot belt grinding/polishing. Gao et al. [15] and Yilmaz and Grindy [16] also published important studies on blade repairing based on the reverse-engineering technique. In their repair process, a CAD model was used to generate motion paths of laser welding and NC machining. Because used blades typically suffer from wear, cracks and other defects, non-contact measuring sensors were applied to locate the defecting regions. Similar to Gao et al., Zheng et al. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Blade repairing The repair of aviation components, particularly the blade, is a highly competitive market and actively supported by well-known engine producers, such as Rolls- Royce, MTU Aero Engines, SNECMA Moteurs and General Electric. Currently, PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 3 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade method is that both energy minimization and distance minimization are used to smooth the point cloud and generate the curve to improve reconstruction accuracy and parameter extraction accuracy as quickly as possible. First, the mathematical morphology (MM) is expanded from image processing to point cloud processing with the objective of restraining the effect of measuring defects, such as holes, uneven density and miss-registration, generating an ordered sequence of 2D measured points, and providing an initial value for the point cloud smoothing and curve reconstruction process. Next, the energy and distance are minimized to iteratively smoothen the measured points, approximate the section curve and extract the mean-camber parameter of the blade surface. The remainder of the paper is organized as follows. Section 2 introduces an implementation of the MM operation to a point cloud, an energy minimization process for smoothing, and a distance minimization process for curve reconstruction. Section 3 introduces the mean-camber curve extraction process via the distance minimization method. Section 4 presents the experiments and analysis. Section 5 contains our conclusion and discusses the potential applications of the proposed method. method is that both energy minimization and distance minimization are used to smooth the point cloud and generate the curve to improve reconstruction accuracy and parameter extraction accuracy as quickly as possible. First, the mathematical morphology (MM) is expanded from image processing to point cloud processing with the objective of restraining the effect of measuring defects, such as holes, uneven density and miss-registration, generating an ordered sequence of 2D measured points, and providing an initial value for the point cloud smoothing and curve reconstruction process. Next, the energy and distance are minimized to iteratively smoothen the measured points, approximate the section curve and extract the mean-camber parameter of the blade surface. The remainder of the paper is organized as follows. Section 2 introduces an implementation of the MM operation to a point cloud, an energy minimization process for smoothing, and a distance minimization process for curve reconstruction. Section 3 introduces the mean-camber curve extraction process via the distance minimization method. Section 4 presents the experiments and analysis. Section 5 contains our conclusion and discusses the potential applications of the proposed method. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 [17] used the reverse-engineering technique to digitize the blade surface, perform point-to-surface alignment, identify the worn area and undamaged area, and generate a laser welding procedure. Berger et al. [18] proposed an integrated process for the multi-axis milling of aviation hard-cutting materials, such as titanium and nickel alloys, which is applied to the intelligent equipment and control of the ESPRIT project. Rong et al. [19] proposed a deformable-template-based method to recover the blade surface from section profiles. This method can automatically deform the nominal curve to best fit the measured points of a blade section. Robotic grinding/polishing has attracted considerable attention in blade repairing due to its advantages of automation, flexible contact and width-line machining. Zhang et al. [20] proposed a local grinding model to simulate robot belt grinding, particularly for free-form surfaces, such as blades. Huang et al. [21] reported a successful development of an automated SMART robotic system to grind/polish vane airfoils. The system layout, section profile fitting, robot path planning and tool wear compensation during the repairing process were introduced. Chen et al. [22] proposed a rail-free robot scheme in on-site welding repair for hydraulic turbine blades with large-scale surfaces. Real-time images of the blade state from a CCD camera were used to determine the damaged area; therefore, the operator could control the robot system to perform the repair. Researchers at the ABB Corporate Research Center in Shanghai [23] briefly reviewed the robotic techniques in industrial application and noted that force control and machine vision were enabling technologies of robotic automation. Because of its high speed and simple operation, non-contact measurement is widely applied in blade manufacturing (in addition to medical orthopedics [24] and surgical changing assessment [25]), and point cloud has received increasing attention as a representation of the blade model. The motivation of this study is to improve section curve reconstruction and mean-camber curve extraction in blade inspection and repairing. This paper introduces a new method of section curve reconstruction and mean-camber extraction. One important characteristic of this 4 / 30 Curve Reconstruction Based on Distance Function Minimization Mathematical morphology operation to a point cloud The development and application of mathematical morphology (MM) [26, 27] originates from image processing and pattern recognition. The computation of MM is a simple combination of adding and subtracting operations based on a defined structure element. Assuming that F is a binary image and B is a structure element to perform the MM operation, the dilation and corrosion can be described by F+B~ [ b[B Fb,FHB~ [ b[B F{b ð1Þ ð1Þ The MM operation is illustrated in Fig. 2. The dilation operation can enlarge the objective and compress the hole regions, and the corrosion operation can control overlapping regions and uneven-density regions. In the following, the MM operation is expanded from a 2D image to 3D measured points. The main objectives are to construct a well-organized point set in a section plane of the blade and provide a fast computation method of the initial value for point cloud smoothing, section curve reconstruction and mean-camber curve extraction. The execution object of the MM algorithm is a binary image, and it should first interpret the measured points into 3D grids. Assume that the measured points from the blade model are described by P and that the mean value of sampling resolution is d. The extrema of the XYZ coordinates from P are defined as (xmax,ymax,zmax) and (xmin,ymin,zmin). Then, the numbers of grids along the XYZ PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 5 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 2. Mathematical morphology operation of a binary image. (a) dilation, (b) corrosion. / Fig. 2. Mathematical morphology operation of a binary image. (a) dilation, (b) corrosion. directions are Nx~int( (xmaxzt){(xmin{t) h ), Ny~int( (ymaxzt){(ymin{t) h ), Nz~int( (zmaxzt){(zmin{t) h ) ð2Þ ð2Þ h where int(N) is an integer operation, t is an allowance (tw3h), and h (equal to d) is the width of the 3D grids. For Vpi[P, the position in the 3D grids is calculated as px~floor( xi{xmin h ),py~floor( yi{ymin h ),pz~floor( zi{zmin h ) ð3Þ ð3Þ where floor(N) is a floor integer operation. If there is a measured point in the grid (px,py,pz), define the value of grid (px,py,pz) as ‘‘1’’; otherwise, define it as ‘‘0’’. Therefore, all measured points of P can be described using a binary image. Fig. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Curve Reconstruction Based on Distance Function Minimization 3(a) presents a point-sampled turbine surface, where section planes that are parallel to the reference plane are used to intercept the point cloud, and Fig. 3(b) shows the 2D points in one section plane. It is difficult to obtain the real intersection points between the section planes and point-sampled surface. In our implementation, the section plane pi (Fig. 3(c)) is set from the reference plane with a relative distance di (which is determined based on the required number of blade inspections), and all points between planes pz i and p{ i are projected onto PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 6 / 30 115471 December 31, 2014 7 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade section, the obtained initial curves and normal vector are used to implement the section curve reconstruction and parameter extraction. 2.2 Point cloud smoothing by energy minimization The initial section curve in Fig. 4(c) is not smooth (folding line) because the position of the measured points are affected by the dilation/erosion operation and measuring noise. Assume that Q0~fq0 1,q0 2, ,q0 ng and Q~fq1,q2, ,qng denote the measured points in one section plane before and after smoothing, respectively, and that k denotes the section curve curvature. Then, the strain energy EC of a curve s is EC~ 1 2 ð k2ds ð4Þ ð4Þ The curvature of a discrete point qi can be approximated by The curvature of a discrete point qi can be approximated by ki< 2 lizliz1 qiz1{qi liz1 { qi{qi{1 li ð5Þ ð5Þ where li~ qi{qi{1 k k2. Because ds~(lizliz1)=2, (4) can be represented by EC~ 1 2 X in i~i0 k2 i : lizliz1 2 ~ X in i~i0 1 lizliz1 qiz1{qi liz1 { qi{qi{1 li 2 2 ð6Þ where li~ qi{qi{1 k k2. Because ds~(lizliz1)=2, (4) can be represented by qi qi{1 k k2. Because ds (lizliz1)=2, (4) can be represented by EC~ 1 2 X in i~i0 k2 i : lizliz1 2 ~ X in i~i0 1 lizliz1 qiz1{qi liz1 { qi{qi{1 li 2 2 ð6Þ ð6Þ S between point qi and point q0 i is P n i~1 qi{q0 i 2 2. Then, the total energy function of point cloud smoothing is defined by According to Zhu’s work [29], the spring energy E E~aECzbES ð7Þ ð7Þ where a[½0:1,0:5,b[½1,1:2. The strain energy EC expresses the relationship between two adjacent points and is used to control the fairness of adjacent points qi{1,qi,qiz1 . The spring energy ES is used to control the distance deviation between unsmoothed point q0 i and smoothed point qi, which avoids a major change in q0 i . where a[½0:1,0:5,b[½1,1:2. The strain energy EC expresses the relationship between two adjacent points and is used to control the fairness of adjacent point qi{1,qi,qiz1 . The spring energy ES is used to control the distance deviation between unsmoothed point q0 i and smoothed point qi, which avoids a major change in q0 i . 1) If point set Q0 is located in an unclosed curve, i0~2,in~n{1; 2) If point set Q0 is located in a closed curve, q0~qn,q{1~qn{1 and qnz1~q1,qnz2~q2, then i0~1,in~n. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 3. Searching for projecting points by section planes. (a) initial 3D measured points and the reference/section planes, (b) measured points in one section plane, (c) search for projecting points in a section plane pi. Fig. 3. Searching for projecting points by section planes. (a) initial 3D measured points and the reference section plane, (c) search for projecting points in a section plane pi. Fig. 3. Searching for projecting points by section planes. (a) initial 3D measured points and the reference/section planes, (b) measured points in one section plane, (c) search for projecting points in a section plane pi. doi:10.1371/journal.pone.0115471.g003 plane pi. The MM operation, which includes dilation, flood filling and erosion, is implemented toward well-ordered points in Fig. 4(b). As shown in Fig. 4(c), an initial profile curve in one section plane can be generated by linking the well- ordered points after erosion, an initial mean-camber curve is rapidly generated by extracting the morphological skeleton from the well-ordered points, and the normal vector at each point is obtained by computing the gradient vector field of the binary image [28]. MM is advantageous because it can reduce the effect of the measured defects, such as holes, uneven density and miss-registration, and confirm the oriented normal information of the measured points. In the following Fig. 4. Mathematical morphology operation of a binary image from the blade model. (a) ‘‘+’’ structure element, (b) MM operation, (c) initial curves and normal vector. Fig. 4. Mathematical morphology operation of a binary image from the blade model. (a) ‘‘+’’ structure element, (b) MM operation, (c) initial curves and normal vector. doi:10.1371/journal.pone.0115471.g004 doi:10.1371/journal.pone.0115471.g004 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 7 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade The coefficients in (8) are ai,i~{(ai,i{2zai,i{1zai,iz1zai,iz2), ai,i{2~a½(lizli{1)lili{1{1, ai,i{1~{a(liz1zli{1)½li{1l2 i liz1{1, ai,iz1~{a(liz2zli)½lil2 iz1liz2{1, ai,iz1~{a(liz2zli)½lil2 iz1liz2{1 The coefficients in (8) are ts in (8) are ai,i~{(ai,i{2zai,i{1zai,iz1zai,iz2), ai,i{2~a½(lizli{1)lili{1{1, ai,i{1~{a(liz1zli{1)½li{1l2 i liz1{1, ai,iz1~{a(liz2zli)½lil2 iz1liz2{1, ai,iz1~{a(liz2zli)½lil2 iz1liz2{1 There are n equations with n variables qi, and the solution is unique. However, if n is high, the calculation and storage of equations will consume large amounts of memory space. In this paper, an iterative solving method is used to calculate the smoothed points in Q. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 2.2 Point cloud smoothing by energy minimization 1) If point set Q0 is located in an unclosed curve, i0~2,in~n{1; ) p Q , 0 , n ; 2) If point set Q0 is located in a closed curve, q0~qn,q{1~qn{1 and qnz1~q1,qnz2~q2, then i0~1,in~n. To minimize the energy function E in (7), one differentiates energy E on variable qi; then, ð8Þ ai,i{2qi{2zai,i{1qi{1z(1zaii)qizai,iz1qiz1zai,iz2qiz2~q0 i ð 8 / 30 For a smoothed point qi, there exists 0 0 0 ð9Þ (1zaii)qi~q0 i {ai,iz1q0 iz1{ai,iz2q0 iz2{ai,i{1qi{1{ai,i{2qi{2 ð9Þ Therefore, during each iteration, there is 1) For an unclosed curve 1) For an unclosed curve N The first smoothed point: (1za11)q1~q0 1{a12q0 2{a13q0 3 N The first smoothed point: (1za11)q1~q0 1{a12q0 2{a13q0 3 N The second smoothed point: (1za22)q2~q0 2{a23q0 3{a24q0 4{a21q1 N The (n{1)th smoothed point: (1zan{1,n{1)qn{1~q0 n{1{an{1,nq0 n{a {an{1,n{3qn{3 N Th h h d i (1 ) 0 qn{2 n{2 n{1, N The first smoothed point: (1za11)q1~q0 1{a12q0 2{a13q0 3 N The second smoothed point: (1za22)q2~q0 2{a23q0 3{a24q0 4{a21q1 N The (n{1)th smoothed point: (1zan{1,n{1)qn{1~q0 n{1{an{1,nq0 n{a {an 1 n 3q 3 q 2 n{2 n{1, p ( 11)q1 q1 12q2 13q3 N The second smoothed point: (1za22)q2~q0 2{a23q0 3{a24q0 4{a21q1 N The second smoothed point: (1za22)q2~q0 2{a23q0 3{a24q0 4{a21q1 N The (n{1)th smoothed point: (1zan{1,n{1)qn{1~q0 n{1{an{1,nq0 n{a {an{1,n{3qn{3 qn{2 n{2 n{1, The second smoothed point: (1za22)q2 q2 a23q3 a24q4 a21q1 N The (n{1)th smoothed point: (1zan{1,n{1)qn{1~q0 n{1{an{1,nq0 n{a {an{1,n{3qn{3 qn{2 n{2 n{1, N The nth smoothed point: (1zann)qn~q0 n{an,n{1qn{1{an,n{2qn{2 2) For a closed curve (q0~qn,q{1~qn{1 and qnz1~q1,qnz2~q2) N The first smoothed point: (1za11)q1~q0 1{a12q0 2{a13q0 3{a1,nq0 n{a1,n{1q0 n{1 N The second smoothed point: (1za22)q2~q0 2{a23q0 3{a24q0 4{a2,nq0 n{a21q1 N The (n{1)th smoothed point: (1zan{1,n{1)qn{1~q0 n{1{an{1,n{3q0 n{3 {an{1,nq0 n{an{1,n{2qn{2{an{1,1q1 N The nth smoothed point: (1zann)qn~q0 n{an,n{2qn{2{an,n{1qn{1{an,1q1 {an,2q2 N The first smoothed point: (1za11)q1~q0 1{a12q0 2{a13q0 3{a1,nq0 n{a1,n{1q0 n{1 N The second smoothed point: (1za22)q2~q0 2{a23q0 3{a24q0 4{a2,nq0 n{a21q1 N The second smoothed point: (1za22)q2~q0 2{a23q0 3{a24q0 4{a2,nq0 n{a21q1 N The (n{1)th smoothed point: (1zan{1,n{1)qn{1~q0 n{1{an{1,n{3q0 n{3 {an{1,nq0 n{an{1,n{2qn{2{an{1,1q1 Th h h d i ( ) 0 3 n N The (n{1)th smoothed point: (1zan{1,n{1)qn{1~q0 n{1{an{1,n{3q0 n{3 {an{1,nq0 n{an{1,n{2qn{2{an{1,1q1 N The nth smoothed point: (1zann)qn~q0 n{an,n{2qn{2{an,n{1qn{1{an,1q1 {an,2q2 N The nth smoothed point: (1zann)qn~q0 n{an,n{2qn{2{an,n{1qn{1{an,1q1 {an,2q2 N The nth smoothed point: (1zann)qn~q0 n{an,n{2qn{2{an,n{1qn{1{an,1q1 {an,2q2 Because the measured points in the section plane are derived from a closed line, the second strategy above is used to iteratively calculate the smoothed points Q~fq1,q2, ,qng. Curve Reconstruction and Mean-Camber Curve Extraction of Blade where q(ti)~qi and t(t1vt2v vtn) is a sequence of real numbers. The symbol Ni,3(t) denotes the basis function of the B-spline, and bi(i~0,1,2, ,l) denotes the control points of the B-spline. The calculation of the curve q(t) requires the interception of planar points, a dilation/erosion operation and energy- where q(ti)~qi and t(t1vt2v vtn) is a sequence of real numbers. The symbol Ni,3(t) denotes the basis function of the B-spline, and bi(i~0,1,2, ,l) denotes the control points of the B-spline. The calculation of the curve q(t) requires the interception of planar points, a dilation/erosion operation and energy- minimization smoothing. In particular, in the interception process, two section planes with a width of d are used to surround the initially measured points from the blade model, and the surrounded points are subsequently projected onto a section plane. The surround points may not be real ‘‘intersection points’’ in the section plane, and the curve q(t) in (10) is only a fitting curve that passes through the smoothed points in Section 3. Therefore, the curve q(t) is considered an initial section curve and is iteratively updated to approximate the measured points in P. I Fi h S(P) d h d i f f h bl d d d minimization smoothing. In particular, in the interception process, two section planes with a width of d are used to surround the initially measured points from the blade model, and the surrounded points are subsequently projected onto a section plane. The surround points may not be real ‘‘intersection points’’ in the section plane, and the curve q(t) in (10) is only a fitting curve that passes through the smoothed points in Section 3. Therefore, the curve q(t) is considered an initial section curve and is iteratively updated to approximate the measured points in P. y p pp p In Fig. 5, assume that S(P) denotes the design surface of the blade and v denotes the normal direction perpendicular to the section plane; for Vq[q(w,t), the foot projecting point q\[S(P) with respect to q is calculated. The design surface S(P) is unknown, and the foot point q\ can be approximately replaced by the closest point of q in P. Curve reconstruction by distance minimization Curve reconstruction by distance minimization Using the smoothed point q[Q, a cubic B-spline q(t) is constructed by q(t)~ X l i~0 Ni,3(t)bi ð10Þ ð10Þ PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 9 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 ð17Þ ð17Þ The implementation steps are as follows: The implementation steps are as follows: The implementation steps are as follows: (1) Perform MM and energy minimization to obtain a group of smoothed points and generate an initial cubic B-spline curve q(t); (1) Perform MM and energy minimization to obtain a group of smoothed points and generate an initial cubic B-spline curve q(t); (2) Scatter q(t) into N points qi and search for the foot-projecting point q\ i of qi in P; (2) Scatter q(t) into N points qi and search for the foot-projecting point q\ i of qi in P; (3) Minimize (17) and obtain the offset Dw of the control variable during each iteration, where the value of l is set as 4; (4) Update the control variable by (wzDw) and obtain an updated curve q( N (4) Update the control variable by (wzDw) and obtain an updated curve q(t); N (4) Update the control variable by (wzDw) and obtain an updated curve q(t); (5) Repeat steps (2)-(4) until 1 N P N i~1 qi{qi \ ƒd=100 or the number of iterations exceeds 20. y (5) Repeat steps (2)-(4) until 1 N P N i~1 qi{qi \ ƒd=100 or the number of iterations exceeds 20. Then, the directed distance function between point q and P is defined by dq,P~ min q[Q,p[P q{p k k2~ q{q\ 2 ð11Þ ð11Þ For point q(w), dq,P is a distance function with variable w. Ideally, if dq,P~0, point q(w) is located at S(P). When a differential perturbation with respect to Dw appears, q(w) becomes q(wzDw). Assume that the foot projecting point of q is still q\; then, dq,P(wzDw)~ q(wzDw){q\ 2 ð12Þ ð12Þ The first-order Taylor expansion at w is The first-order Taylor expansion at w is The first-order Taylor expansion at w is dq,P(wzDw)< q(w){q\ 2z X m j~1 qwj(w):½q(w){q\ q(w){q\ k k2 Dwj ð13Þ dq,P(wzDw)< q(w){q\ 2z X m j~1 qwj(w):½q(w){q\ q(w){q\ k k2 Dwj ð13Þ Fig. 5. Point q(w) moves to the position of q(wzDw) when there is a perpendicular Dw. ð13Þ Fig. 5. Point q(w) moves to the position of q(wzDw) when there is a perpendicular Dw. doi:10.1371/journal.pone.0115471.g005 doi:10.1371/journal.pone.0115471.g005 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 10 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade where m~2(lz1) and qwj(w) denotes the first-order derivative with respect to the variable wj. Then, dq,P(wzDw)<dq,P(w)z X m j~1 qwj(w):½q(w){q\ q(w){q\ k k2 Dwj ð14Þ ð14Þ To simplify the calculation process, one scatters the initial B-spline curve into qi(i~1,2, ,N) if the distance between two adjacent points is not larger than d. Then, the problem of constructing the section curve q(t) of the point-sampled blade surface becomes min w[R2(lz1) X N i~1 ½dqi,P(w)2 ð15Þ ð15Þ According to (14), the nonlinear least-square problem of (15) corresponds to min Dw[Rm X N i~1 ½dqi,P(w)z X m j~1 qwj(w):½q(w){q\ q(w){q\ k k2 Dwj2 ð16Þ According to (14), the nonlinear least-square problem of (15) corresponds to min Dw[Rm X N i~1 ½dqi,P(w)z X m j~1 qwj(w):½q(w){q\ q(w){q\ k k2 Dwj2 ð16Þ min Dw[Rm X N i~1 ½dqi,P(w)z X m j~1 qwj(w):½q(w){q\ q(w){q\ k k2 Dwj2 ð16Þ ð16Þ To limit the excessive offset of the variable w, the coefficient l is added to the constraint. The final optimization model is To limit the excessive offset of the variable w, the coefficient l is added to the constraint. The final optimization model is min Dw[Rm X N i~1 ½dqi,P(w)z X m j~1 qwj(w):½q(w){q\ q(w){q\ k k2 Dwj2zl X m j~1 Dw2 j ! Mean-camber curve extraction of the point-sampled blade surface An aviation blade has many geometric parameters, such as the mean-camber curve, maximum gauge, leading/trailing edge, chord length, chord inclination, PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 11 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 doi:10.1371/journal.pone.0115471.g007 Curve Reconstruction and Mean-Camber Curve Extraction of Blade torsion resistance, and tortuosity. The manufacturing accuracy is important in the service performance of the blade. The mean-camber curve is a continuous curve of in-circle centers, where the maximum gauge corresponds to the maximal inscribed circle. The mean-camber curve is an important design basis, and a marginal shift may decrease the aviation aerodynamic performance considerably. Calculation and inspection of the mean-camber curve are vital to blade manufacturing. In the following, we introduce the method to extract the mean- camber curve. A group of point set M from the mean-camber curve is initially generated using the skeleton MM implementation presented in Section 2. For Vmj[M, the two nearest points qj1 and qj2 are calculated in the suction and pressure surfaces of the constructed curve q(t), respectively. Assume that dj1~ mj{qj1 ,dj2~ mj{qj2 , and define rj~(dj1zdj2) 2 as the inscribed circle radius at point mj. Then, Mr~f(mj,rj),j~1,2, ,nMg constitutes a group of new point sets and yields an envelope curve mr(w,t)~(m(w,t),r(w,t)) in Fig. 6, where m(w,t) denotes the mean-camber curve and r(w,t) denotes the radius of the inscribed circle. For the convenience of calculation, the control point set is expressed as a column vector w[Ra below. First, the reconstructed curve q(t) is scattered into N points. For Vq[Q, the normal vector is defined as n, and a straight line Lq is generated using point q and vector n. The intersection point between line Lq and the mean-camber curve is m(w); then, the directed distance from point q to the envelope curve mr(w,t) is dq,m(w)~½q{m(w):n{r(w) ð18Þ ð18Þ When a differential perturbation with respect to Dw appears, q(w) becomes q(wzDw), and the first-order Taylor expansions at w are Fig. 6. Calculation process of the mean-camber curve. doi:10.1371/journal.pone.0115471.g006 Fig. 6. Calculation process of the mean-camber curve. doi:10.1371/journal.pone.0115471.g006 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 12 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 doi:10.1371/journal.pone.0115471.g008 Curve Reconstruction and Mean-Camber Curve Extraction of Blade m(wzDw)~m(w)z X a j~1 mwj(w):Dwj, r(wzDw)~r(w)z X a j~1 rwj(w):Dwj ð19Þ ð19Þ j~1 r(wzDw)~r(w)z X a j~1 rwj(w):Dwj ð19Þ where mwj(w) and rwj(w) denote the first-order derivatives with respect to the variable wj. Because dq,m(wzDw)~½q{m(wzDw):v{r(wzDw) ~ q{m(w){ X a j~1 mwj(w):Dwj " # :v{ r(w)z X a j~1 rwj(w):Dwj " # ~ q{m(w) ½ :v{r(w){ X a j~1 mwj(w):vz X a j~1 rwj(w) " # :Dwj ~dq,m(w){ X a j~1 mwj(w):vz X a j~1 rwj(w) " # :Dwj ð20Þ ð20Þ Fig. 7. Scanning a turbine blade and intercepting its point cloud. (a) Turbine blade and scanning sensor, (b) Section planes and obtained measured points. Fig. 7. Scanning a turbine blade and intercepting its point cloud. (a) Turbine blade and scanning sensor, (b) Section planes and obtained measured points. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 13 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 doi:10.1371/journal.pone.0115471.g009 471 December 31, 2014 15 / 30 A cubic B-spline curve is used to construct an envelope curve mr(w,t); (3) Calculate the offset Dw of the control point set during each iteration according to (22); (3) Calculate the offset Dw of the control point set during each iteration according to (22); (4) Apply the calculated wzDw and update the envelope curve mr(w,t); (5) Calculate the distance dq,m(w) between q and the curve mr(w,t) using (18); (5) Calculate the distance dq,m(w) between q and the curve mr(w,t) using (18); (6) Repeat steps (2)–(5) until "ƒd=100 or the number of iterations exceeds 20. (6) Repeat steps (2)–(5) until "ƒd=100 or the number of iterations exceeds 20. Experiments and Analysis In Fig. 7(a), a turbine blade (150 mm6100 mm640 m) is machined for the experimental analysis using a 5-axis Mikron CNC machine (UCP 800 Duro). The blade is placed on an anti-vibration platform and scanned using a Hexagon laser- scanning equipment (Infinite SC 2.4 m). The laser-scanning equipment has 6 rotational degrees of freedom and can scan the integral blade in one coordinate system without moving the blade or scanning equipment. The data scale of the obtained point cloud is 358,748, and the sampling space of the measured points after uniform simplification is 0.3 mm. Three section planes are used to intercept the point cloud and obtain three groups of 2D measured points (red color) in Fig. 7(b). Curve Reconstruction and Mean-Camber Curve Extraction of Blade The first-order differential increment of dq,m(w) is The first-order differential increment of dq,m(w) is Ddq,m(w)~{ X a j~1 mwj(w):vz X a j~1 rwj(w) " # :Dwj ð21Þ ð21Þ Therefore, the calculation process of the mean-camber curve becomes a nonlinear least-squares problem Fig. 8. Measured points (blue) before smoothing and its folding line (violet) after smoothing in Section 1–3. Set a~0:2,b~1, and perform 20 iterations. Fig. 8. Measured points (blue) before smoothing and its folding line (violet) after smoothing in Section 1–3. Set a~0:2,b~1, and perform 20 iterations. 14 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 9. Mean-camber points (blue) from the morphological skeleton and its folding line (violet) after smoothing in Section 1–3. Fig 9 Mean camber points (blue) from the morphological skeleton and its folding line (violet) after Fig. 9. Mean-camber points (blue) from the morphological skeleton and its folding line (violet) after smoothing in Section 1–3. Fig. 9. Mean-camber points (blue) from the morphological skeleton and its folding line (violet) after smoothing in Section 1–3. doi:10.1371/journal.pone.0115471.g009 min Dw[Ra X N i~1 dqi,m(w)zDdqi,m(w) 2zl X a j~1 Dw2 j ! ð22Þ ð22Þ The calculation error of the mean-camber curve is defined by "~ 1 N X N i~1 dqi,m(w) ð23Þ ð23Þ The implementation steps are as follows: The implementation steps are as follows: The implementation steps are as follows: PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 15 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Curve Reconstruction and Mean Camber Curve Extraction of Blade e.0115471 December 31, 2014 16 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 16 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 10. Energy variation of the section curves in Section 1–3 with an increasing number of iterations. Set a~0:2,b~1, and perform 20 iterations. doi:10.1371/journal.pone.0115471.g010 (1) Scatter q(t) into N points qi (i~1,2, ,N) and maintain the dense points in the regions of the leading and trailing edges; (1) Scatter q(t) into N points qi (i~1,2, ,N) and maintain the dense points in the regions of the leading and trailing edges; (2) For each point mj, calculate its inscribed circle radius rj and obtain a new point set Mr. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Fig. 10. Energy variation of the section curves in Section 1–3 with an increasing number of iterations. Set a~0:2,b~1, and perform 20 iterations. Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 11. Energy variation of the mean-camber curves in Section 1–3 with an increasing iterations. Fig. 11. Energy variation of the mean-camber curves in Section 1–3 with an increasing iterations. doi:10.1371/journal.pone.0115471.g011 doi:10.1371/journal.pone.0115471.g011 maintains a fine fairness of the bolding line. The energy variations of the strain energy and spring energy are shown in Figs. 10 and 11, respectively. The strain energy, which controls the fairness of adjacent points, gradually decreases, and 15 iterations are sufficient to attain a stable value. From the 15th iteration to the 20th iteration, the strain energy slowly decreases, but the spring energy rapidly increases. Thus, the distance deviation between an unsmoothed point q0 i and a smoothed point qi varies considerably to adapt to the fairness requirement. In the experiment, if the strain energy is approximately stable, it should stop the subsequent iterations to control the large movement of 2D measured points. Figs. 12 and 13 present the variation in the curvature of the section curves and mean-camber curves, respectively. The blue polyline denotes the 2D measured points before smoothing, and the violet polyline denotes the folding line after smoothing. The variations in the curvature of the blue polyline are choppy, and the absolute value in some regions is relatively large. However, the curvature of the points smoothed via energy minimization nearly surrounds the X-axis, except for two high values at the leading/trailing edges. In addition, the plus-minus direction of curvature in the regions between the leading and trailing edges is not constant and does not fit with practical applications. The section curve is only represented by the folding line when energy-minimization smoothing is implemented, and it is not considered to maintain a direction consistency during each iteration. This problem can be solved using the distance minimization method. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Measured-point smoothing First, the strain energy in (4) and spring energy in (6) are calculated to construct a total energy function in (7), which is minimized to calculate the new position of the measured points. To obtain a stable strain energy of the 2D measured points, 20 iterations were run for convergence. The experimental results are shown in Figs. 8–13. In Fig. 8, the measured points are marked with blue circles, and the measured points after smoothing are marked with red folding lines. To better graphically display the smoothing results, no more than 200 measured points are shown. The blue circles are uniformly distributed on two sides of the folding line. The same experimental results with respect to the mean-camber line are observed in Fig. 9. However, in some regions of the section curve, there is a large distance deviation (shown with red circles) between the measured points and folding line. The existing Gaussian noise is extremely high in these regions. The energy minimization in Section 3 suppresses the mutation of 2D measured points and g y g g gy minimization in Section 3 suppresses the mutation of 2D measured points and PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 17 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Curve Reconstruction and Mean-Camber Curve Extractio .pone.0115471 December 31, 2014 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 18 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 18 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 12. Variation in the curvature of the measured points after MM (blue) and the folding line (violet) that corresponds to Fig. 8. doi:10.1371/journal.pone.0115471.g012 Fig. 12. Variation in the curvature of the measured points after MM (blue) and the folding line (violet) that corresponds to Fig. 8. Fig. 12. Variation in the curvature of the measured points after MM (blue) and the folding line (violet) that corresponds to Fig. 8. doi:10.1371/journal.pone.0115471.g012 doi:10.1371/journal.pone.0115471.g012 doi:10.1371/journal.pone.0115471.g012 of the pressure surface is steadily negative and provides an increasing curvature value when approaching leading/trailing edges, which is consistent with practice. of the pressure surface is steadily negative and provides an increasing curvature value when approaching leading/trailing edges, which is consistent with practice. The constructed B-spline curves and control points after 20 iterations are shown in Figs. 15–17(a). To observe the constructed results in a quantifiable manner, the geometric errors of the average distance, distance standard error and mini/max error are calculated and shown in Figs. 15–17(b-c). Three continuous B-spline curves are obtained in Section 1–3. In Figs. 15–17(b), the value of the average distance Ave j j is approximately 0.03 mm. The error mainly originates from the measuring uncertainty of the Hexagon laser-scanning equipment. However, in Figs. 15–17(c), the value of Ave j j is reduced to approximately 0.01 mm, which benefits from the energy minimization in Section 3. The strain energy is used to smooth the measured points, and the spring energy is used to prevent a large deviation between an unsmoothed point and smoothed point, and provide a good initial value to iteratively construct a B-spline curve in Section 4. Mean-camber curve extraction Finally, the implementation steps in Section 5 are performed to calculate the mean-camber curves of Section 1–3. The variations in the control points of each cubic B-spline curve are iteratively calculated and revised by minimizing the objective function in (22). Similarly, in this experiment, 20 iterations are performed to obtain the optimal value. The obtained mean-camber curves (blue), envelope circles (black) and maximum gauge circles (violet) are graphically shown in Fig. 18. To accurately calculate the maximum gauge circler, each B-spline curve of the mean-camber curve is scattered into 1,000 points, which are used to generate the envelope circles. To better display the generated envelope circles, approximately 50 points (from 1,000 points) and their corresponding circles are selected and shown in Fig. 18. In Figs. 18(a–c), the radii of the maximum gauge circles are 10.0011, 10.9182 and 11.8530 mm. Three mean-camber curves are successfully extracted, and the envelope circles are sufficiently close to the surrounding section curves. In addition, the curvature distributions of the three mean-camber curves are also calculated and displayed in Fig. 19. From the leading edge to the trailing edge, the curvature variation is rather consistent, which further demonstrates variable of parameter extraction. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Section curve reconstruction The smoothed points in Section 1–3 are used to create three cubic B-spline curves according to (10). By minimizing the objective function in (17), the variations in the control points of the cubic B-spline curve are iteratively calculated and revised. In this experiment, 20 iterations are performed to obtain the optimal variable. The curvature values of the measured points before performing the MM operation (blue), the continuous B-spline curve before the distance minimization (jasper) and the continuous B-spline curve after the distance minimization (red) are obtained. The curvature variations in Section 1–3 are shown in Figs. 14 (a–c). To better display the curvature variation, the region of pressure surface is selected for further analysis. The curvature variation of the measured points oscillates and is unordered, and the curvature variation of the continuous curve before distance minimization (but after energy minimization) is stable around the X-axis. The main problem is that the plus-minus direction of curvature in the pressure region or suction region oscillates and does not maintain a consistent direction in the concave or convex surface. The continuous B-spline curve after distance minimization has the most realistic curvature variation. The curvature direction p minimization has the most realistic curvature variation. The curvature direction 19 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade 0115471 December 31 2014 20 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 20 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 20 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Conclusion Rapid advancements in 3D scanning techniques have led to dense and accurate point clouds from real objects. Point cloud processing, curve approximation and parameter analysis based on 3D scanning have become a topic of considerable 21 / 30 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Fig. 13. Variation in the curvature of the mean-camber points after MM (blue) and the foldi (violet) that corresponds to Fig. 9. doi:10.1371/journal.pone.0115471.g013 Curve Reconstruction and Mean-Camber Curve Extractio Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 13. Variation in the curvature of the mean-camber points after MM (blue) and the folding line (violet) that corresponds to Fig. 9. doi:10.1371/journal.pone.0115471.g013 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 22 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade interest in blade manufacturing. This paper proposes Fig. 14. Curvature variation of the measured points (blue), continuo minimization (green) and continuous curve after distance function doi:10.1371/journal.pone.0115471.g014 Fig. 14. Curvature variation of the measured points (blue), continuous curve before distance function minimization (green) and continuous curve after distance function minimization (red). doi:10.1371/journal.pone.0115471.g014 Fig. 14. Curvature variation of the measured points (blue), continuous curve before distance function minimization (green) and continuous curve after distance function minimization (red). doi:10.1371/journal.pone.0115471.g014 interest in blade manufacturing. This paper proposes a new method to address two common tasks in blade manufacturing: section curve reconstruction and mean-camber curve extraction. The main contributions of the proposed method interest in blade manufacturing. This paper proposes a new method to address two common tasks in blade manufacturing: section curve reconstruction and mean-camber curve extraction. The main contributions of the proposed method PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 23 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 15. Curve reconstruction result in Section 1 after distance minimization: (a) reconstructed B- spline curve and its control points; (b) geometric errors between the measured points and B-spline curve; (c) geometric errors between the energy-smoothed points and B-spline curve. The displays of (b–c) are provided by iCloud3D software, which is developed by our team. doi:10.1371/journal.pone.0115471.g015 Fig. 15. Curve reconstruction result in Section 1 after distance minimization: (a) reconstructed B- spline curve and its control points; (b) geometric errors between the measured points and B-spline curve; (c) geometric errors between the energy-smoothed points and B-spline curve. The displays of (b–c) are provided by iCloud3D software, which is developed by our team. Conclusion doi:10.1371/journal.pone.0115471.g015 doi:10.1371/journal.pone.0115471.g015 include the following. First, the MM is expanded and applied to measured-point processing to restrain the effect of scanning defects. The results demonstrate that the MM implementation can also generate an ordered point sequence and provide an initial value for the curve approximation and parameter extraction. Second, the energy functions of the strain energy and spring energy are built to smooth the 2D measured points while preventing a large distance deviation between the unsmoothed and smoothed points. This implementation process is based on PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 24 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 16. Curve reconstruction result in Section 2 after distance minimization: (a) reconstructed B- spline curve and its control points; (b) geometric errors between the measured points and B-spline curve; (c) geometric errors between the energy-smoothed points and B-spline curve. The displays of (b–c) are provided by iCloud3D software, which is developed by our team. doi:10.1371/journal.pone.0115471.g016 Fig. 16. Curve reconstruction result in Section 2 after distance minimization: (a) reconstructed B- spline curve and its control points; (b) geometric errors between the measured points and B-spline curve; (c) geometric errors between the energy-smoothed points and B-spline curve. The displays of (b–c) are provided by iCloud3D software, which is developed by our team. doi:10.1371/journal.pone.0115471.g016 points and lines and is thus easy to implement. Third, a directed distance function from a measured point to its foot point or an envelope curve is defined and used to build a constrained nonlinear least-squares function. The purpose of this function is to iteratively approximate the cubic B-spline curve and extract the mean-camber curve using distance minimization. In addition, a turbine blade is machined and scanned to implement the experiments. The values of curvature PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 25 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 17. Curve reconstruction result in Section 3 after distance minimization: (a) reconstructed B- spline curve and its control points; (b) geometric errors between the measured points and B-spline curve; (c) geometric errors between the energy-smoothed points and B-spline curve. The displays of (b–c) are provided by iCloud3D software, which is developed by our team. doi:10.1371/journal.pone.0115471.g017 Fig. 17. Conclusion Curve reconstruction result in Section 3 after distance minimization: (a) reconstructed B- spline curve and its control points; (b) geometric errors between the measured points and B-spline curve; (c) geometric errors between the energy-smoothed points and B-spline curve. The displays of (b–c) are provided by iCloud3D software, which is developed by our team. doi:10.1371/journal.pone.0115471.g017 variation, energy variation and approximation error are obtained and analyzed. The experimental results demonstrate the availability of the proposed method. Curve reconstruction and parameter extraction are common problems in blade manufacturing, and the proposed method can find specific applications in the following fields. Future work will focus on practical applications of the proposed method. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 26 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 18. Graphical representation of the mean-camber curves and maximum gauge circles in Section 1–3, respectively. The displays of (a–c) are provided by iCloud3D software, which is developed by our team. doi:10.1371/journal.pone.0115471.g018 Fig. 18. Graphical representation of the mean-camber curves and maximum gauge circles in Section 1–3, respectively. The displays of (a–c) are provided by iCloud3D software, which is developed by our team. Fig. 18. Graphical representation of the mean-camber curves and maximum gauge circles in Section 1–3, respectively. The displays of (a–c) are provided by iCloud3D software, which is developed by our team. doi:10.1371/journal.pone.0115471.g018 doi:10.1371/journal.pone.0115471.g018 1) Aviation casting-blade finish inspection. A turbine blade is typically short (300–600 mm), and a casting technique can be used to directly form its geometric shape. Measurements via a CMM are a common but inefficient inspection strategy in blade manufacturing. In practice, casting-blade finish inspection can be performed based on a 3D scanning method because there is nearly no reflective problem from the casting part. In this application, the calculation of the section curve and parameters is an important task. 1) Aviation casting-blade finish inspection. A turbine blade is typically short (300–600 mm), and a casting technique can be used to directly form its geometric shape. Measurements via a CMM are a common but inefficient inspection strategy in blade manufacturing. In practice, casting-blade finish inspection can be performed based on a 3D scanning method because there is nearly no reflective problem from the casting part. In this application, the calculation of the section curve and parameters is an important task. 2) Large forging-blade allowance inspection. A nuclear blade is rather long (600–2,000 mm). PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 Conclusion A general manufacturing process includes hammer forging, transformation straightening, milling and profile inspection. Mold wear and thermal stress releasing after forging typically result in uncertain blade transformation. Before milling, the uniformity of machining allowance in selected section planes should be inspected. The existing contact snap-gauge inspection based on naked eye/light transmission is not accurate and can be replaced by a 3D scanning method. In this application, the dislocation, mean- camber line and maximum gauge are three main parameters that must be calculated. PLOS ONE \| DOI:10.1371/journal.pone.0115471 December 31, 2014 27 / 30 Curve Reconstruction and Mean-Camber Curve Extraction of Blade Fig. 19. Graphical representation of the curvature value of the mean-camber curves in Section 1–3. doi:10.1371/journal.pone.0115471.g019 Fig. 19. Graphical representation of the curvature value of the mean-camber curves in Section 1–3. doi:10.1371/journal.pone.0115471.g019 doi:10.1371/journal.pone.0115471.g019 3) Visual-guided robot grinding localization. Robot grinding has the advantages of flexible contacting and wide-line machining and can lead to high surface roughness and quality consistency compared to traditional artificial grinding. One important task is to construct a relationship between the blade workpiece in the robot coordinate system and the grinding path in the design coordinate system. The workpiece can be scanned using a laser scanning sensor, and the obtained point cloud is used to register the design model. The registering results can be evaluated using the mean square error and section parameter error to determine whether the grinding allowance is satisfactory. 3) Visual-guided robot grinding localization. Robot grinding has the advantages of flexible contacting and wide-line machining and can lead to high surface roughness and quality consistency compared to traditional artificial grinding. One important task is to construct a relationship between the blade workpiece in the robot coordinate system and the grinding path in the design coordinate system. The workpiece can be scanned using a laser scanning sensor, and the obtained point cloud is used to register the design model. The registering results can be evaluated using the mean square error and section parameter error to determine whether the grinding allowance is satisfactory. Author Contributions Conceived and designed the experiments: WLL HX. Performed the experiments: WLL HX ZPY. Analyzed the data: QDL HX LPZ. Contributed reagents/materials/ analysis tools: WLL HX QDL. Wrote the paper: WLL HX. 1. Hsu TH, Lai JY, Ueng WD, Hwang JZ (2005) An iterative coordinate setup algorithm for airfoil blades inspection. 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https://openalex.org/W2977755584	http://eprints.whiterose.ac.uk/154936/1/ece3.5713.pdf	English	null	Rapid redistribution of agricultural land alters avian richness, abundance, and functional diversity	Ecology and evolution	2,019	cc-by	11,779	Stephen Pringle1 \| Ngoni Chiweshe2 \| Peter R. Steward3 \| Peter J. Mundy2 \| Martin Dallimer3 Martin Dallimer3 1Durrell Institute of Conservation and Ecology, University of Kent, Canterbury, UK 2Forest Resources and Wildlife Management, National University of Science and Technology, Bulawayo, Zimbabwe 3Sustainability Research Institute, School of Earth and Environment, University of Leeds, Leeds, UK O R I G I N A L R E S E A R C H O R I G I N A L R E S E A R C H Abstract The conversion of natural, or seminatural, habitats to agricultural land and changes in agricultural land use are significant drivers of biodiversity loss. Within the con‐ text of land‐sharing versus land‐sparing debates, large‐scale commercial agriculture is known to be detrimental to biodiversity, but the effects of small‐scale subsist‐ ence farming on biodiversity are disputed. This poses a problem for sustainable land‐use management in the Global South, where approximately 30% of farmland is small‐scale. Following a rapid land redistribution program in Zimbabwe, we evalu‐ ated changes in avian biodiversity by examining richness, abundance, and functional diversity. Correspondence Martin Dallimer, Sustainability Research Institute, School of Earth and Environment, University of Leeds, Leeds LS2 9JT, UK. Email: m.dallimer@leeds.ac.uk Funding information Rufford Small Grants Foundation, Grant/ Award Number: 109151‐1 and 14873-2 Correspondence Martin Dallimer, Sustainability Research Institute, School of Earth and Environment, University of Leeds, Leeds LS2 9JT, UK. Email: m.dallimer@leeds.ac.uk Funding information Rufford Small Grants Foundation, Grant/ Award Number: 109151‐1 and 14873-2 Rapid land redistribution has, in the near term, resulted in increased avian abun‐ dance in newly farmed areas containing miombo woodland and open habitat. Conversion of seminatural ranched land to small‐scale farms had a negative impact on larger‐bodied birds, but species richness increased, and birds in some feeding guilds maintained or increased abundance. We found evidence that land‐use change caused a shift in the functional traits of the communities present. However, func‐ tional analyses may not have adequately reflected the trait filtering effect of land redistribution on large species. Whether newly farmed landscapes in Zimbabwe can deliver multiple benefits in terms of food production and habitat for biodiversity in the longer term is an open question. When managing agricultural land transitions, relying on taxonomic meas‐ ures of diversity, or abundance‐weighted measures of function diversity, may ob‐ scure important information. If the value of smallholder‐farmed land for birds is to be maintained or improved, it will be essential to ensure that a wide array of habitat types is retained alongside efforts to reduce hunting and persecution of large bird species. Received: 25 November 2018 \| Revised: 20 August 2019 \| Accepted: 30 August 2019 Received: 25 November 2018 \| Revised: 20 August 2019 \| Accepted: 30 August 2019 DOI: 10.1002/ece3.5713 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Rapid redistribution of agricultural land alters avian richness, abundance, and functional diversity Stephen Pringle1 \| Ngoni Chiweshe2 \| Peter R. Steward3 \| Peter J. Mundy2 \| Martin Dallimer3 Stephen Pringle1 \| Ngoni Chiweshe2 \| Peter R. Steward3 \| Peter J. Mundy2 \| Martin Dallimer3 12260 \| 1 \| INTRODUCTION Furthermore, the theoretical benefits of land sparing have rarely been demonstrated in the field (Fischer et al., 2014). Partial trade‐off analyses that provide support for land sparing ignore real‐ world complexity in terms of the scale or type of farming undertaken (for instance, smallholder‐farmed landscapes form the backbone of global food security (Samberg, Gerber, Ramankutty, Herrero, & West, 2016)). These analyses also fail to account for regional vari‐ ations in how agriculture expands and the associated implications for persistence of biodiversity (Fischer et al., 2014; Tscharntke et al., 2012). Even if land sparing can reduce habitat loss within a system, retention of biodiversity may be less than expected if mechanisms to prevent anthropogenic disturbance are lacking (Barlow et al., 2016; Fischer et al., 2014). Global avian abundance has declined by around a quarter since agriculture became widespread (Gaston, Blackburn, & Goldewijk, 2003). This decline is strongest in intensively farmed areas (Newton, 2004). Where forest is converted to agriculture, diverse or small‐ scale agricultural landscapes may help to mitigate declines in tax‐ onomic measures of bird biodiversity, such as species richness or diversity (Frishkoff et al., 2014; Plexida & Sfougaris, 2015), but this is inconsistently observed (Sinclair, Mduma, & Arcese, 2002; Sinclair, Nkwabi, Mduma, & Magige, 2014; Tscharntke et al., 2008). Even in diverse agricultural landscapes, land‐use change differentially af‐ fects bird species according to their traits. For example, human ac‐ tivity in tropical and subtropical forests reduces the abundance and occurrence of long‐lived, large, nonmigratory, primarily frugivorous or insectivorous forest species (Newbold et al., 2013). It has also been shown to reduce avian functional diversity (Edwards, Edwards, Hamer, & Davies, 2013) and avian phylogenetic diversity (Frishkoff et al., 2014). As the impacts of land‐use change on biodiversity can be context‐specific, it is critical to consider more than just taxo‐ nomic measures of biodiversity. To provide insights into trade‐offs between sub‐Saharan African farming systems and biodiversity, we assess the effects of land‐use change on both taxonomic diversity and functional diversity of avian communities in a Zimbabwean con‐ text. The study system is a land redistribution area where commer‐ cial livestock farming with large seminatural areas (land sparing) has been redistributed and replaced by small‐scale farming where agri‐ cultural production and unfarmed habitats are intermixed (land shar‐ ing). 12260 \| 1 \| INTRODUCTION 12260 PRINGLE et al. (FTLRP) led to the resettlement of eight million hectares between 2000 and 2007 (Moyo & Matondi, 2008). Over the same period, total Zimbabwean agricultural output decreased by 44%, largely due to lower large‐scale commercial production (Clover & Eriksen, 2009). In many areas of Zimbabwe, newly resettled rural commu‐ nities now engage in subsistence agriculture on marginal lands, creating new social, economic, and ecological challenges, such as habitat degradation and loss (Fakarayi, Mashapa, Gandiwa, & Kativu, 2015). In parallel with changes in land tenure and agricul‐ tural practice, by 2007, it was estimated that Zimbabwean wildlife had declined by 60% in national parks, and 50%–80% in conser‐ vancies and game farms (Degeorges & Reilly, 2007). Zimbabwe may illustrate an example of national policies resulting in a shift from land sparing to land sharing, where the displacement of large‐scale commercial production by more mixed smallholder ag‐ riculture has resulted in declines in biodiversity. However, more information is needed to draw robust conclusions. Currently, there is limited information on taxonomic or functional biodiversity re‐ sponses to conversion of natural habitats to small‐scale farming in Africa. This restricts our ability to determine trade‐offs in the re‐ lationship between food production and biodiversity conservation (Tscharntke et al., 2012). A fundamental driver of global biodiversity loss is the conversion of natural habitats to agriculture (Hooper et al., 2012; Vié et al., 2009). It is estimated that the current rate of global human population growth will lead to a 59%–98% rise in food demand between 2005 and 2050 (Valin et al., 2014). Demand for food increases land scar‐ city, which is thought to drive the conversion of natural vegetation to agriculture. This theory has led to much debate, often based on con‐ cepts of land sharing and land sparing, to examine the trade‐offs be‐ tween food production and biodiversity conservation (Phalan, Onial, Balmford, & Green, 2011; Tscharntke et al., 2012). For instance, evidence from Ghana, India, Uganda, and Borneo suggests that “land‐sparing” landscapes, with segregated areas of agriculture and nonfarmed habitat, are more likely to meet both goals as compared to “land‐sharing” landscapes, where agriculture and natural habitats are interspersed (Edwards et al., 2010; Hulme et al., 2013; Phalan et al., 2011). However, the environmental costs of agriculture are often overlooked and the impacts on functional biodiversity across farmed landscapes are often poorly understood (Tscharntke et al., 2012). K E Y W O R D S biodiversity conservation, land sharing, land sparing, land‐use change, smallholder farming, Zimbabwe This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 Th A th E l d E l ti bli h d b J h Wil & S Ltd \| 12259 www.ecolevol.org 2.1 \| Study system and survey method The study area is located on a 91,000‐ha area of central southern Zimbabwe (29°34′E, 20°04′S) that was formerly in private owner‐ ship, and originally used for cattle and game ranching (Figures 1‒3). When in private ownership, the entire ranch was characterized by areas of open habitat, miombo (dominated by Brachystegia spp.) and acacia woodland (dominated by Acacia spp.). During the FTLRP, the Zimbabwean government acquired around 72% of the ranched area for redistribution, leaving the remaining land to the original owners. In a rapid resettlement program during 2001–2002, around 3,000 families were moved into allocated 5‐ to 6‐ha plots spread through‐ out the redistributed area. Retention of land by original owners and the continuation of land management practices on that land were unusual during the FTLRP. The study area therefore offers a unique insight into how the avian community has responded to shifts in land tenure and land use. Land that did not change ownership is still uti‐ lized entirely for cattle and game ranching (hereafter referred to as “ranched areas”). The redistributed land is now used for smallholder mixed livestock and arable subsistence farming (farmed areas). At the time of this study, lands in both ownership categories contin‐ ued to contain extensive areas of open habitat, miombo and acacia woodland (Figure 3). The open habitat within the farmed area con‐ tained both small cropped fields close to homesteads, and grassland; in the ranched area, there was only grassland. In both farmed and ranched areas, livestock are grazed through all habitat types, but feed mainly in the open habitat grasslands and fallow fields. Bird surveys were carried out shortly after sunrise, or before sunset, in fine weather with good visibility. When two sites were counted on the same day, they were selected from different habitat types, and situated as far apart as possible. Each site was surveyed twice, once in winter (June 2012), when resident birds dominate and Palaearctic migrants are absent, and once in summer (January 2013). Two 600‐m parallel transects, spaced 300 m apart, were walked at constant slow speed; mean transect duration was 118 min (SD 16.7). In total, 108 km of transects was surveyed. Data recorded for each bird species observation were as follows: the number of individuals, the angle of deviation from the transect direction, and the distance from the observer (measured using a Leica LRF1200 rangefinder). 2.1 \| Study system and survey method Care was taken to avoid double‐counting birds and to exclude those overflying. Only observations within 100 m of transects were con‐ sidered to represent birds using the survey site. While carrying out transects, the number of people, homesteads, dogs, cut trees, crops, and cattle were also recorded as indicators of the level of human activities. open sites were surveyed, as this was the main habitat type. Wooded sites were categorized as acacia (Acacia spp. dominant) or miombo woodlands (Brachystegia spp. dominant). In total, 45 sites were sur‐ veyed: 23 ranched (acacia n = 5, miombo n = 7, open n = 11) and 22 farmed (acacia n = 5, miombo n = 6, open n = 11). The distances (mean; SD; closest) between ranched and farmed sites of the same habitat type were as follows: acacia, (16.1; 3.2; 3.5) km; miombo, (13.3; 1.8; 3.4) km; and open, (11.2; 1.1; 3.6) km. open sites were surveyed, as this was the main habitat type. Wooded sites were categorized as acacia (Acacia spp. dominant) or miombo woodlands (Brachystegia spp. dominant). In total, 45 sites were sur‐ veyed: 23 ranched (acacia n = 5, miombo n = 7, open n = 11) and 22 farmed (acacia n = 5, miombo n = 6, open n = 11). The distances (mean; SD; closest) between ranched and farmed sites of the same habitat type were as follows: acacia, (16.1; 3.2; 3.5) km; miombo, (13.3; 1.8; 3.4) km; and open, (11.2; 1.1; 3.6) km. 12260 \| 1 \| INTRODUCTION Specifically, we test the hypotheses that: (a) the redistribution of land results in decreased avian species richness and diversity; (b) the redistribution of land results in a decline in large‐bodied species; and (c) changes in (a) and (b) will be reflected in a shift in the func‐ tional traits of communities present. Land‐use change is generally driven by increasing demand for agricultural commodities and land scarcity. However, it may also be driven by policies designed to address major societal issues, such as poverty and fair access to land, through socioeconomic change (Chappell et al., 2013). Major socioeconomic changes are generally accompanied by rapid land‐use change in rural areas. In sub‐Saharan Africa, historical patterns of inequitable land distri‐ bution are key factors linking impoverished rural livelihoods, food security, and food sovereignty (Clover & Eriksen, 2009). Several countries in eastern, central, and southern Africa have imple‐ mented land tenure reform policies (Clover & Eriksen, 2009). In Namibia, semiarid savanna redistributed to land reform beneficia‐ ries is often farmed in small units by settlers with limited farm‐ ing experience (Lohmann, Falk, Geissler, Blaum, & Jeltsch, 2014). Assessment of the ecological implications of Namibian land re‐ settlement by small‐scale farmers suggests it is sustainable in the short term, with no savanna degradation due to bush encroach‐ ment (Lohmann et al., 2014). However, the effects of land tenure changes over longer timescales may be less sustainable (Dougill et al., 2016). In Zimbabwe, the Fast‐Track Land Reform Programme PRINGLE et al. 12261 2.2 A database of traits was compiled using data from publications list‐ ing the relevant Zimbabwean subspecies (Brown, Urban, & Newman, 1982; Fry, Keith, & Urban, 1988, 2000; Fry & Keith, 2004; Keith, Urban, & Fry, 1992; Urban, Fry, & Keith, 1986, 1997). Identification at subspecies level is of importance in selecting the correct mensu‐ ral trait data for our subsequent analyses. The database comprised seven traits for each species: five measurements of morphology (average adult body mass, and lengths of wing, tail, bill, and tar‐ sus), feeding guild (frugivore, granivore, insectivore, nectarivore, or predator), and migratory behavior (resident, intra‐African migrant, or Palaearctic migrant). Species were also classified into primary feed‐ ing guilds (De Graaf, Tilghman, & Anderson, 1985), which added an omnivore category to the above five guilds (Table S1). Bird surveys were carried out along linear transects, which were selected with random start points and orientations from Google Earth images. Transects were chosen to be as homogenous as pos‐ sible between sites, but with no other selection criteria. The relative abundance of different habitat types in ranched and farmed areas was taken into account in selecting sites. Thus, a greater number of FI G U R E 1 Homesteads in the resettled lands of the study area in central southern Zimbabwe. Photo: Ngoni Chiweshe Species richness (SR) was estimated using EstimateS 9.1.0 soft‐ ware (Colwell, 2013) to analyze individual‐based count data by land use (ranched or farmed), season (winter or summer), and habitat type (open habitat, acacia woodland, or miombo woodland). In estimating SR, no corrections were made to take account of species‐ or habitat‐ dependent variations in detection probabilities. Species accumula‐ tion, interpolation (rarefaction), and extrapolation curves were used to evaluate sampling adequacy and to calculate Chao1 estimators of species richness. The ecological significance of differences in spe‐ cies richness between land uses was assessed by comparing 95% confidence intervals (CIs) and effect sizes. FI G U R E 1 Homesteads in the resettled lands of the study area in central southern Zimbabwe. Photo: Ngoni Chiweshe 12262 \| PRINGLE et al. FI G U R E 2 The study area (Debshan) in Africa and location within Zimbabwe showing the location of the survey transect sites FI G U R E 3 Typical transect sites in mid‐summer (a‐b) and mid‐winter (c‐f). 2.2 In both ranched (a, c, f) and farmed areas (b, d, e), livestock and game animals graze freely throughout open habitats (a‐d), acacia woodlands (e), and miombo woodlands (f). In summer, small arable fields adjacent to homesteads in the farmed open habitats (b) are cropped at a subsistence level to provide the annual food supplies for resettled families. Photos: Ngoni Chiweshe (a‐b); Martin Dallimer (c–f) PRINGLE et al. 12262 \| 12262 FI G U R E 2 The study area (Debshan) in Africa and location within Zimbabwe showing the location of the survey transect sites FI G U R E 2 The study area (Debshan) in Africa and location within Zimbabwe showing the location of the survey transect sites Bird population densities, corrected for detection probabilities, were estimated using Distance 7.1 software (Thomas et al., 2010). Conventional Distance Sampling mode was used, with two modeling options: half normal functions with Cosine series expansion and uni‐ form functions with simple polynomial series expansion (Buckland et al., 2001). The most parsimonious model solution was chosen using Akaike's information criterion (Buckland et al., 2001). Density esti‐ mates, stratified by land use, season, and habitat, were calculated from the counts for: (a) all species grouped by migratory behavior; (b) all species grouped by primary feeding guild; (c) all species grouped into adult body mass ranges (as proxies for bird sizes); (d) common species (i.e., those with >60 detections); and (e) rarer species (<60 FI G U R E 3 Typical transect sites in mid‐summer (a‐b) and mid‐winter (c‐f). In both ranched (a, c, f) and farmed areas (b, d, e), livestock and game animals graze freely throughout open habitats (a‐d), acacia woodlands (e), and miombo woodlands (f). In summer, small arable fields adjacent to homesteads in the farmed open habitats (b) are cropped at a subsistence level to provide the annual food supplies for resettled families. Photos: Ngoni Chiweshe (a‐b); Martin Dallimer (c–f) FI G U R E 3 Typic mid‐summer (a‐b) an both ranched (a, c, f (b, d, e), livestock an graze freely through (a‐d), acacia woodlan woodlands (f). 2.2 In sum fields adjacent to ho farmed open habitat a subsistence level t food supplies for res Photos: Ngoni Chiw Dallimer (c–f) FI G U R E 3 Typical transect sites mid‐summer (a‐b) and mid‐winter (c both ranched (a, c, f) and farmed ar (b, d, e), livestock and game animals graze freely throughout open habit (a‐d), acacia woodlands (e), and mio woodlands (f). In summer, small ara fields adjacent to homesteads in th farmed open habitats (b) are cropp a subsistence level to provide the a food supplies for resettled families Photos: Ngoni Chiweshe (a‐b); Mar Dallimer (c–f) FI G U R E 3 Typical transect sites in mid‐summer (a‐b) and mid‐winter (c‐f). In both ranched (a, c, f) and farmed areas (b, d, e), livestock and game animals graze freely throughout open habitats (a‐d), acacia woodlands (e), and miombo woodlands (f). In summer, small arable fields adjacent to homesteads in the farmed open habitats (b) are cropped at a subsistence level to provide the annual food supplies for resettled families. Photos: Ngoni Chiweshe (a‐b); Martin Dallimer (c–f) Bird population densities, corrected for detection probabilities, were estimated using Distance 7.1 software (Thomas et al., 2010). Conventional Distance Sampling mode was used, with two modeling options: half normal functions with Cosine series expansion and uni‐ form functions with simple polynomial series expansion (Buckland et al., 2001). The most parsimonious model solution was chosen using Bird population densities, corrected for detection probabilities, were estimated using Distance 7.1 software (Thomas et al., 2010). Conventional Distance Sampling mode was used, with two modeling options: half normal functions with Cosine series expansion and uni‐ form functions with simple polynomial series expansion (Buckland et al., 2001). The most parsimonious model solution was chosen using Akaike's information criterion (Buckland et al., 2001). Density esti‐ mates, stratified by land use, season, and habitat, were calculated from the counts for: (a) all species grouped by migratory behavior; (b) all species grouped by primary feeding guild; (c) all species grouped into adult body mass ranges (as proxies for bird sizes); (d) common species (i.e., those with >60 detections); and (e) rarer species (<60 Akaike's information criterion (Buckland et al., 2001). 2.2 Density esti‐ mates, stratified by land use, season, and habitat, were calculated from the counts for: (a) all species grouped by migratory behavior; (b) all species grouped by primary feeding guild; (c) all species grouped into adult body mass ranges (as proxies for bird sizes); (d) common species (i.e., those with >60 detections); and (e) rarer species (<60 \| 12263 PRINGLE et al. FI G U R E 4 Avian species richness (SR) by habitats in farmed and ranched sites by season. SR values are individual‐ based rarefaction estimates calculated from count data using EstimateS V9.1.0 software. Error bars represent 95% confidence intervals. Legends in bold are those where the difference in SR between farmed and ranched sites in the same season and habitat type shows an effect size > 1 0 20 40 60 80 100 120 140 Species richness Open habitat farmed - Summer Open habitat ranched - Summer Open habitat farmed - Winter Open habitat ranched - Winter Miombo woodland farmed - Summer Miombo woodland ranched - Summer Miombo woodland farmed - Winter Miombo woodland ranched - Winter Acacia woodland farmed - Summer Acacia woodland ranched - Summer Acacia woodland farmed - Winter Acacia woodland ranched - Winter \| 12263 0 20 40 60 80 100 120 140 Species richness Open habitat farmed - Summer Open habitat ranched - Summer Open habitat farmed - Winter Open habitat ranched - Winter Miombo woodland farmed - Summer Miombo woodland ranched - Summer Miombo woodland farmed - Winter Miombo woodland ranched - Winter Acacia woodland farmed - Summer Acacia woodland ranched - Summer Acacia woodland farmed - Winter Acacia woodland ranched - Winter PRINGLE et al. 12263 FI G U R E 4 Avian species richness (SR) by habitats in farmed and ranched sites by season. SR values are individual‐ based rarefaction estimates calculated from count data using EstimateS V9.1.0 software. Error bars represent 95% confidence intervals. Legends in bold are those where the difference in SR between farmed and ranched sites in the same season and habitat type shows an effect size > 1 traits were not unduly weighted in those species assigned to more than one guild (Laliberté et al., 2014). Abundance‐weighted func‐ tional trait indices for the avian communities for each land use and habitat combination were calculated in R software (R Development Core Team, 2016) using the “fundiv” package. FI G U R E 4 Avian species richness (SR) by habitats in farmed and ranched sites by season. SR values are individual‐ based rarefaction estimates calculated from count data using EstimateS V9.1.0 software. Error bars represent 95% confidence intervals. Legends in bold are those where the difference in SR between farmed and ranched sites in the same season and habitat type shows an effect size > 1 2.2 Trait–species distance calculations were performed using Gower's similarity function to allow incorporation of trait types of mixed scales (Podani & Schmera, 2006). detections) grouped by prominence (defined in Table S2). Biomass estimates for every species were calculated by multiplying species‐ level population density estimates by the average adult body mass of each species. These species‐level biomass estimates were subse‐ quently grouped into biomasses of each feeding guild. The similarity of avian assemblages according to land use, strat‐ ified by habitat, was visualized by nonmetric ordination plots using PAST 3.10 software (Hammer et al., 2001). All species' count data were Log10 (x + 1)‐transformed before running Bray–Curtis distance analyses adjusted for missing data. Bray–Curtis residual distances and projection geometry were used to generate ordination plots onto the first two axes of three‐dimensional fits. For each feeding guild, a correlation coefficient (represented as a vector from the ori‐ gin) was calculated between the guild and the ordination score. One‐ way analyses of similarity (ANOSIM) tests using the Bray–Curtis index were run with 9,999 permutations. The functional diversity (FD) index is closely related to species richness (Petchey & Gaston, 2006) and does not fully represent the multifaceted aspects of community functional diversity (Villéger, Mason, & Mouillot, 2008). To alleviate these limitations, three inde‐ pendent functional indices are often used to quantify the relation‐ ships between species' abundances, their traits, and trait variability (Villéger et al., 2008). For each community, we calculated functional diversity (FD), functional evenness (Feve), and functional divergence (Fdiv). These are, respectively: the dispersion of an assemblage of species in trait space; the regularity of distribution in trait space of species weighted by their abundances; and the proportion of total abundance in the assemblage formed by those species with the most extreme trait values (Mouillot, Graham, Villéger, Mason, & Bellwood, 2013; Petchey & Gaston, 2006; Villéger et al., 2008). The indices are constrained to the range 0–1. Values for a fourth index, functional richness, were not calculated, as species richness was below the ex‐ ponential of the number of traits included in the analyses (Villéger et al., 2008). 2.3 \| Functional traits' analyses The impact of land‐use change on functional traits' diversity was an‐ alyzed using the population density estimates, stratified by land use and habitat (with summer and winter counts combined), and the da‐ tabase of bird traits (Table S1). Traits were selected to reflect key as‐ pects of resource usage that drive ecosystem functions (Şekercioğlu, 2006). Body metrics were used to represent rate of resource con‐ sumption (mass), foraging mode and behavior (bill and tarsus), and flight range for resource access and dispersal (wing and tail). Prior to analyses, values for each morphometric trait were standardized to a mean value of zero and unit standard deviation, thus weighting these traits equally. Morphological metrics were defined using continuous scales of millimeters or grams. Feeding guilds are relevant in terms of ecosystem services (e.g., seed dispersal, pollination), population con‐ trol and resource removal (e.g., insectivore and predator–prey cap‐ ture), and nutrient recycling (e.g., fecal deposition). Binary feeding guilds were each assigned a 1/5 weighting; this ensured that these TA B LE 1 Bird densities by season, habitat, and land use for resident and migrant species, based on counts corrected for detection probabilities Season Migratory behavior Acacia woodland Miombo woodland Open habitat Ranched (120 ha) Farmed (120 ha) Ranched (168 ha) Farmed (144 ha) Ranched (264 ha) Farmed (264 ha) Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI TA B LE 1 Bird densities by season, habitat, and land use for resident and migrant species, based on counts corrected for detection probabilities Season Migratory behavior Acacia woodland Miombo woodland Open habitat Ranched (120 ha) Farmed (120 ha) Ranched (168 ha) Farmed (144 ha) Ranched (264 ha) Farmed (264 ha) Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Summer Resident 6.32 5.29–7.55 7.59 6.27–9.18 3.70 2.74–5.00 3.75 2.96–4.74 3.84 3.23–4.57 4.97 4.23–5.85 Effect size 1.75 0.08 2.74 Intra‐African migrant 0.22 0.11–0.45 0.37 0.13–1.08 0.28 0.09–0.92 0.13 0.07–0.26 0.33 0.17–0.64 0.08 0.04–0.17 Effect size 0.56 0.61 2.15 Palaearctic migrant 0.83 0.49–1.38 0.84 0.53–1.31 0.51 0.27–0.97 0.48 0.22–1.04 1.21 0.88–1.65 1.02 0.72–1.45 Effect size 0.04 0.12 0.86 Winter Resident 5.38 4.37–6.61 8.66 6.85–10.95 1.87 1.37–2.57 4.77 3.83–5.93 1.83 1.47–2.28 4.92 3.88–6.25 Effect size 3.50 6.06 6.10 Intra‐African migrant 0 0 0 0 0 0 0 0 0.07 0.02–0.29 0.81 0.17–3.90 Effect size na na 0.66 Palaearctic migrant 0 0 0 0 0 0 0 0 0 0 0 0 Effect size na na na Note: In winter, intra‐African migrants were present only in the open habitat, and Palaearctic migrants were absent. Effect size (ES) values for each category of migratory behavior are calculated for bird densities in the same season and habitat type, but different land uses. Values that may indicate ecologically significant differences in densities are highlighted (blue, ES = 0.8–1.0; red, ES > 1.0). homesteads (100 vs. 4); and dogs (64 vs. 1). In addition, cut trees were more common (evidence seen on 21 farmed sites vs. 1 ranched site). All ranched sites were uncultivated, whereas diverse crops in‐ cluding maize, sugar beans, groundnuts, roundnuts, finger millet, and gourds were present in 20 farmed sites. Similar total numbers of cat‐ tle were observed in each land‐use type (509 vs. 495). homesteads (100 vs. 4); and dogs (64 vs. 1). In addition, cut trees were more common (evidence seen on 21 farmed sites vs. 1 ranched site). All ranched sites were uncultivated, whereas diverse crops in‐ cluding maize, sugar beans, groundnuts, roundnuts, finger millet, and gourds were present in 20 farmed sites. Similar total numbers of cat‐ tle were observed in each land‐use type (509 vs. 495). In ranched sites, a total of 3,066 individuals of 136 species were counted, compared with 3,702 individuals of 155 species in farmed sites. TA B LE 1 Bird densities by season, habitat, and land use for resident and migrant species, based on counts corrected for detection probabilities Season Migratory behavior Acacia woodland Miombo woodland Open habitat Ranched (120 ha) Farmed (120 ha) Ranched (168 ha) Farmed (144 ha) Ranched (264 ha) Farmed (264 ha) Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Rarefaction curves of birds recorded in each season, habitat, and land‐use type (Figure S1) indicated that adequate sampling was achieved. The potential ecological significance of changes in SR in relation to land use was assessed in terms of 95% CIs and where effect sizes > 1 (Figure 4). Species richness estimates indicated that, in austral winter, open habitats in the farmed land held significantly more species than ranched open habitats, while the reverse was true for acacia woodlands. During austral summer, species richness in open habitats and miombo woodlands in farmed areas was higher than in these habitats on ranched land. Densities of resident and migrant birds varied according to hab‐ itat and land use (Table 1). With one exception (miombo woodlands, in summer), farmed habitats of all types held significantly higher den‐ sities of resident birds in both winter and summer than were present during these seasons in ranched habitats. In contrast, intra‐African migrants showed a significant preference for ranched, compared with farmed, open habitat in summer. Palaearctic migrants were present only in summer; these birds showed no land‐use affinities. 3 \| RESULTS While walking transects, we observed that farmed sites (n = 22) showed substantially higher impacts from human use than those that were ranched (n = 23). The following indications of impact are for all habitats combined: higher numbers of people (totals of 193 vs. 10 on all farmed sites and all ranched sites, respectively); 12264 PRINGLE et al. TA B LE 2 Estimated bird densities for winter and summer combined, categorized by primary feeding guild, habitat and land use, based on counts corrected for detection probabilities Feeding guild Acacia woodland Miombo woodland Open habitat Ranched (120 ha) Farmed (120 ha) Ranched (168 ha) Farmed (144 ha) Ranched (264 ha) Farmed (264 ha) Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Frugivore 1.02 0.75–1.40 1.10 0.80–1.53 0.39 0.25–0.62 0.46 0.30–0.71 0.35 0.24–0.52 0.46 0.33–0.63 Effect size 0.39 0.57 1.27 Granivore 0.79 0.57–1.09 2.11 1.40–3.18 0.43 0.30–0.63 1.36 0.96–1.93 0.86 0.59–1.25 1.41 1.07–1.86 Effect size 3.29 4.43 2.56 Insectivore 3.40 2.86–4.05 3.85 3.23–4.59 1.87 1.47–2.40 2.23 1.77–2.82 2.07 1.75–2.42 2.80 2.43–3.23 Effect size 1.27 1.26 3.65 Nectarivore 0.16 0.09–0.27 0.15 0.07–0.30 0.32 0.17–0.61 0.14 0.07–0.26 0.09 0.04–0.18 0.06 0.04–0.11 Effect size 0.15 1.54 0.81 Omnivore 1.37 1.03–1.81 1.05 0.69–1.60 0.35 0.25–0.49 0.58 0.41–0.82 0.38 0.28–0.53 0.84 0.60–1.16 Effect size 1.26 2.35 3.61 Predator 0.05 0.03–0.11 0.01 0.00–0.04 0.05 0.02–0.14 0.16 0.05–0.49 0.04 0.02–0.08 0.08 0.05–0.13 Effect size 1.65 0.93 1.73 Note: Effect size (ES) values for each feeding guild are calculated for bird densities in the same habitat type, but different land uses. Values that may indicate ecologically significant differences in densities are highlighted (blue, ES = 0.8–1.0; red, ES > 1.0). eding guild are calculated for bird densities in the same habitat type, but different land uses. Values that may indicate ecologically significant differences in densities are highlighted (blue, Effect size 1.65 0.93 1.73 Note: Effect size (ES) values for each feeding guild are calculated for bird densities in the same habitat type, but different land uses. Values that may indicate ecologically significant differences in densities are highlighted (blue, ES = 0.8–1.0; red, ES > 1.0). Estimated bird densities for winter and summer combined, categorized by primary feeding guild, habitat and land use, based on counts corrected for detection probabilities Acacia woodland Miombo woodland Open habitat Ranched (120 ha) Farmed (120 ha) Ranched (168 ha) Farmed (144 ha) Ranched (264 ha) Farmed (264 ha) Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI eding guild, habitat and land use, based on counts corrected for detection probabilities Nonmetric ordination plots show distinct groupings of sites when stratified by land use, suggesting dissimilarities between avian assemblages present on farmed and ranched land (Figure 7). ANOSIM tests confirm that these differences in the bird commu‐ nities were significant in all habitats combined (top left, R = .069, p = .036), in miombo woodland (bottom left, R = .156, p = .042), and open habitat (bottom right, R = .223, p = .002), but not in acacia woodland (top‐right, R = .056, p = .373). The feeding guild arrows overlaid onto the ordination plots show predators were more asso‐ ciated with ranched, not farmed, habitats. The arrows also highlight the differential impact of land transformation on avian commu‐ nities in acacia woodland compared with miombo woodland and open habitats. Whereas the abundance of frugivores, insectivores, nectarivores, and omnivores in miombo and open areas increased in farmed land, the reverse was true of acacia woodland birds. However, R‐statistic values (.056–.223) are low, indicating relatively even dissimilarities within and between the land uses (Clarke, 1993). 3.1 \| Functional traits' index responses Land‐use change from ranched to farmed management appears to have impacted functional traits' indices most strongly in bird com‐ munities in acacia woodlands (Table 4). Effect sizes > 1 suggest that ecologically significant changes have occurred in the diversity (de‐ creased), and evenness (increased), of bird traits in farmed acacia woodland that had previously been ranched. There are also weaker indications (ES < 1) of change in the divergence of traits in birds re‐ corded in farmed acacia woodlands (increased) and open habitats (decreased), compared with those ranched habitats. Miombo woodland In general, densities stratified by primary feeding guild (Table 2) were maintained or increased in farmed land that had previously been ranched. This was observed for granivores and insectivores in all habitat types; for frugivores, omnivores, and predators in open habitat; and for omnivores in miombo woodland. This trend was re‐ versed for nectarivores in miombo woodland, and for omnivores and predators in ranched acacia woodland. Avian biomass stratified by habitat and land use showed numer‐ ous significant differences according to feeding guilds, with differ‐ ences in population densities of larger birds being a major factor (Table 3). Despite being present in relatively low numbers (which precluded detailed analyses of their data at species level), density variation (or absence) of six large species of omnivores and preda‐ tors dominated the total avian biomass in each habitat and land use (Table S3). No breeding colonies, or roosts, of any bird species were en‐ countered in our transect counts. With the exception of Red‐billed Quelea Quelea quelea, no other species recorded in our counts oc‐ curred in large flocks (>30 individuals). Even in this species, only 164 individuals were counted across all transects in both seasons combined. Of the 179 bird species recorded, 32 were present in large enough numbers (>60 individuals) to permit analyses of pop‐ ulation density differences by land‐use category at species level (Figure 5). These common birds represented 67.6% of the total number of individuals counted. Fourteen species occurred at higher densities (effect sizes > 1) in farmed sites, five species showed the opposite pattern (effect sizes > 1), and densities were unchanged PRINGLE et al. 12265 TA B LE 2 Estimated bird densities for winter and summer combined, categorized by primary feeding guild, habitat and land use, based on counts corrected for detection probabilities Feeding guild Acacia woodland Miombo woodland Open habitat Ranched (120 ha) Farmed (120 ha) Ranched (168 ha) Farmed (144 ha) Ranched (264 ha) Farmed (264 ha) Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Birds/ha 95% CI Frugivore 1.02 0.75–1.40 1.10 0.80–1.53 0.39 0.25–0.62 0.46 0.30–0.71 0.35 0.24–0.52 0.46 0.33–0.63 Effect size 0.39 0.57 1.27 Granivore 0.79 0.57–1.09 2.11 1.40–3.18 0.43 0.30–0.63 1.36 0.96–1.93 0.86 0.59–1.25 1.41 1.07–1.86 Effect size 3.29 4.43 2.56 Insectivore 3.40 2.86–4.05 3.85 3.23–4.59 1.87 1.47–2.40 2.23 1.77–2.82 2.07 1.75–2.42 2.80 2.43–3.23 Effect size 1.27 1.26 3.65 Nectarivore 0.16 0.09–0.27 0.15 0.07–0.30 0.32 0.17–0.61 0.14 0.07–0.26 0.09 0.04–0.18 0.06 0.04–0.11 Effect size 0.15 1.54 0.81 Omnivore 1.37 1.03–1.81 1.05 0.69–1.60 0.35 0.25–0.49 0.58 0.41–0.82 0.38 0.28–0.53 0.84 0.60–1.16 Effect size 1.26 2.35 3.61 Predator 0.05 0.03–0.11 0.01 0.00–0.04 0.05 0.02–0.14 0.16 0.05–0.49 0.04 0.02–0.08 0.08 0.05–0.13 Effect size 1.65 0.93 1.73 Note: Effect size (ES) values for each feeding guild are calculated for bird densities in the same habitat type, but different land uses. Values that may indicate ecologically significant differences in densities are highlighted (blue, ES = 0.8–1.0; red, ES > 1.0). (effect sizes < 1) in 13 species. In terms of body mass dependence (Figure 6), larger birds with mass >150 g were negatively affected by land‐use change, while smaller birds were present at higher densities (all effect sizes > 1). TA B LE 3 Estimated avian biomass for winter and summer combined, categorized by primary feeding guild, habitat and land use, based on counts corrected for detection probabilities Acacia woodland Miombo woodland Open habitat Major factors determining change in biomass Ranched Farmed Ranched Farmed Ranched Farmed Frugivore (n) 163 153 70 81 145 206 Frugivore bio‐ mass (g/ha) 94.8 118.1 45.4 41.1 37.0 48.0 Frugivore bio‐ mass (95% CI) 81.7–110.1 103.1–135.3 33.1–62.9 30.6–56.8 30.2–46.2 40.4–57.8 Effect size 2.81 0.50 2.27 Granivore (n) 122 213 66 157 224 569 Granivore bio‐ mass (g/ha) 40.4 73.4# 33.7 51.0# 43.1 60.2# More waxbills, doves, bishops, widowbirds# Granivore bio‐ mass (95% CI) 33.3–49.4 61.9–87.6 23.1–48.9 35.3–75.8 31.6–59.0 51.6–71.0 Effect size 5.44 1.68 2.47 Insectivore (n) 493 432 314 316 853 1,022 Insectivore bio‐ mass (g/ha) 145.7 138.9 58.4 67.9 94.7 123.9 Most small–medium size spe‐ cies increased Insectivore bio‐ mass (95% CI) 118.9–189.1 115.9–167.7 44.5–78.8 53.3–91.3 81.4–112.7 102.0–162.0 Effect size 0.36 0.85 1.92 Nectarivore (n) 24 19 31 17 20 26 Nectarivore bio‐ mass (g/ha) 2.0 2.1 1.7 1.2 0.6 0.9 Nectarivore bio‐ mass (95% CI) 1.8–2.3 1.9–2.4 1.5–2.0 1.0–1.4 0.5–0.7 0.8–1.0 Effect size 0.65 3.78 5.88 Omnivore (n) 241 119 81 105 176 233 Kori Bustard (n = 3) only in ranched area† Omnivore bio‐ mass (g/ha) 721.5† 55.5†† 47.5 44.3 108.5 45.0†† Far fewer francolins, spurfowl, guineafowl†† Omnivore bio‐ mass (95% CI) 592.1–895.7 48.3–63.7 33.9–67.1 32.8–61.4 103.8–115.0 39.2–53.7 Effect size 10.59 0.34 16.21 Predator (n) 10 0 16 8 17 26 Detection probability = 1 from Distance 7.1* Predator biomass (g/ha) 85.5 0.0‡ 221.8 41.6‡ 28.7‡‡ 69.1 No White‐backed Vulture, African Fish Eagle‡ Predator biomass (95% CI) 85.5–85.5* 0.0–0.0 221.8– 221.8* 23.3–74.7 28.7–28.7 52.1–91.6 No Black‐headed Heron, secretarybird‡‡ Effect size na 15.83 4.98 Note: Effect size (ES) values for each feeding guild are calculated for avian biomasses in the same habitat type, but different land uses. Values that may indicate ecologically significant differences in densities are highlighted (blue, ES = 0.8–1.0; red, ES > 1.0). TA B LE 3 Estimated avian biomass for winter and summer combined, categorized by primary feeding guild, habitat and land use, based on counts corrected for detection probabilities Note: Effect size (ES) values for each feeding guild are calculated for avian biomasses in the same habitat type, but different land uses. 4 \| DISCUSSION Land‐use change, sometimes driven by land reform programs, can have substantial impacts on biodiversity (Chappell et al., 2013). We found significant changes (as indicated by effect sizes) in the bird communities in an area where land previously used for cattle and wildlife grazing was converted to arable and mixed livestock farm‐ ing by smallholders. The effects on species richness, population densities, diversity, and functional trait distribution were complex. Most, but not all, species and functional groups were positively, or neutrally, affected by land‐use change. In farmed open habitats (ar‐ able areas and grasslands), and in miombo woodlands, there were increases in species richness, population densities, and biomasses of most feeding guilds. The impact on bird communities in farmed acacia woodland areas that had been ranched was more varied; den‐ sities of resident birds increased, but while densities of some feed‐ ing guilds increased, others decreased. In both summer and winter, farmed open sites hosted more species than equivalent ranched 12266 PRINGLE et al. Values that may indicate ecologically significant differences in densities are highlighted (blue, ES = 0.8–1.0; red, ES > 1.0). Note: Effect size (ES) values for each feeding guild are calculated for avian biomasses in the same habitat type, but different land uses. Values that may indicate ecologically significant differences in densities are highlighted (blue, ES = 0.8–1.0; red, ES > 1.0). sites; however, ranched, not farmed, acacia woodlands hosted more species in winter. for pollinators, see Hagen & Kraemer, 2010; Kasina, 2007; Otieno et al., 2011, but see Gemmill‐Herren & Ochieng, 2008; Sande, Crewe, Raina, Nicolson, & Gordon, 2009). In our study, it thus far appears that these mixed systems, which are representative of a land‐sharing scenario, are allowing bird biodiversity to persist within the agricul‐ tural matrix. However, agricultural yields are likely to be low com‐ pared to conventional practices, potentially meaning that more land would be required for the same amount of commodity production. The introduction of smallholder mixed farming to previously ranched land may have increased the complexity of the landscape at a spatial scale that benefits most bird species. High landscape complexity in African agroecosystems can maintain species' den‐ sities, richness, and abundance in relation to natural habitats (for birds, see Gove, Hylander, Nemomissa, Shimelis, & Enkossa, 2013; 12267 PRINGLE et al. \| 12267 PRINGLE et al. FI G U R E 5 Ratios of densities, for all seasons and habitats combined, of common (n > 60 individuals) species recorded in farmed sites compared with ranched sites. Ratios > 1 indicate higher density in farmed sites. Error bars represent 95% confidence intervals around the mean ratio values. Species' labels in red are those where the density difference between farmed and ranched sites shows an effect size > 1. Photos: Stephen Pringle (left: Swainson's Spurfowl; right: Yellow‐fronted Canary) FI G U R E 5 Ratios of densities, for all seasons and habitats combined, of common (n > 60 individuals) species recorded in farmed sites compared with ranched sites. Ratios > 1 indicate higher density in farmed sites. Error bars represent 95% confidence intervals around the mean ratio values. Species' labels in red are those where the density difference between farmed and ranched sites shows an effect size > 1. Photos: Stephen Pringle (left: Swainson's Spurfowl; right: Yellow‐fronted Canary) While the effect of land redistribution in this study area was pos‐ itive based on species richness and diversity, this may be mislead‐ ing from the perspectives of conservation and functional diversity. Species composition and functional traits of the avian communities differed between sites in different areas of land use. Comparing bird densities by feeding guild in farmed and ranched sites shows that the former supported higher, or similar, densities of most guilds in most habitats. Exceptions were omnivores and predators in acacia woodlands, and nectarivores in miombo woodlands and open habi‐ tats. Indeed, guild associations within the NMDS community space showed that there was a different association of guilds following land‐use change in acacia woodland bird communities, compared with other habitats (Figure 6). The differential impact of land‐use change on acacia woodland birds was also highlighted by the func‐ tional traits' analyses. Effect sizes indicate that changes in possi‐ ble ecological significance occurred in the diversity and evenness of their traits, with a smaller effect in traits' divergence. This was likely due to greater number and/or diversity of distinctive large species of omnivores and predators. Indeed, large‐bodied bird spe‐ cies (>150 g) were less abundant in farmed sites, while smaller spe‐ cies (<150 g) were more abundant. Of the larger species, 44% were predators, 33% omnivores, and 11% insectivores. While the majority were Least Concern, White‐backed Vulture (Critically Endangered), and Bateleur Terathopius ecaudatus and Kori Bustard (both Near Threatened) are all of conservation concern (IUCN, 2017). Numbers of these three species were low, so evidence for population change was weak. The reduction in large bird species in the farmed sites echoes similar widespread declines across the tropics and in Africa (Newbold et al., 2013). Drivers of these declines include hunting for food (Thiollay, 2006a, 2006b), poisoning (Ogada & Keesing, 2010; Virani, Kendall, Njoroge, & Thomsett, 2011), and habitat loss (Thiollay, 2006a). A positive association between a species' size and its influence on ecosystem function is usually found, largely because biomass is directly related to the amount of energy and resources assimilated within a species (Grime, 1998; Villéger et al., 2008). Therefore, the impact of the loss of large species may not be captured by abundance‐ weighted analyses of community function. PRINGLE et al. \| FI G U R E 6 Ratios of densities, for all seasons and habitats combined, of species grouped by mean adult body mass, in farmed sites compared with ranched sites. Each horizontal bar represents the ratio of densities for all species within the body mass range shown in grams; each range contains approximately equal numbers of species. Ratios > 1 indicate higher density in farmed sites. Error bars represent 95% confidence intervals around the mean ratio values. For all mass ranges, the density difference between farmed and ranched sites shows an effect size > 1 Frugivores Granivores Insectivores Omnivores Nectarivores Predators –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 0.4 Coordinate 1 –0.2 –0.1 0.0 0.1 Coordinate 2 Frugivores Granivores Insectivores Omnivores Nectarivores Predators –0.4 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 Coordinate 1 –0.3 –0.2 –0.1 0.0 0.1 0.2 2 e t a nid r o o C Granivores Insectivores Omnivores Nectarivores Predators –0.2 –0.1 0.0 0.1 0.2 0.3 0.4 Coordinate 1 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 2 e t a nid r o o C Stress = 0.17 Stress = 0.16 Stress = 0.22 Stress = 0.15 Frugivores (a) All habitats (d) Open (c) Miombo (b) Acacia Frugivores Granivores Insectivores Omnivores Nectarivores Predators –0.4 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 Coordinate 1 –0.3 –0.2 –0.1 0.0 0.1 0.2 Coordinate 2 Stress = 0.16 (b) Acacia Frugivores Granivores Insectivores Omnivores Nectarivores Predators –0.4 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 Coordinate 1 –0.3 –0.2 –0.1 0.0 0.1 0.2 Coordinate 2 Frugivores Granivores Insectivores Omnivores Nectarivores Predators –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 0.4 Coordinate 1 –0.2 –0.1 0.0 0.1 Coordinate 2 Stress = 0.17 (a) All habitats FI G U R E 7 Nonmetric ordination plots of the avian communities for all seasons combined, showing (a) all habitats combined, (b) acacia woodland, (c) miombo woodland, and (d) open habitat. Each symbol represents a transect site in the farmed (filled circles) and ranched (filled triangles) areas. The convex polygons (farmed: red; ranched: gray) connect the outermost site points of each land‐ use type, highlighting the dissimilarity between the avian communities. ANOSIM tests on land‐use type: (a) R = .069, p = .036; (b) R = .056, p = .373; (c) R = 0.156, p = .042; (d) R = .223, p = .002. For example, one male Kori Bustard weighs approximately as much as 1,500 Yellow‐fronted Canaries, the species that benefited most in terms of increased population density in sites that were transformed to farming. We found that the biomass of different feeding guilds differed in farmed areas and ranched areas, something which might impact ecosystem function (Şekercioğlu, 2006). For example, omnivores were most negatively affected by land‐use change, with reduced or absent pop‐ ulations of four species, namely Swainson's Spurfowl Pternistis swain‐ sonii, Helmeted Guineafowl Numida meleagris, Shelley's Francolin Scleroptila shelleyi, and Kori Bustard. All of these species have varied diets, which include the seeds of indigenous plants, weeds and pi‐ oneer grasses, and some crop pests, such as locusts (Urban et al., 1986). These species provide the ecosystem function of seed dis‐ persal as well as being trophic process linkers that impact on popu‐ lations of invertebrates and small vertebrates (Şekercioğlu, 2006). In addition, the Kori Bustard provides a cultural ecosystem service as 12268 \| PRINGLE et al. it has an iconic status in African culture. White‐backed Vulture and African Fish Eagle were also large species absent from the farmed land redistribution on bird communities appear less severe than elsewhere in Africa. For example, agricultural lands bordering the FI G U R E 6 Ratios of densities, for all seasons and habitats combined, of species grouped by mean adult body mass, in farmed sites compared with ranched sites. Each horizontal bar represents the ratio of densities for all species within the body mass range shown in grams; each range contains approximately equal numbers of species. Ratios > 1 indicate higher density in farmed sites. Error bars represent 95% confidence intervals around the mean ratio values. For all mass ranges, the density difference between farmed and ranched sites shows an effect size > 1 FI G U R E 7 Nonmetric ordination plots of the avian communities for all seasons combined, showing (a) all habitats combined, (b) acacia woodland, (c) miombo woodland, and (d) open habitat. Each symbol represents a transect site in the farmed (filled circles) and ranched (filled triangles) areas. The convex polygons (farmed: red; ranched: gray) connect the outermost site points of each land‐ use type, highlighting the dissimilarity between the avian communities. ANOSIM tests on land‐use type: (a) R = .069, p = .036; (b) R = .056, p = .373; (c) R = 0.156, p = .042; (d) R = .223, p = .002. Vector lengths are proportional to the correlation between feeding guilds and the two main ordination axes; arrows indicate the direction of change in feeding guild densities Frugivores Granivores Insectivores Omnivores Nectarivores Predators –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 0.4 Coordinate 1 –0.2 –0.1 0.0 0.1 Coordinate 2 Frugivores Granivores Insectivores Omnivores Nectarivores Predators –0.4 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 Coordinate 1 –0.3 –0.2 –0.1 0.0 0.1 0.2 2 e t a nid r o o C Granivores Insectivores Omnivores Nectarivores Predators –0.2 –0.1 0.0 0.1 0.2 0.3 0.4 Coordinate 1 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 2 e t a nid r o o C Stress = 0.17 Stress = 0.16 Stress = 0.22 Stress = 0.15 Frugivores (a) All habitats (d) Open (c) Miombo (b) Acacia Frugivores Granivores Insectivores Omnivores Nectarivores Predators –0.4 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 Coordinate 1 –0.3 –0.2 –0.1 0.0 0.1 0.2 Coordinate 2 12268 \| 12268 PRINGLE et al. PRINGLE et al. Vector lengths are proportional to the correlation between feeding guilds and the two main ordination axes; arrows indicate the direction of change in feeding guild densities Coordinate 1 Coordinate 1 Coordinate 1 Granivores Insectivores Omnivores Nectarivores Predators –0.2 –0.1 0.0 0.1 0.2 0.3 0.4 Coordinate 1 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 2 e t a nid r o o C Stress = 0.15 Frugivores (c) Miombo Frugivores Granivores Insectivores Omnivores Nectarivores Predators –0.4 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 Coordinate 1 –0.3 –0.2 –0.1 0.0 0.1 0.2 2 e t a nid r o o C Stress = 0.22 (d) Open 2 e t a nid r o o C 2 0.1 0.0 Coordinate 1 Coordinate 1 Coordinate 1 it has an iconic status in African culture. White‐backed Vulture and African Fish Eagle were also large species absent from the farmed sites, which had a major impact on estimates of predator biomass. As scavengers, vultures provide a range of ecosystem services, in‐ cluding disease limitation, and maintenance of food‐web energy flow (Şekercioğlu, 2006). While a declining vulture population could have ecosystem consequences, our survey methods were not optimal for large, wide‐ranging species, so drawing firm conclusions is difficult. However, it is unlikely that farming activities were directly attribut‐ able to the absence of African Fish Eagle (an obligate piscivore) from farmed sites, but human disturbance may have been a factor. land redistribution on bird communities appear less severe than elsewhere in Africa. For example, agricultural lands bordering the Serengeti reserve had greatly reduced avian species richness, with insectivores and large terrestrial feeders most affected (Sinclair et al., 2002, 2014). Although these Serengeti studies also found a reduction in large‐bodied birds, many of our other find‐ ings are in marked contrast to those of Sinclair et al. (2002, 2014). There is a considerable overlap between the avifauna of western Zimbabwe and western Tanzania, and in both study sites, subsis‐ tence farming is carried out beside protected areas. In Serengeti however, the protected area is a wildlife conservation reserve, not a ranched area for livestock and game animals. PRINGLE et al. Major differ‐ ences in methodologies used in the two studies could account for Despite the loss of large species of potentially high func‐ tional and cultural value, the effects of land‐use change from \| 12269 Metric Acacia woodland Miombo woodland Open habitat Ranched Farmed Ranched Farmed Ranched Farmed Functional diversity (FD) 0.739 0.409 0.810 0.755 0.429 0.419 SD 0.188 0.152 0.206 0.209 0.114 0.199 Effect size 1.93 0.27 0.06 Functional diver‐ gence (Fdiv) 0.695 0.719 0.689 0.701 0.708 0.660 SD 0.033 0.022 0.056 0.032 0.062 0.042 Effect size 0.86 0.26 0.91 Functional even‐ ness (Feve) 0.470 0.544 0.544 0.539 0.535 0.488 SD 0.070 0.036 0.045 0.063 0.061 0.062 Effect size 1.33 0.09 0.76 Note: Effect sizes that may indicate ecologically significant differences in the distribution of traits within the bird communities are highlighted (blue, ES = 0.8–1.0; red, ES > 1.0). PRINGLE et al. 12269 TA B LE 4 Functional traits' indices for bird communities recorded in ranched and farmed sites, with both seasons combined. Effect size (ES) values for each functional index are calculated for the bird communities in the same habitat type, but different land uses Note: Effect sizes that may indicate ecologically significant differences in the distribution of traits within the bird communities are highlighted (blue, ES = 0.8–1.0; red, ES > 1.0). higher abundances of smaller inconspicuous birds in our surveys (entirely walked transects in our study vs. driven transects with stops in Serengeti; no correction for detection probabilities in Serengeti). We also included all birds recorded along farmed tran‐ sects, not just birds seen within the cultivated or habituated parts. Other important factors are likely to be differences in the natural vegetation, human population density, agricultural intensity, and types of crops grown in the farmed mosaics. The elapsed times‐ cale between the commencement of farming in the area and the study was also at least five times longer in the case of Serengeti (Sinclair et al., 2002). Bird species richness and abundance were lower outside protected areas at three sites across South Africa, and insectivore richness was much higher inside protected areas, with the converse true for granivores (Greve, Chown, Rensburg, Dallimer, & Gaston, 2011). However, the redistributed lands in this Zimbabwean case study are dynamic and young, with rel‐ atively low human population density. PRINGLE et al. Conservation of the ex‐ tensive remaining miombo and acacia woodlands that are now interspersed with arable fields and grasslands in the farmed area will be critical to maintaining biodiversity. Efforts to reduce hunt‐ ing or persecution of large bird species will also play an important role. Research from other parts of Africa may provide an indica‐ tion of the future trajectories for Zimbabwe's bird communities; in the absence of conservation interventions, widespread species loss and a decline in abundance across all guilds and body sizes can accompany land‐use changes (Greve et al., 2011; Sinclair et al., 2002, 2014). indicating that numerous species and several functional groups have benefited, although a few have not. This may be a temporary effect; only one decade has passed since small‐scale farming commenced on the ranched lands. Changes in the functional traits of birds present in the transformed land suggest that the diversity of traits has reduced, and these are now more evenly distributed across the community. However, our analyses may not have fully captured the paucity of larger species in the farmed sites. Relying on taxonomic measures of diversity and abundance‐weighted functional traits may, therefore, obscure functional changes in bird communities and, by extension, important information required for avian conservation. It is unknown whether smallholder‐farmed landscapes in the Global South can deliver multiple benefits, for example, in terms of food production and habitat for biodiversity (e.g., Brussaard et al., 2010; Chappell & LaValle, 2011). If we are to facilitate the uptake of biodiversity‐friendly agricultural practices through evidence‐ based prioritization, it is essential to quantify the relationships between farming, land management practices, and biodiversity. Similarly, we must recognize the central role that improving in‐ come and yields has in a small‐scale farming setting across the developing world. Here, we provide an insight into how avian abundance and diversity differs between newly established small‐ scale farms and commercial ranched land. 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https://openalex.org/W4382862480	https://www.mdpi.com/2504-3110/7/7/515/pdf?version=1688032954	English	null	Maximum Principle for Nonlinear Fractional Differential Equations with the Hilfer Derivative	Fractal and fractional	2,023	cc-by	5,579	fractal and fractional fractal and fractional fractal and fractional f Abu Bakr Elbukhari 1,2,* , Zhenbin Fan 1 and Gang Li 1 Abu Bakr Elbukhari 1,2,* , Zhenbin Fan 1 and Gang Li 1 1 School of Mathematical Sciences, Yangzhou University, Yangzhou 225002, China; zbfan@yzu.edu.cn (Z.F.); gli@yzu.edu.cn (G.L.) g y ( ) 2 Department of Mathematics, Faculty of Education, University of Khartoum, Omdurman 406, Sudan * C d b k i123@h il b b k lb kh i@ fk d T l 86 13270 5513 72 g y 2 Department of Mathematics, Faculty of Education, University of Khartoum, Omdurman 406, Sudan * Correspondence: mrbakri123@hotmail.com or abubakr.elbukhari@uofk.edu; Tel.: +86-13270-5513-72 Department of Mathematics, Faculty of Education, University of Khartoum, Omdurman 406, Sudan * Correspondence: mrbakri123@hotmail.com or abubakr.elbukhari@uofk.edu; Tel.: +86-13270-5513-72 * Correspondence: mrbakri123@hotmail.com or abubakr.elbukhari@uofk.edu; Tel.: +86-13270-5513-72 Abstract: In this paper, two signiﬁcant inequalities for the Hilfer fractional derivative of a function in the space ACγ([0, b], Rn), 0 ≤γ ≤1 are obtained. We ﬁrst veriﬁed the extremum principle for the Hilfer fractional derivative. In addition, we estimated the Hilfer derivative of a function at its extreme points. Furthermore, we derived and proved a maximum principle for a nonlinear Hilfer fractional differential equation. Finally, we analyzed the solutions of a nonlinear Hilfer fractional differential equation. Our results generalize and extend some obtained theorems on this topic. Keywords: fractional differential equations; Hilfer fractional derivative; extremum principle for Hilfer derivative; maximum principle 1. Introduction Recently, the subject of fractional calculus has been extensively developed and studied by many academics due to its applications in numerous branches of applied sciences, technology, and engineering. Examples include engineering [1], ecology [2], biology [3], medicine [4], chemistry [5], animal science [6], ﬁnance [7], control theory [8], and other branches. In recent decades, Hilfer deﬁned a generalized fractional derivative of Riemann– Liouville (R-L), which includes fractional derivatives of Caputo and R-L [9]. Later on, this derivative became known as the Hilfer fractional derivative and has been used widely in modeling in many ﬁelds of science, see, e.g., [10–15]. Brief Report Maximum Principle for Nonlinear Fractional Differential Equations with the Hilfer Derivative Abu Bakr Elbukhari 1,2,* , Zhenbin Fan 1 and Gang Li 1 Citation: Elbukhari, A.B.; Fan, Z.; Li, G. Maximum Principle for Nonlinear Fractional Differential Equations with the Hilfer Derivative. Fractal Fract. 2023, 7, 515. https://doi.org/ 10.3390/fractalfract7070515 Citation: Elbukhari, A.B.; Fan, Z.; Li, G. Maximum Principle for Nonlinear Fractional Differential Equations with the Hilfer Derivative. Fractal Fract. 2023, 7, 515. https://doi.org/ 10.3390/fractalfract7070515 In recent years, maximum principles have been regarded as one of a few efﬁcient ways for obtaining preliminary information about solutions of ordinary or partial differential equations without having a good understanding of the solutions themselves. Furthermore, the maximum principle is considered to be an essential analytical tool in the study of existence and uniqueness results for linear and nonlinear fractional differential equations, we refer to the books [16,17] and survey [18]. Academic Editors: Carlo Cattani and Adem Kilicman Academic Editors: Carlo Cattani and Adem Kilicman Received: 6 June 2023 Revised: 21 June 2023 Accepted: 26 June 2023 Published: 29 June 2023 Numerous academics have recently focused on the formulation and implementation of maximum principles for diffusion equations. In [19], Luchko proposed and proved for the ﬁrst time a maximum principle for a generalized fractional diffusion equation with the Caputo derivative over an open bounded domain ΩT := G × (0, T), G ∈Rn in the form: cDρ 0+y(ν, ϱ) = ∆y(ν, ϱ) + F(ν, ϱ), (ν, ϱ) ∈ΩT , 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/). with the conditions ( yϱ=0 = y0(x), x ∈¯G, yS = v(x, ϱ), (x, ϱ) ∈S × [0, T ], where cDρ 0+ is the Caputo derivative of order 0 < ρ ≤1, F is a smooth function, S is open and bounded in Rn, and ∆y(ν, ϱ) is in fact a linear elliptic differential operator of https://www.mdpi.com/journal/fractalfract Fractal Fract. 2023, 7, 515. https://doi.org/10.3390/fractalfract7070515 Fractal Fract. 2023, 7, 515 2 of 9 the second order. Luchko [20] demonstrated the existence and uniqueness of solutions for initial-boundary value problems of the multidimensional time fractional diffusion equation, and also formulated and discussed the maximum principle for the generalized time fractional diffusion equations, including the multi-term diffusion equation and the diffusion equation of distributed order [21]. Al-Refai [22] applied the maximum principle to Caputo fractional boundary value problems of order 1 < ρ < 2. Ye et al. [23] presented a maximum principle for multi-term time-space fractional differential equations utilizing the modiﬁed Riesz space-fractional derivative. In [24], Al-Refai and Luchko obtained weak and strong maximum principles for one-dimensional linear and non-linear fractional diffusion equations with the R-L time-fractional derivative RLDρ 0+y(ν, ϱ) = ∆y(ν, ϱ) + F(ν, ϱ), (ν, ϱ) ∈(0, l) × (0, T ], where the initial and boundary conditions are the sides of a given rectangle, RLDρ 0+ is the R-L derivative of order 0 < ρ ≤1, and ∆y(ν, ϱ) is a second order differential equation. Al-Refai and Luchko [25] investigated the initial boundary value problems for linear and nonlinear multi-term fraction diffusion equations with the R-L derivative. Cao et al. [26] proposed maximum and minimum principles for time-fractional Caputo–Katugampola diffusion equations. In [27], Al-Refai and Pal developed a maximum principle for a general fractional linear boundary value problem with a Caputo–Fabriaio fractional deriva- tive. Al-Refai [28] established and veriﬁed weak and strong maximum principles for frac- tional nonlinear differential equations with R-L fractional derivatives of order 0 < ρ < 1. Al-Refai and Baleanu [29] derived and proved a comparison principle for linear fractional differential equations via a non-local derivative in the form:            NLDρ a,by(ϱ) = F(y, ϱ), ϱ ∈(a, b), NLDρ a,by(ϱ) + w(ϱ)y(ϱ) = r(ϱ), ϱ ∈(a, b), y(a) = ya, y(b) = yb, , where NLDρ a,b is the non-local fractional derivative of order 0 < ρ < 1 and w, r ∈C[a, b]. Luchko et al. [30] proved a maximum principle for the general multi-term space-time fractional transport equation. Kamocki [31] presented a new method of expressing the Hilfer derivative, which is deﬁned as a modiﬁed R-L derivative, and then proved the importance of the expression in practical applications of fractional differential equations. Inspired by the above discussion of maximum principles, we shall formulate and prove an extremum principle for the Hilfer derivative as well as a maximum principle for the nonlinear Hilfer fractional system. 2. Preliminaries In this section, we will review the R-L fractional integral and derivative and the Hilfer fractional derivative, all of which are used in the proof of the theorems in this paper. p p p We begin by introducing the R-L fractional integral and the R-L and Hilfer derivatives. Deﬁnition 1 (see [14]). The R-L fractional integral of order ρ > 0 for a function f : [0, +∞) →R is given by is given by Iρ 0+ f (ϱ) = 1 Γ(ρ) Z ϱ 0 (ϱ −s)ρ−1 f (s)ds. Iρ 0+ f (ϱ) = 1 Γ(ρ) Z ϱ 0 (ϱ −s)ρ−1 f (s)ds. Deﬁnition 2 (see [32]). The R-L fractional derivative of order 0 < ρ < 1 for a function f ∈Cm([0, +∞), R) is given by RLDρ 0+ f (ϱ) = 1 Γ(1 −ρ) dm dϱm Z ϱ 0 (ϱ −s)−ρ f (s)ds. Deﬁnition 3 (see [15]). The Hilfer fractional derivative (left-sided) HDρ,η a+ of order 0 < ρ < 1, type 0 ≤η ≤1 for a function f ∈L1([0, T ], Rn) is deﬁned as Deﬁnition 3 (see [15]). The Hilfer fractional derivative (left-sided) HDρ,η a+ of order 0 < ρ < 1, type 0 ≤η ≤1 for a function f ∈L1([0, T ], Rn) is deﬁned as HDρ,η 0+ f (ϱ) =(Iη(1−ρ) 0+ D(I(1−η)(1−ρ) 0+ f ))(ϱ) = Iη(1−ρ) 0+ DI1−γ 0+ f (ϱ) = Iη(1−ρ) 0+ RLDγ 0+ f (ϱ), HDρ,η 0+ f (ϱ) =(Iη(1−ρ) 0+ D(I(1−η)(1−ρ) 0+ f ))(ϱ) = Iη(1−ρ) 0+ DI1−γ 0+ f (ϱ) = Iη(1−ρ) 0+ RLDγ 0+ f (ϱ), where D := d dϱ and γ = ρ + η −ρη. where D := d dϱ and γ = ρ + η −ρη. 3. Maximum Principle In this paper, we consider the following nonlinear fractional system involving a Hilfer derivative of the form:      HDρ,η 0+ y(ν, ϱ) = ∆y(ν, ϱ) + F(ν, ϱ), (ν, ϱ) ∈(0, π) × (0, T ] := ΩT , y(0, ϱ) = y(π, ϱ) = 0, ϱ ∈(0, T ], I1−γ 0+ y(ν, 0) = y0, ν ∈[0, π], (1) (1) where HDρ,η 0+ stands for the Hilfer fractional derivative of order 0 < ρ < 1 and type 0 ≤η ≤1, the operator ∆y(ν, ϱ) stands for a second order differential operator, the nonlinear term F(ν, ϱ) : ΩT →Rn, and γ = ρ + η −ρη, 0 < γ ≤1. We assume the operator ∆y(ν, ϱ), deﬁned as ∆y(ν, ϱ) = p(ν, ϱ)yνν(ν, ϱ) + q(ν, ϱ)yν(ν, ϱ) + r(ν, ϱ)y(ν, ϱ), (ν, ϱ) ∈ΩT . (2) (2) We assume the functions p(ν, ϱ), q(ν, ϱ), and r(ν, ϱ) are continuous functions on Ωb = [0, π] × [0, T ], and p(ν, ϱ) > 0, r(ν, ϱ) < 0 for (ν, ϱ) ∈ΩT . We assume the functions p(ν, ϱ), q(ν, ϱ), and r(ν, ϱ) are continuous functions on Ωb = [0, π] × [0, T ], and p(ν, ϱ) > 0, r(ν, ϱ) < 0 for (ν, ϱ) ∈ΩT . Fractal Fract. 2023, 7, 515 3 of 9 This paper is organized as follows: some essential deﬁnitions are introduced in Section 2. In Section 3, we estimate the Hilfer fractional derivative of a function f ∈ACγ([0, T ], Rn) at its extreme points. Moreover, maximum and minimum princi- ples are established and applied to system (1). Section 4 is devoted to the conclusion. 3. Maximum Principle In this section, we ﬁrst present some remarks that are helpful in the proofs of our theorems. Moreover, to show that a function in the representation (3) has maximum points, two examples are presented. Then, we estimate the Hilfer derivative of a function in the space f ∈ACγ([0, T ], Rn), 0 ≤γ ≤1 at its extreme points. Finally, we derive and prove the maximum principle for system (1). Remark 1. We denote ACµ([0, T ], Rn), µ ∈(0, 1), in short, ACµ is the set of all functions f : [0, T ] →Rn that have the following representation, f (ϱ) = c Γ(µ)ϱµ−1 + Iµ 0+ψ (ϱ), a.e. ϱ ∈[0, T ], (3) (3) where c ∈Rn and ψ ∈L1([0, T ], Rn). The integral representation (3) was given in [14,31]. where c ∈Rn and ψ ∈L1([0, T ], Rn). The integral representation (3) was given in [14,31]. Remark 2. In the following examples, we show that the function f in Remark 1 has a maximum value at the maximum point ϱ0 ∈(0, T ]. Let us choose T = 1. Fractal Fract. 2023, 7, 515 4 of 9 Example 1. Let ψ(ϱ) = 1 for all ϱ ∈(0, 1], and c = −1. Then, by applying the convolution integrals to ψ(ϱ) and gµ(ϱ) = ϱµ−1 Γ(µ) , ϱ ∈(0, 1], 0 < µ < 1, we have Example 1. Let ψ(ϱ) = 1 for all ϱ ∈(0, 1], and c = −1. Then, by applying the convolution integrals to ψ(ϱ) and gµ(ϱ) = ϱµ−1 Γ(µ) , ϱ ∈(0, 1], 0 < µ < 1, we have f (ϱ) = −1 Γ(µ)ϱµ−1 + g1+µ(ϱ) = ϱµ Γ(1 + µ) −ϱµ−1 Γ(µ). Since f is non-decreasing function on (0, 1], then one can easily see that f attains its maximum at the right boundary of (0, 1], that is, fmax = f (1) = 1−µ Γ(1+µ). Since f is non-decreasing function on (0, 1], then one can easily see that f attains its maximum at the right boundary of (0, 1], that is, fmax = f (1) = 1−µ Γ(1+µ). Example 2. Let ψ(ϱ) = 2ϱ −eϱ for all ϱ ∈(0, 1], and c = −1, we have f (ϱ) = Iµ 0+(2ϱ −eϱ) − 1 Γ(µ)ϱµ−1. 3. Maximum Principle (4) (4) Due to difﬁculty in obtaining the exact values of the function f in (4) manually, particularly the term Iµ 0+eϱ, we have computed the maximum point numerically by using a MATLAB tool called Fractional Trapezoidal Formula to obtain that f attains its maximum point in (0, 1]. Remark 3. We note that the function f in (3) might have no maximum points when c > 0. Thus, we assume that the initial point c = y0 < 0, where y0 is the initial state of system (1). However, we later will impose a condition on y0 in the proof of our maximum and minimum principles, that is, y0 < 0 and y0 > 0, respectively. Next, we present the new expression of the Hilfer derivative that was presented in [31]. 2023, 7, 515 Now, utilizing Deﬁnition 3 and the semigroup property, we have Now, utilizing Deﬁnition 3 and the semigroup property, we have (HDρ,η 0+ f )(ϱ) = Iη(1−ρ) 0+ DI1−γ 0+ f (ϱ) = DIη(1−ρ) 0+ I1 0+DI1−γ 0+ f (ϱ) =DIη(1−ρ) 0+ I1−γ 0+ f −(I1−γ 0+ f )(0) (ϱ) = RLDρ 0+ f (ϱ) − I1−γ 0+ f (0) Γ(γ −ρ) ϱγ−ρ−1. In the following, let us recall the extremum principle for the R-L derivative. In the following, let us recall the extremum principle for the R-L derivative Lemma 2 (see [24]). The R-L derivative of the function f ∈ACγ([0, T ], Rn) at the maximum point ϱ0 ∈(0, T ] satisﬁes the following inequality RLDρ 0+ f (ϱ0) ≥ ϱ−ρ 0 Γ(1 −ρ) f (ϱ0). Proof of Lemma 2. The proof is almost identical to that in [24]; thus, it is omitted here. Proof of Lemma 2. The proof is almost identical to that in [24]; thus, it is omitted here. Now we are able to demonstrate the extremum principle for the Hilfer derivative and our maximum principle as well. Theorem 1 (Extremum principle). Let f ∈ACγ([0, T ], Rn) attain its maximum at a point ϱ0 ∈(0, T ], γ = ρ + η −ρη, 0 < γ ≤1, 0 < ρ < 1, 0 ≤η ≤1, γ ≥ρ, γ > η and 1 −γ < 1 −η(1 −ρ). Then, the following inequality holds, (HDρ,η 0+ f )(ϱ0) ≥ ϱ−ρ 0 Γ(1 −ρ) f (ϱ0) − I1−γ 0+ f (0) Γ(γ −ρ) ϱγ−ρ−1 0 . Proof of Theorem 1. It follows from Lemmas 1 and 2 that Proof of Theorem 1. It follows from Lemmas 1 and 2 that (HDρ,η 0+ f )(ϱ0) = RLDρ 0+ f (ϱ0) − I1−γ 0+ f (0) Γ(γ −ρ) ϱγ−ρ−1 0 ≥ ϱ−ρ 0 Γ(1 −ρ) f (ϱ0) − I1−γ 0+ f (0) Γ(γ −ρ) ϱγ−ρ−1 0 . Now, we will demonstrate our maximum principle. Next, we present the new expression of the Hilfer derivative that was presented in [31]. Next, we present the new expression of the Hilfer derivative that was presented in [31]. Remark 4 (see [31]). Let f ∈L1([0, T ], Rn), µ ∈(0, 1), then the function f has the left-sided R-L derivative of order µ if and only if f ∈ACµ([0, T ]; Rn). Moreover, for ψ ∈L1([0, T ]; Rn), c ∈Rn, we have RLDµ 0+ f (ϱ) = ψ(ϱ), a.e. ϱ ∈[0, T ], and I1−µ 0+ f (0) = c. Remark 5 (see [31]). Let f ∈L1([0, T ]; Rn), γ = ρ + η −ρη, 0 < γ ≤1, 0 < ρ < 1, 0 < η ≤1, f has the left-sided Hilfer fractional derivative of order ρ and type η if and only if f ∈ACγ([0, T ], Rn). Moreover, HDρ,η 0+ f (ϱ) = Iη(1−ρ) 0+ ψ (ϱ), a.e. ϱ ∈[0, T ] and I1−γ 0+ f (0) = c, c ∈Rn. (5) Lemma 1 (see [31]). Let f ∈ACγ([0, T ], Rn), then f has the left-sided Hilfer fractional derivative of order ρ and type η as follows: Lemma 1 (see [31]). Let f ∈ACγ([0, T ], Rn), then f has the left-sided Hilfer fractional derivative of order ρ and type η as follows: (HDρ,η 0+ f )(ϱ) = RLDρ 0+ f (ϱ) − I1−γ 0+ f (0) Γ(γ −ρ) ϱγ−ρ−1, a.e. ϱ ∈[0, T ]. Proof of Lemma 1. The proof was given in [31], which is summarized here because it is required for establishing Theorem 1. Let f ∈ACγ([0, T ], Rn) be introduced as the same as in Remark 1 with µ = γ. Then, c = I1−γ 0+ f (0) and ψ(ϱ) = d dϱ I1−γ 0+ f (ϱ), I1−γ 0+ f ∈ACγ([0, T ], Rn), we get I1−γ 0+ f (ϱ) = I1−γ 0+ f (0) + Z ϱ 0 d ds h I1−γ 0+ f (ϱ) i dϱ, ϱ ∈[0, T ]. Thus, we get Z ϱ 0 d dϱ h I1−γ 0+ f (ϱ) i dϱ = I1−γ 0+ f (ϱ) − I1−γ 0+ f (0). 5 of 9 Fractal Fract. Now, we will demonstrate our maximum principle. Now, we will demonstrate our maximum principle. Now, we will demonstrate our maximum principle. heorem 2 (Maximum principle). Let y(ν, ϱ) ∈ACγ([0, T ], Rn) such that y0 < 0 and Pρ,ηy(ν, ϱ) = HDρ,η 0+ y(ν, ϱ) −∆y(ν, ϱ) ≤0, ∀(ν, ϱ) ∈ΩT . Then, we get max (ν,ϱ)∈ΩT y(ν, ϱ) ≤max n max (ν,ϱ)∈∂ΩT y(ν, ϱ), 0 o , (6) (6) where ∂ΩT is the boundary of the domain ΩT . Proof of Theorem 2. If y attains its maximum at the boundary ∂ΩT , then the inequality (6) holds. On the other hand, we will consider the case where y attains its maximum in the interior of ΩT . Fractal Fract. 2023, 7, 515 6 of 9 Let us assume y attains its positive maximum, which is y(ν0, ϱ0), (ν0, ϱ0) ∈ΩT , then we have the following inequality y(ν0, ϱ0) > max n max (ν,ϱ)∈∂ΩT y(ν, ϱ), 0 o = M ≥0. Assume 0 < ϵ := y(ν0, ϱ0) −M. Since yν(ν0, ϱ0) = 0, yνν(ν0, ϱ0) ≤0, p(ν0, ϱ0) > 0, and r(ν0, ϱ0) < 0, we have ∆y(ν0, ϱ0) = p(ν0, ϱ0)yνν(ν0, ϱ0) + q(ν0, ϱ0)yν(ν0, ϱ0) + r(ν0, ϱ0)y(ν0, ϱ0). (7) (7) Suppose that w(ν, ϱ) = y(ν, ϱ) + ϵ 2 (T −ϱ) T , (ν, ϱ) ∈ΩT , (8) (8) we can easily get wν(ν, ϱ) = yν(ν, ϱ), and wνν(ν, ϱ) = yνν(ν, ϱ), (ν, ϱ) ∈ΩT . (9) (9) Also, we obtain w(ν, ϱ) ≤y(ν, ϱ) + ϵ 2, (ν, ϱ) ∈ΩT . Then, w(ν0, ϱ0) > y(ν0, ϱ0) = M + ϵ ≥ϵ + y(ν, ϱ) ≥w(ν, ϱ) + ϵ 2, (ν, ϱ) ∈∂ΩT . Thus, w cannot attain its maximum at ∂ΩT . Therefore, we presume w attains its maximum at the point (ν1, ϱ1) ∈ΩT , then one can see that w(ν1, ϱ1) ≥w(ν0, ϱ0) > M + ϵ ≥ϵ > 0. w(ν1, ϱ1) ≥w(ν0, ϱ0) > M + ϵ ≥ϵ > 0. We derive the Hilfer derivative for Equation (8) as follows: HDρ,η 0+ w(ν, ϱ) = HDρ,η 0+ y(ν, ϱ) + HDρ,η 0+ ϵ 2 (T −ϱ) T = HDρ,η 0+ y(ν, ϱ) + ϵ 2Γ(1 −ρ)ϱ−ρ −ϵ 2T ϱ1−ρ Γ(2 −ρ) = HDρ,η 0+ y(ν, ϱ) − ϵϱ−ρ 2Γ(1 −ρ) ϱ T (1 −ρ) −1 ! . Now, we will demonstrate our maximum principle. (10) (10) In view of the semigroup property and applying the convolution to Equation (8), we have In view of the semigroup property and applying the convolution to Equation (8), we have I1−γ 0+ w(ν, ϱ) ϱ=0 = I1−γ 0+ y(ν, ϱ) + ϵ 2 (T −ϱ) T (ϱ) ϱ=0 = y0 + I1−γ 0+ ϵ 2 (T −ϱ) T ϱ=0 = y0 + I1−γ 0+ ϵ 2 (ϱ) ϱ=0 − I1−γ 0+ ϵϱ 2T (ϱ) ϱ=0 = y0. Calculating (10) at the maximum point (ν1, ϱ1) yields Calculating (10) at the maximum point (ν1, ϱ1) yields HDρ,η 0+ y(ν1, ϱ1) = HDρ,η 0+ w(ν1, ϱ1) + ϵϱ−ρ 1 2Γ(1 −ρ) ϱ1 T (1 −ρ) −1 . Fractal Fract. 2023, 7, 515 7 of 9 Using Theorem 1 and the second equation of (1), we get Using Theorem 1 and the second equation of (1), we get HDρ,η 0+ y(ν1, ϱ1) ≥ ϱ−ρ 1 Γ(1 −ρ)w(ν1, ϱ1) − I1−γ 0+ w(ν1, 0) Γ(γ −ρ) ϱγ−ρ−1 1 + ϵϱ−ρ 1 2Γ(1 −ρ) ϱ1 T (1 −ρ) −1 = ϱ−ρ 1 Γ(1 −ρ)w(ν1, ϱ1) − y0 Γ(γ −ρ)ϱγ−ρ−1 1 + ϵϱ−ρ 1 2Γ(1 −ρ) ϱ1 T (1 −ρ) −1 ≥ ϵϱ−ρ 1 Γ(1 −ρ) − y0 Γ(γ −ρ)ϱγ−ρ−1 1 + ϵϱ−ρ 1 2Γ(1 −ρ) ϱ1 T (1 −ρ) −1 . (11) (11) On the other hand, we have ∆y(ν1, ϱ1) =∆ w(ν1, ϱ1) −ϵ 2 (T −ϱ1) T =p(ν1, ϱ1)wνν(ν1, ϱ1) + q(ν1, ϱ1)wν(ν1, ϱ1) + r(ν1, ϱ1) w(ν1, ϱ1) −ϵ 2 (T −ϱ1) T ≤r(ν1, ϱ1) w(ν1, ϱ1) −ϵ 2 (T −ϱ1) T ≤r(ν1, ϱ1) ϵ −ϵ 2 (T −ϱ1) T . (12) + r(ν1, ϱ1) w(ν1, ϱ1) −ϵ 2 (T −ϱ1) T ≤r(ν1, ϱ1) w(ν1, ϱ1) −ϵ 2 (T −ϱ1) T ≤r(ν1, ϱ1) ϵ −ϵ 2 (T −ϱ1) T . Remark 6. An analogous result that follows from Theorem 2 can be obtained for the Hilfer fractional derivative at minimum points by replacing y with −y. Speciﬁcally, introducing the minimum principle, this can be illustrated in the following theorem. Proof of Theorem 4. We can simply obtain the proof using Theorem 2. Theorem 5. Suppose that F(ν, ϱ) ≥0 for any (ν, ϱ) ∈ΩT , y0 > 0. If y(ν, ϱ) ∈ACγ([0, T ], Rn) is a solution of system (1), then y(ν, ϱ) ≥0, (ν, ϱ) ∈ΩT . Consequently, the nonlinear Hilfer fractional system (1) has no negative solutions in ACγ([0, T ], Rn). Proof of Theorem 5. One can directly obtain the proof of this theorem by applying Theorem 3. Proof of Theorem 5. One can directly obtain the proof of this theorem by applying Theorem 3. Data Availability Statement: No data were used to support this study. Data Availability Statement: No data were used to support this study. Acknowledgments: The authors are extremely thankful to the anonymous referees for their insightful comments and suggestions that signiﬁcantly enhanced the quality of this manuscript. This work was supported by the National Natural Science Foundation of China (grant numbers 11871064 and 11571300). Conﬂicts of Interest: The authors declare that there are no conﬂicts of interest regarding the publica- tion of this article. Now, we will demonstrate our maximum principle. (12) (12) Noting that (ν1, ϱ1) ∈Ωb, by using the results in (11) and (12), we have Noting that (ν1, ϱ1) ∈Ωb, by using the results in (11) and (12), we have Pρ,ηy(ν1, ϱ1) = HDρ,η 0+ y(ν1, ϱ1) −∆y(ν1, ϱ1) ≥ ϵϱ−ρ 1 Γ(1 −ρ) − y0 Γ(γ −ρ)ϱγ−ρ−1 1 + ϵϱ−ρ 1 2Γ(1 −ρ) ϱ1 T (1 −ρ) −1 −r(ν1, ϱ1) ϵ −ϵ 2 (T −ϱ1) T = ϵϱ−ρ 1 Γ(1 −ρ) − y0 Γ(γ −ρ)ϱγ−ρ−1 1 + ϵϱ1−ρ 1 2T Γ(2 −ρ) − ϵϱ−ρ 1 2Γ(1 −ρ) −r(ν1, ϱ1)ϵ + r(ν1, ϱ1)ϵ 2 (T −ϱ1) T ≥ ϵϱ−ρ 1 2Γ(1 −ρ) − y0 Γ(γ −ρ)ϱγ−ρ−1 1 + ϵϱ1−ρ 1 2T Γ(2 −ρ) −r(ν1, ϱ1)ϵ + r(ν1, ϱ1)ϵ 2 (T −ϱ1) T ≥ ϵϱ−ρ 1 2Γ(1 −ρ) − y0 Γ(γ −ρ)ϱγ−ρ−1 1 + ϵϱ1−ρ 1 2T Γ(2 −ρ) −r(ν1, ϱ1)ϵ 2 > 0 which contradicts the fact that Pρ,ηy(ν, ϱ) ≤0 for all (ν, ϱ) ∈ΩT . Consequently, (6) holds. One can note that the last inequality holds only when y0 < 0 and r(ν1, ϱ1) < 0. which contradicts the fact that Pρ,ηy(ν, ϱ) ≤0 for all (ν, ϱ) ∈ΩT . Consequently, (6) holds. One can note that the last inequality holds only when y0 < 0 and r(ν1, ϱ1) < 0. Remark 6. An analogous result that follows from Theorem 2 can be obtained for the Hilfer fractional derivative at minimum points by replacing y with −y. Speciﬁcally, introducing the minimum principle, this can be illustrated in the following theorem. Fractal Fract. 2023, 7, 515 8 of 9 Theorem 3 (Minimum principle). Let y(ν, ϱ) ∈ACγ([0, T ], Rn) such that y0 > 0 and Pρ,ηy(ν, ϱ) = HDρ,η 0+ y(ν, ϱ) −∆y(ν, ϱ) ≥0, ∀(ν, ϱ) ∈ΩT . Theorem 3 (Minimum principle). Let y(ν, ϱ) ∈ACγ([0, T ], Rn) such that y0 > 0 and Pρ,ηy(ν, ϱ) = HDρ,η 0+ y(ν, ϱ) −∆y(ν, ϱ) ≥0, ∀(ν, ϱ) ∈ΩT . Theorem 3 (Minimum principle). Let y(ν, ϱ) ∈ACγ([0, T ], Rn) such that y0 > 0 and Pρ,ηy(ν, ϱ) = HDρ,η 0+ y(ν, ϱ) −∆y(ν, ϱ) ≥0, ∀(ν, ϱ) ∈ΩT . Then, min (ν,ϱ)∈ΩT y(ν, ϱ) ≥min n min (ν,ϱ)∈∂ΩT y(ν, ϱ), 0 o , thus, the minimum principle is established. The following theorems are the direct results of Theorems 2 and 3. thus, the minimum principle is established. 1. Bohaienko, V.; Gladky, A.; Romashchenko, M.; Matiash, T. Identiﬁcation of fractional water transport model with ψ-Caputo derivatives using particle swarm optimization algorithm. Appl. Math. Comput. 2021, 390, 125665. [CrossRef] g p p g pp p [ ] 2. Li, Y. Ecological balance model of effective utilization of agricultural water resources based on fractional differential equations. Appl. Math. Nonlinear Sci. 2021, 7, 371–378. [CrossRef] Now, we will demonstrate our maximum principle. thus, the minimum principle is established. The following theorems are the direct results of Theorems 2 and 3. The following theorems are the direct results of Theorems 2 and 3. Theorem 4. SupposethatF(ν, ϱ) ≤0 for every (ν, ϱ) ∈ΩT , y0 < 0. If y(ν, ϱ) ∈ACγ([0, T ], Rn) is a solution of system (1), then y(ν, ϱ) ≤0, (ν, ϱ) ∈ΩT . Theorem 4. SupposethatF(ν, ϱ) ≤0 for every (ν, ϱ) ∈ΩT , y0 < 0. If y(ν, ϱ) ∈ACγ([0, T ], Rn) is a solution of system (1), then y(ν, ϱ) ≤0, (ν, ϱ) ∈ΩT . y(ν, ϱ) ≤0, (ν, ϱ) ∈ΩT . Therefore, the nonlinear Hilfer fractional system (1) has no positive solutions in ACγ([0, T ], Rn). 4. Conclusions In this paper, we ﬁrst have proved the extremum principle for the Hilfer fractional derivative. Then, we have derived and proved maximum and minimum principles for a nonlinear fractional differential equation with the Hilfer derivative. Moreover, we have used this maximum principle to prove the existence of the solutions to system (1). For future research, we shall use the results of this paper to investigate existence results for Hilfer fractional integrodifferential equations with fractional Brownian motion. Author Contributions: Methodology, Z.F.; formal analysis, writing—original draft preparation A.B.E.; conceptualization, writing—review and editing, A.B.E. and Z.F.; Supervision, project adminis- tration, G.L. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Natural Science Foundation of China (grant numbers 11871064 and 11571300). Data Availability Statement: No data were used to support this study. References The Maximum Principle; Springer Science and Business Media: Oakland, CA, USA, 2 17. Pucci, P.; Serrin, J. The Maximum Principle; Springer Science and Business Media: Oakland, CA, USA, 2007. hk l f h f l l ff l 18. Luchko, Y.; Yamamoto, M. Maximum principle for the time-fractional PDEs. In Fractional Differential Luchko, Y., Eds.; Walter de Gruyter: Berlin, Germany; Boston, MA, USA, 2019; pp. 299–326. 19. Luchko, Y. Maximum principle for the generalized time-fractional diffusion equation. J. Math. Anal. 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https://openalex.org/W1995517411	https://europepmc.org/articles/pmc4170709?pdf=render	Latin	null	Equivalent Pairs of Words and Points of Connection	The scientific world journal/TheScientificWorldjournal	2,014	cc-by	5,858	1. Introduction There is a well-known relationship between the action of PSL(2, Z) on R and continued fractions. There are many articles on the connection between the geodesics on the mod- ular surface and continued fractions which are particularly important in the theory of approximation of real numbers by rational [4, 5]. Important results have been obtained using these ideas in a very well-written article [6]. A good account on relationship between continued fractions and indefinite binary quadratic forms is also given in [7]. In [8], Mushtaq has shown a relationship between reduced indefinite binary quadratic forms and orbits of the modular group. There is another interesting article [9] of Mushtaq and Hayat on the topic of Pell numbers, Pell-Lucas numbers, and modular group. In [10], Bong and Mushtaq determine the Fibonacci and Locus numbers through the action of modular group on real quadratic fields. The modular group PSL(2, Z) [1–3] an interesting group of hyperbolic isometries and has a finite presentation ⟨𝑥, 𝑦: 𝑥2 = 𝑦3 = 1⟩, (1) (1) where 𝑥and 𝑦are the linear fractional transformations defined by 𝑧→−1/𝑧and 𝑧→(𝑧−1)/𝑧, respectively. The modular group PSL(2, Z) is of great interest in many fields of mathematics, for example, group theory, graph theory, and number theory. By adjoining a new element 𝑡: 𝑧→1/𝑧with 𝑥and 𝑦, we obtain a presentation ⟨𝑥, 𝑦, 𝑡: 𝑥2 = 𝑦3 = 𝑡2 = (𝑥𝑡)2 = (𝑦𝑡)2 = 1⟩ (2) (2) of the extended modular group PGL(2, Z).h The Hecke group 𝐻(𝜆𝑞) is the discrete group generated by 𝑧→−1/𝑧, 𝑧→−1/(𝑧+𝜆𝑞), where 𝜆𝑞= 2 cos(𝜋/𝑞). It is well known that the modular group can be generalized to the Hecke groups. For 𝑞= 3, the resulting Hecke group 𝐻(𝜆3) is the modular group PSL(2, Z). In 1978, G. Higman introduced a new type of graph called a coset diagram for PGL(2, Z). The three cycles of 𝑦 are denoted by small triangles whose vertices are permuted counterclockwise by 𝑦and any two vertices which are interchanged by 𝑥are joined by an edge. The fixed points of 𝑥and 𝑦are denoted by heavy dots. Notice (𝑦𝑡)2 = 1 is equivalent to 𝑡𝑦𝑡= 𝑦−1, which means that 𝑡reverses the orientation of the triangles representing the three cycles of 𝑦 (as reflection does); because of this, there is no need to make the diagram more complicated by introducing 𝑡-edges. For details about coset diagrams, one can refer to [11–14]. Qaiser Mushtaq and Abdul Razaq Department of Mathematics, Quaid-i-Azam University, Islamabad, Pakistan Department of Mathematics, Quaid-i-Azam University, Islamabad, Pakistan Correspondence should be addressed to Abdul Razaq; makenqau@gmail.com Received 2 May 2014; Revised 26 June 2014; Accepted 10 July 2014; Published 8 September 2014 Copyright © 2014 Q. Mushtaq and A. Razaq. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Higman has defined coset diagrams for PGL(2, Z). The coset diagrams are composed of fragments, and the fragments are further composed of two or more circuits. A condition for the existence of a certain fragment 𝛾in a coset diagram is a polynomial f in Z[𝑧], obtained by choosing a pair of words 𝐹[𝑤𝑖, 𝑤𝑗] such that both 𝑤𝑖and 𝑤𝑗fix a vertex v in 𝛾. Two pairs of words are equivalent if and only if they have the same polynomial. In this paper, we find distinct pairs of words that are equivalent. We also show there are certain fragments, which have the same orientations as those of their mirror images. Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 505496, 8 pages http://dx.doi.org/10.1155/2014/505496 Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 505496, 8 pages http://dx.doi.org/10.1155/2014/505496 Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2014, Article ID 505496, 8 pages http://dx.doi.org/10.1155/2014/505496 1. Introduction If 𝑞is a power of a prime 𝑝, then the projective line over the finite field 𝐹𝑞, which is 𝐹𝑞∪{∞}, is denoted by PL(𝐹𝑞). The group PGL(2, 𝑞) has its customary meaning, a group of all linear fractional transformations 𝑧→(𝑎𝑧+ 𝑏)/(𝑐𝑧+ 𝑑) such that 𝑎, 𝑏, 𝑐, 𝑑∈𝐹𝑞and 𝑎𝑑−𝑏𝑐 ̸= 0, while PSL(2, 𝑞) is its subgroup consisting of all those, where 𝑎𝑑−𝑏𝑐is a non-zero square in 𝐹𝑞. 2 2 The Scientific World Journal The Scientific World Journal Two homomorphisms 𝛼and 𝛽from PGL(2, Z) to PGL(2, 𝑞) are called conjugate if 𝛽= 𝛼𝜌for some inner auto- morphism 𝜌on PGL(2, 𝑞). We call 𝛼to be non-degenerate if neither of 𝑥, 𝑦lies in the kernel of 𝛼. In [15], it has been shown that the conjugacy classes of non-degenerate homomorphisms from PGL(2, Z) to PGL(2, 𝑞) are in one to one correspondence with the elements 𝜃 ̸= 0, 3 of 𝐹𝑞under the correspondence which maps each class to its parameter 𝜃. As in [15], the coset diagram corresponding to the action of PGL(2, Z) on PL(𝐹𝑞) via a homomorphism 𝛼with parameter 𝜃is denoted by 𝐷(𝜃, 𝑞). Each conjugacy class is represented by a unique coset diagram, unique in the sense that the diagram remains invariant except that labels of vertices change from 𝛼 to 𝛽. The coset diagram 𝐷(𝜃, 𝑞) is made of fragments. It is therefore necessary to ask when a fragment exists in 𝐷(𝜃, 𝑞). In [16], this question is answered in the following way. Theorem 2. Given a fragment, there is a polynomial 𝑓in Z[𝑧] such that (i) if the fragment occurs in 𝐷(𝜃, 𝑞), then 𝑓(𝜃) = 0; (ii) if 𝑓(𝜃) = 0, then the fragment, or a homomorphic image of it occurs in 𝐷(𝜃, 𝑞) or in PL(𝐹𝑞). (i) if the fragment occurs in 𝐷(𝜃, 𝑞), then 𝑓(𝜃) = 0; (ii) if 𝑓(𝜃) = 0, then the fragment, or a homomorphic image of it occurs in 𝐷(𝜃, 𝑞) or in PL(𝐹𝑞). In [16], the method of calculating a polynomial from a fragment is given. Here, we describe this method briefly. Since a fragment is composed of two non-periodic and connected circuits (𝑛1, 𝑛2, . . . , 𝑛2𝑘) and (𝑚1, 𝑚2, . . . 2. Joining of Circuits where 𝑘1, 𝑘2 > 0. By using (3.1) to (3.7) of [16], the matrices 𝑊𝑖and 𝑊𝑗can be expressed linearly as Consider two non-periodic and simple circuits (𝑛1, 𝑛2, . . . , 𝑛2𝑘) and (𝑚1, 𝑚2, . . . , 𝑚2𝑘). Let V𝑖be any vertex in (𝑛1, 𝑛2, . . . , 𝑛2𝑘) fixed by a word 𝑤𝑖and let V𝑗be any vertex in (𝑚1, 𝑚2, . . . , 𝑚2𝑘) fixed by a word 𝑤𝑗. In order to connect these two circuits at V𝑖and V𝑗, we choose, without loss of generality (𝑛1, 𝑛2, . . . , 𝑛2𝑘), and apply 𝑤𝑗on V𝑖in such a way that 𝑤𝑗ends at V𝑖. Consequently, we get a fragment denoted by 𝛾. In other words, by 𝛾, we will mean a nonsimple fragment whose one vertex V = V𝑖= V𝑗is fixed by a pair of words 𝑤𝑖, 𝑤𝑗, dented by 𝐹[𝑤𝑖, 𝑤𝑗]. 𝑊𝑖= 𝜆0𝐼+ 𝜆1𝑋+ 𝜆2𝑌+ 𝜆3𝑋𝑌, 𝑊𝑗= 𝜇0𝐼+ 𝜇1𝑋+ 𝜇2𝑌+ 𝜇3𝑋𝑌 (6) (6) such that 𝜆𝑖and 𝜇𝑖, for 𝑖= 0, 1, 2, 3, are polynomials in 𝑟and Δ, where 𝑟is the trace of 𝑋𝑌and Δ is its determinant. Since V is a common fixed vertex of 𝑤𝑖and 𝑤𝑗, therefore the 2 × 2 matrices 𝑊𝑖and 𝑊𝑗have a common eigenvector. Then, by Lemma 3.1 of [16], the algebra generated by 𝑊𝑖and 𝑊𝑗has dimension 3. The algebra contains 𝐼, 𝑊𝑖, 𝑊𝑗, and 𝑊𝑖𝑊𝑗and so these are linearly dependent. Using (3.1) to (3.7) of [16], the matrix 𝑊𝑖𝑊𝑗is expressed as such that 𝜆𝑖and 𝜇𝑖, for 𝑖= 0, 1, 2, 3, are polynomials in 𝑟and Δ, where 𝑟is the trace of 𝑋𝑌and Δ is its determinant. Since V is a common fixed vertex of 𝑤𝑖and 𝑤𝑗, therefore the 2 × 2 matrices 𝑊𝑖and 𝑊𝑗have a common eigenvector. Then, by Lemma 3.1 of [16], the algebra generated by 𝑊𝑖and 𝑊𝑗has dimension 3. The algebra contains 𝐼, 𝑊𝑖, 𝑊𝑗, and 𝑊𝑖𝑊𝑗and so these are linearly dependent. Using (3.1) to (3.7) of [16], the matrix 𝑊𝑖𝑊𝑗is expressed as Example 1. We connect the vertex V, fixed by (𝑥𝑦)(𝑥𝑦−1) 3 (𝑥𝑦)3, in (4, 3) with the vertex 𝑢, fixed by (𝑥𝑦)3(𝑥𝑦−1) 2 of (3, 2), and compose a fragment 𝛾as shown in Figures 1, 2, and 3. 1. Introduction , 𝑚2𝑘) with a com- mon fixed vertex, say, V, then there is a pair of words, for instance, 𝛽 By a circuit in a coset diagram for an action of PGL(2, Z) on PL(𝐹𝑞), we will mean a closed path of triangles and edges. Let 𝑛1, 𝑛2, . . . , 𝑛2𝑘be a sequence of positive inte- gers, the circuit which contains a vertex, fixed by 𝑤 = (𝑥𝑦)𝑛1(𝑥𝑦−1)𝑛2 ⋅⋅⋅(𝑥𝑦−1)𝑛2𝑘∈PSL(2, Z) for some 𝑘≥1; we will mean the circuit in which 𝑛1 triangles have one vertex inside the circuit and 𝑛2 triangles have one vertex outside the circuit and so on. Since it is a cycle (𝑛1, 𝑛2, . . . , 𝑛2𝑘), so it does not make any difference if 𝑛1 triangles have one vertex outside the circuit and 𝑛2 triangles have one vertex inside the circuit and so on. The circuit of the type (𝑛1, 𝑛2, . . . , 𝑛2𝑘󸀠, 𝑛1, 𝑛2, . . . , 𝑛2𝑘󸀠, . . . , 𝑛1, 𝑛2, . . . , 𝑛2𝑘󸀠) is called a periodic circuit and the length of its period is 2𝑘󸀠. A circuit that is not of this type is non-periodic. A circuit is called simple, if each vertex of the circuit is fixed by a unique word 𝑤or its inverse 𝑤−1. 𝐹[𝑤𝑖, 𝑤𝑗] = 𝐹[(𝑥𝑦)𝑙1(𝑥𝑦−1) 𝑙2 ⋅⋅⋅(𝑥𝑦−1) 𝑙2𝑘1, (𝑥𝑦)𝑚1(𝑥𝑦−1) 𝑚2 ⋅⋅⋅(𝑥𝑦−1) 𝑚2𝑘2 ] (4) (4) such that (V)𝑤𝑖= V and (V)𝑤𝑗= V. Let 𝑋and 𝑌be the matrices corresponding to 𝑥and 𝑦of PGL(2, 𝑞). Then, 𝑤𝑖and 𝑤𝑗can be expressed as 𝑊𝑖= (𝑋𝑌)𝑙1(𝑋𝑌−1) 𝑙2 ⋅⋅⋅(𝑋𝑌−1) 𝑙2𝑘1 , 𝑊𝑗= (𝑋𝑌)𝑚1(𝑋𝑌−1) 𝑚2 ⋅⋅⋅(𝑋𝑌−1) 𝑚2𝑘2 , (5) (5) 3. Main Results Theorem 3. In the above notation, the polynomial obtained from a fragment 𝛾is unique. Proof. Let V𝑖and V𝑘be any two vertices of a fragment 𝛾, such that V𝑖is fixed by a pair of words 𝐹[𝑤𝑖, 𝑤𝑗] and V𝑘is fixed by a pair of words 𝐹[𝑤𝑘, 𝑤𝑙]. Suppose 𝑓(𝜃) is a polynomial obtained by choosing 𝐹[𝑤𝑖, 𝑤𝑗] and 𝑔(𝜃) is a polynomial obtained by choosing 𝐹[𝑤𝑘, 𝑤𝑙]. Now, if 𝑓(𝜃) = 0, then, by Theorem 2, the fragment 𝛾or its homomorphic image occurs in 𝐷(𝜃, 𝑞) or in PL(𝐹𝑞). So, there exists a vertex in 𝐷(𝜃, 𝑞) or in PL(𝐹𝑞) which is fixed by 𝐹[𝑤𝑘, 𝑤𝑙]. Again, by Theorem 2, we have 𝑔(𝜃) = 0. Similarly, it can be proved that if 𝑔(𝜃) = 0, then 𝑓(𝜃) = 0. Hence, 𝑓(𝜃) = 𝑔(𝜃). Figure 2 Figure 2 Remark 4. If a vertex V is fixed by a word 𝑤𝑖∈PSL(2, Z), then the vertex (V)𝑤is fixed by the conjugate 𝑤−1𝑤𝑖𝑤of 𝑤𝑖. We know that 𝛾is a fragment created by joining a vertex V𝑖of (𝑛1, 𝑛2, . . . , 𝑛2𝑘) with the vertex V𝑗of (𝑚1, 𝑚2, . . . , 𝑚2𝑘). Since the same polynomial is evolved for all the points of connection for the fragment 𝛾, therefore it is important to know all the points of connection for the fragment 𝛾. Follow- ing theorem is useful in finding all points of connection of (𝑛1, 𝑛2, . . . , 𝑛2𝑘) and (𝑚1, 𝑚2, . . . , 𝑚2𝑘) for the fragment 𝛾. Figure 3 Theorem 5. Let fragment 𝛾be constructed by joining a vertex V𝑖of (𝑛1, 𝑛2, . . . , 𝑛2𝑘) with the vertex V𝑗of (𝑚1, 𝑚2, . . . , 𝑚2𝑘). Then, 𝛾is obtainable also, if the vertex (V𝑖)𝑤of (𝑛1, 𝑛2, . . . , 𝑛2𝑘) is joined with the vertex (V𝑗)𝑤of (𝑚1, 𝑚2, . . . , 𝑚2𝑘). If we calculate V1, V2, V3 in terms of 𝜆𝑖and 𝜇𝑖and substitute in (8), we find that it is equivalent to If we calculate V1, V2, V3 in terms of 𝜆𝑖and 𝜇𝑖and substitute in (8), we find that it is equivalent to (𝜆2𝜇3 −𝜇2𝜆3) 2 + Δ(𝜆3𝜇1 −𝜇3𝜆1) 2 + (𝜆1𝜇2 −𝜇1𝜆2) 2 + 𝑟(𝜆2𝜇3 −𝜇2𝜆3) (𝜆3𝜇1 −𝜇3𝜆1) + (𝜆2𝜇3 −𝜇2𝜆3) (𝜆1𝜇2 −𝜇1𝜆2) = 0. (9) Proof. Suppose vertex V𝑖is fixed by a word 𝑤𝑖and vertex V𝑗is fixed by a word 𝑤𝑗. 2. Joining of Circuits 𝑊𝑖𝑊𝑗= ]0𝐼+ ]1𝑋+ ]2𝑌+ ]3𝑋𝑌, (7) (7) One can see that the vertex V = 𝑢in 𝛾(Figure 3) is fixed by a pair of words where V𝑖, for 𝑖= 0, 1, 2, 3, can be calculated in terms of the 𝜆𝑖 and 𝜇𝑖, using (3.1) to (3.7) of [16]. The condition that 𝐼, 𝑊𝑖, 𝑊𝑗, and 𝑊𝑖𝑊𝑗are linearly dependent is expressed as 𝐹[(𝑥𝑦) (𝑥𝑦−1) 3(𝑥𝑦)3, (𝑥𝑦)3(𝑥𝑦−1) 2] . (3) (3) 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 𝜆1 𝜆2 𝜆3 𝜇1 𝜇2 𝜇3 ]1 ]2 ]3 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 = 0. (8) 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 𝜆1 𝜆2 𝜆3 𝜇1 𝜇2 𝜇3 ]1 ]2 ]3 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 = 0. (8) n of PGL(2, Z) on PL(𝐹𝑞2) yields two compo- y, PL(𝐹𝑞) and PL(𝐹𝑞2) \ PL(𝐹𝑞). For sake of PL(𝐹𝑞) denote the complement PL(𝐹𝑞2)\PL(𝐹𝑞). 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 𝜆1 𝜆2 𝜆3 𝜇1 𝜇2 𝜇3 ]1 ]2 ]3 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 = 0. (8) The action of PGL(2, Z) on PL(𝐹𝑞2) yields two compo- nents, namely, PL(𝐹𝑞) and PL(𝐹𝑞2) \ PL(𝐹𝑞). For sake of simplicity, let PL(𝐹𝑞) denote the complement PL(𝐹𝑞2)\PL(𝐹𝑞). (8) 3 3 The Scientific World Journal Figure 1 Figure 1 Figure 1 u Figure 2 3. Main Results Then, one vertex of the fragment 𝛾is fixed by 𝑤𝑖and 𝑤𝑗. Now, we join the vertex (V𝑖)𝑤of (𝑛1, 𝑛2, . . . , 𝑛2𝑘) with the vertex (V𝑗)𝑤of (𝑚1, 𝑚2, . . . , 𝑚2𝑘) and compose a fragment 𝛿. By Remark 4, the vertex (V)𝑤of fragment 𝛿is fixed by 𝑤−1𝑤𝑖𝑤and 𝑤−1𝑤𝑗𝑤, whereas the vertex ((V)𝑤)𝑤−1 = V of fragment 𝛿is fixed by 𝑤(𝑤−1𝑤𝑖𝑤)𝑤−1 = 𝑤𝑖 and 𝑤(𝑤−1𝑤𝑗𝑤)𝑤−1 = 𝑤𝑗. This shows that 𝛾and 𝛿are the same fragments. (9) This gives a homogeneous equation in Δ and 𝑟. By substituting Δ𝜃for 𝑟2, we get an equation in 𝜃. Two pairs of words 𝐹[𝑤𝑖, 𝑤𝑗] and 𝐹[𝑤𝑘, 𝑤𝑙] are equivalent if and only if they have the same polynomial. If two pairs of words 𝐹[𝑤𝑖, 𝑤𝑗] and 𝐹[𝑤𝑘, 𝑤𝑙] are equivalent, then we write 𝐹[𝑤𝑖, 𝑤𝑗] ∼𝐹[𝑤𝑘, 𝑤𝑙].i Since 𝛾and 𝛿are the same fragments, and a unique polynomial is obtained from a fragment, hence 𝐹[𝑤𝑖, 𝑤𝑗] ∼ 𝐹[𝑤−1𝑤𝑖𝑤, 𝑤−1𝑤𝑗𝑤]. 𝑗 In this paper, we find distinct pairs of words that are equivalent. We also show there are certain fragments, which have the same orientations as those of their mirror images. 4 The Scientific World Journal Corollary 6. Let 𝑃be the set of words such that for any 𝑤∈𝑃, both vertices (V𝑖)𝑤and (V𝑗)𝑤lie on the circuits (𝑛1, 𝑛2, . . . , 𝑛2𝑘) and (𝑚1, 𝑚2, . . . , 𝑚2𝑘). Let 𝑠be the total number of points of connection of the circuits to compose 𝛾. Then, 𝑠= \|𝑃\|. respectively. Let the words 𝑤𝑖, 𝑤𝑗, and 𝑤𝑖𝑤𝑗fix the vertices V𝑖, V𝑗, and V𝑘, respectively. We join a vertex V𝑖fixed by 𝑤𝑖of the circuit (𝑛1, 𝑛2, . . . , 𝑛2𝑘) with the vertex V𝑗fixed by 𝑤𝑗of the circuit (𝑚1, 𝑚2, . . . , 𝑚2𝑘) to compose 𝛾. Also, we join a vertex V𝑖fixed by 𝑤𝑖of the circuit (𝑛1, 𝑛2, . . . , 𝑛2𝑘) with the vertex fixed by 𝑤𝑖𝑤𝑗of the (𝑚󸀠 1, 𝑚󸀠 2, . . . , 𝑚󸀠 2𝑘) to compose a fragment 𝛿. So, in 𝛿, V𝑖is a common fixed vertex of 𝐹[𝑤𝑖, 𝑤𝑖𝑤𝑗]. The fragment 𝛿is different from the fragment 𝛾if and only if, in fragment 𝛿, the word 𝑤𝑖𝑤𝑗fixes the vertex V𝑖in the following way: 𝑤𝑖maps the vertex V𝑖to some vertex 𝑎 ̸= V𝑖, and then 𝑤𝑖 𝑤𝑗 respectively. Let the words 𝑤𝑖, 𝑤𝑗, and 𝑤𝑖𝑤𝑗fix the vertices V𝑖, V𝑗, and V𝑘, respectively. 3. Main Results We join a vertex V𝑖fixed by 𝑤𝑖of the circuit (𝑛1, 𝑛2, . . . , 𝑛2𝑘) with the vertex V𝑗fixed by 𝑤𝑗of the circuit (𝑚1, 𝑚2, . . . , 𝑚2𝑘) to compose 𝛾. Also, we join a vertex V𝑖fixed by 𝑤𝑖of the circuit (𝑛1, 𝑛2, . . . , 𝑛2𝑘) with the vertex fixed by 𝑤𝑖𝑤𝑗of the (𝑚󸀠 1, 𝑚󸀠 2, . . . , 𝑚󸀠 2𝑘) to compose a fragment 𝛿. So, in 𝛿, V𝑖is a common fixed vertex of 𝐹[𝑤𝑖, 𝑤𝑖𝑤𝑗]. The fragment 𝛿is different from the fragment 𝛾if and only if, in fragment 𝛿, the word 𝑤𝑖𝑤𝑗fixes the vertex V𝑖in the following way: 𝑤𝑖maps the vertex V𝑖to some vertex 𝑎 ̸= V𝑖, and then 𝑤𝑖 𝑤𝑗 Example 7. As in Example 1, the vertex V in (4, 3) is connected with the vertex 𝑢in (3, 2), and a fragment 𝛾is evolved. Then, one can see that 𝑃= {𝑦, 𝑦−1, 𝑒, 𝑥, 𝑥𝑦, 𝑥𝑦−1, 𝑥𝑦𝑥, 𝑥𝑦𝑥𝑦, 𝑥𝑦𝑥𝑦−1} (10) (10) 𝑤𝑗maps the vertex 𝑎to the vertex V𝑖; that is, V𝑖 𝑤𝑖 󳨀󳨀→𝑎 𝑤𝑗 󳨀󳨀→V𝑖. Since 𝑤𝑖is a composition of linear fractional transformations 𝑥and 𝑦, therefore 𝑎= V𝑖. This implies that 𝛾and 𝛿are the same fragments, since a unique polynomial is obtained from a fragment. Hence, 𝐹[𝑤𝑖, 𝑤𝑗] ∼𝐹[𝑤𝑖, 𝑤𝑖𝑤𝑗]. is the set of words such that, for any 𝑤∈𝑃, both vertices (V) 𝑤 and (𝑢) 𝑤lie on (4, 3) and (3, 2). Since \|𝑃\| = 9, there are nine points of connection of (4, 3) and (3, 2) composing 𝛾. Hence, 9 vertices of one circuit are merged in another circuit. In other words, 3 triangles of one circuit are merged in another circuit. g 𝑖 𝑗 𝑖 Similarly, it is easy to prove that 𝐹[𝑤𝑖, 𝑤𝑗] ∼𝐹[𝑤𝑖, 𝑤𝑖𝑤−1 𝑗] , 𝐹[𝑤𝑖, 𝑤𝑗] ∼𝐹[𝑤𝑖, 𝑤𝑗𝑤𝑖] , 𝐹[𝑤𝑖, 𝑤𝑗] ∼𝐹[𝑤𝑖, 𝑤𝑗𝑤−1 𝑖] . (12) The number of points of connection of the circuits for a fragment is always multiple of three and plays a significant role because they are directly related to the structure of the fragment. The following theorem illustrates this relation. (12) Since ∼is an equivalence relation, hence Since ∼is an equivalence relation, hence Since ∼is an equivalence relation, hence Theorem8. Let 𝑟be the total number of triangles in a fragment 𝛾and 𝑠the total number of points of connection of the circuits (𝑛1, 𝑛2, . . . , 𝑛2𝑘) and (𝑚1, 𝑚2, . . . , 𝑚2𝑘). Then, 𝑟= ∑2𝑘 𝑗=1 𝑛𝑗+ ∑2𝑘 𝑗=1 𝑚𝑗−(1/3)𝑠. 𝐹[𝑤𝑖, 𝑤𝑗] ∼𝐹[𝑤𝑖, 𝑤𝑖𝑤𝑗] ∼𝐹[𝑤𝑖, 𝑤𝑖𝑤−1 𝑗] ∼𝐹[𝑤𝑖, 𝑤𝑗𝑤𝑖] ∼𝐹[𝑤𝑖, 𝑤𝑗𝑤−1 𝑖] . (13) (13) Proof. Since circuits (𝑛1, 𝑛2, . . . , 𝑛2𝑘) and (𝑚1, 𝑚2, . . . , 𝑚2𝑘) have ∑2𝑘 𝑗=1 𝑛𝑗and ∑2𝑘 𝑗=1 𝑚𝑗number of triangles, respec- tively, and there are 𝑠number of points of connection of (𝑛1, 𝑛2, . . . , 𝑛2𝑘) and (𝑚1, 𝑚2, . . . , 𝑚2𝑘) composing 𝛾, there- fore, by Corollary 6, there are 𝑠number of words 𝑤such that (V𝑖) 𝑤, (V𝑗) 𝑤lie on (𝑛1, 𝑛2, . . . , 𝑛2𝑘) and (𝑚1, 𝑚2, . . . , 𝑚2𝑘), respectively. So, 𝑠number of vertices of (𝑚1, 𝑚2, . . . , 𝑚2𝑘) are merged in (𝑛1, 𝑛2, . . . , 𝑛2𝑘), and remaining 3 ∑2𝑘 𝑗=1 𝑚𝑗− 𝑠number of vertices are included in (𝑛1, 𝑛2, . . . , 𝑛2𝑘) to compose 𝛾. So, the total number of vertices in 𝛾is (3 ∑2𝑘 𝑗=1 𝑚𝑗− 𝑠+ 3 ∑2𝑘 𝑗=1 𝑛𝑗). This implies that 𝑟= ∑2𝑘 𝑗=1 𝑛𝑗+ ∑2𝑘 𝑗=1 𝑚𝑗− (1/3)𝑠. Example 11. Theorem 10 shows that the fragment 𝛾, which has a vertex fixed by 𝐹[𝑤𝑖, 𝑤𝑗], and the fragment Example 11. Theorem 10 shows that the fragment 𝛾, which has a vertex fixed by 𝐹[𝑤𝑖, 𝑤𝑗], and the fragment 𝛿󸀠, which has a vertex fixed by 𝐹[𝑤𝑖, 𝑤𝑗𝑤𝑖], are the same. So, the fragment, which has a vertex fixed by 𝐹[(𝑥𝑦)2(𝑥𝑦−1), (𝑥𝑦−1)(𝑥𝑦)3], and the fragment, which has a vertex fixed by 𝐹[(𝑥𝑦)2(𝑥𝑦−1), (𝑥𝑦−1)(𝑥𝑦)5(𝑥𝑦−1)], are the same. Since the words (𝑥𝑦)2(𝑥𝑦−1), (𝑥𝑦−1)(𝑥𝑦)3, and (𝑥𝑦−1)(𝑥𝑦)5(𝑥𝑦−1) correspond to the circuits (2, 1), (3, 1), and (5, 2), so the fragment (Figure 4) is obtained, not only by joining the vertex V1 in (2, 1) (Figure 5) with the vertex V2 in (3, 1) (Figure 6) but also by joining the vertex V1 in (2, 1) (Figure 7) with the vertex 𝑢2 in (5, 2) (Figure 8). Remark 9. Consider a fragment 𝛾such that one vertex V of 𝛾is fixed by 𝐹[𝑤𝑖, 𝑤𝑗]. Since ∼is an equivalence relation, hence In Figure 9, one can see that the vertex 𝑢1 is fixed by (𝑥𝑦)𝑛1(𝑥𝑦−1)𝑛2 and 𝑢3𝑛2 is fixed by (𝑥𝑦−1)𝑛1(𝑥𝑦)𝑛2. So, 𝑢3𝑛2 = 𝑢∗ 1. Also, the vertex 𝑢2 is fixed by 𝑦(𝑥𝑦)𝑛1(𝑥𝑦−1)𝑛2−1𝑥𝑦and 𝑢3𝑛2−1 is fixed by 𝑦−1(𝑥𝑦−1)𝑛1(𝑥𝑦)𝑛2−1𝑥𝑦−1. So, 𝑢3𝑛2−1 = 𝑢∗ 2. Similarly, 𝑢3𝑛2−2 = 𝑢∗ 3, 𝑢3𝑛2−3 = 𝑢∗ 4, . . . . The vertices V1 and V3𝑛1 are fixed by (𝑥𝑦)𝑛2(𝑥𝑦−1)𝑛1 and (𝑥𝑦−1)𝑛2(𝑥𝑦)𝑛1, respectively, implying that V3𝑛2 = V∗ 1. Also, the vertices V2 and V3𝑛1−1 are fixed by 𝑦(𝑥𝑦)𝑛2(𝑥𝑦−1)𝑛1−1𝑥𝑦and 𝑦−1(𝑥𝑦−1)𝑛2(𝑥𝑦)𝑛1−1𝑥𝑦−1, respectively, implying that V3𝑛1−1 = V∗ 2. Similarly, V3𝑛1−2 = V∗ 3, V3𝑛2−3 = V∗ 4, . . . . Let the homomorphic image of the fragment (Figure 10) occur in the coset diagram 𝐷(𝜃, 𝑞). Since 𝐷(𝜃, 𝑞) admits the axis of symmetry, the mirror image of the fragment under the permutation 𝑡will also occur as shown in Figure 11. Proposition 14. If the fragment 𝛾has one vertex V fixed by 𝐹[𝑤𝑖, 𝑤𝑗], then its mirror image 𝛾∗has one vertex fixed by 𝐹[𝑤∗ 𝑖, 𝑤∗ 𝑗]. Proof. Let 𝛾be the fragment with one vertex V fixed by 𝐹[𝑤𝑖, 𝑤𝑗]. Since 𝛾∗is the mirror image of 𝛾, therefore 𝛾∗has hi Figure 4 1 Figure 5 2 Figure 6 1 Figure 7 Figure 4 u2 Figure 8 Figure 8 Figure 4 u3n2 u3n2−2 u3n2−1 u1 u2 u3 u4 u5 u6 1 2 3 4 5 6 3n1 3n1−2 3n1−1 Figure 9 1 Figure 5 1 Figure 5 Figure 9 2 Figure 6 1 Figure 7 Theorem 13. If a vertex V is fixed by a word 𝑤in a circuit (𝑛1, 𝑛2), then there exists a vertex V∗in (𝑛1, 𝑛2) such that V∗ is fixed by 𝑤∗. Proof. Consider a simple circuit (𝑛1, 𝑛2). In Figure 9, one can see that the vertex 𝑢1 is fixed by (𝑥𝑦)𝑛1(𝑥𝑦−1)𝑛2 and 𝑢3𝑛2 is fixed by (𝑥𝑦−1)𝑛1(𝑥𝑦)𝑛2. So, 𝑢3𝑛2 = 𝑢∗ 1. Also, the vertex 𝑢2 is fixed by 𝑦(𝑥𝑦)𝑛1(𝑥𝑦−1)𝑛2−1𝑥𝑦and 𝑢3𝑛2−1 is fixed by 𝑦−1(𝑥𝑦−1)𝑛1(𝑥𝑦)𝑛2−1𝑥𝑦−1. So, 𝑢3𝑛2−1 = 𝑢∗ 2. Similarly, 𝑢3𝑛2−2 = 𝑢∗ 3, 𝑢3𝑛2−3 = 𝑢∗ 4, . . . . The vertices V1 and V3𝑛1 are fixed by (𝑥𝑦)𝑛2(𝑥𝑦−1)𝑛1 and (𝑥𝑦−1)𝑛2(𝑥𝑦)𝑛1, respectively, implying that V3𝑛2 = V∗ 1. Also, the vertices V2 and V3𝑛1−1 are fixed by 𝑦(𝑥𝑦)𝑛2(𝑥𝑦−1)𝑛1−1𝑥𝑦and 𝑦−1(𝑥𝑦−1)𝑛2(𝑥𝑦)𝑛1−1𝑥𝑦−1, respectively, implying that V3𝑛1−1 = V∗ 2. Similarly, V3𝑛1−2 = V∗ 3, V3𝑛2−3 = V∗ 4, . . . . Since ∼is an equivalence relation, hence Then, obviously, V is fixed by 𝐹[𝑤−1 𝑖, 𝑤−1 𝑗]. So, 𝐹[𝑤𝑖, 𝑤𝑗] ∼𝐹[𝑤−1 𝑖, 𝑤−1 𝑗]. Also, it is trivial that 𝐹[𝑤𝑖, 𝑤𝑗] ∼ 𝐹[𝑤𝑗, 𝑤𝑖]. Remark 9. Consider a fragment 𝛾such that one vertex V of 𝛾is fixed by 𝐹[𝑤𝑖, 𝑤𝑗]. Then, obviously, V is fixed by 𝐹[𝑤−1 𝑖, 𝑤−1 𝑗]. So, 𝐹[𝑤𝑖, 𝑤𝑗] ∼𝐹[𝑤−1 𝑖, 𝑤−1 𝑗]. Also, it is trivial that 𝐹[𝑤𝑖, 𝑤𝑗] ∼ 𝐹[𝑤𝑗, 𝑤𝑖]. Theorem 12. In a coset diagram for the action of PGL(2, Z) on PL(𝐹𝑞), if a vertex V is fixed by 𝑤, then the vertex (V)𝑡is fixed by 𝑤∗. The following Theorem shows that (𝑛1, 𝑛2, . . . , 𝑛2𝑘) and (𝑚1, 𝑚2, . . . , 𝑚2𝑘) are not a unique pair of circuits for the fragment 𝛾. Proof. Let 𝑤= 𝑥𝑦𝜂1𝑥𝑦𝜂2 ⋅⋅⋅𝑥𝑦𝜂𝑛−1𝑥𝑦𝜂𝑛be a word such that (V) 𝑥𝑦𝜂1𝑥𝑦𝜂2 ⋅⋅⋅𝑥𝑦𝜂𝑛−1𝑥𝑦𝜂𝑛= V. (14) Since (𝑥𝑡)2 = (𝑦𝑡)2 = 1, this implies that 𝑦𝑡= 𝑡𝑦−1, 𝑦−1𝑡= 𝑡𝑦, 𝑥𝑡= 𝑡𝑥. (15) Proof. Let 𝑤= 𝑥𝑦𝜂1𝑥𝑦𝜂2 ⋅⋅⋅𝑥𝑦𝜂𝑛−1𝑥𝑦𝜂𝑛be a word such that (V) 𝑥𝑦𝜂1𝑥𝑦𝜂2 ⋅⋅⋅𝑥𝑦𝜂𝑛−1𝑥𝑦𝜂𝑛= V. (14) (14) Theorem 10. The following pairs of words are equivalent: Since (𝑥𝑡)2 = (𝑦𝑡)2 = 1, this implies that Since (𝑥𝑡)2 = (𝑦𝑡)2 = 1, this implies that 𝐹[𝑤𝑖, 𝑤𝑗] , 𝐹[𝑤𝑖, 𝑤𝑖𝑤𝑗] , 𝐹[𝑤𝑖, 𝑤𝑖𝑤−1 𝑗] , 𝐹[𝑤𝑖, 𝑤𝑗𝑤𝑖] , 𝐹[𝑤𝑖, 𝑤𝑗𝑤−1 𝑖] . (11) 𝑦𝑡= 𝑡𝑦−1, 𝑦−1𝑡= 𝑡𝑦, 𝑥𝑡= 𝑡𝑥. (15) (15) (11) Now, by using (14) and (15), we have (V)𝑡 = ((V)𝑥𝑦𝜂1𝑥𝑦𝜂2 ⋅⋅⋅𝑥𝑦𝜂𝑛−1𝑥𝑦𝜂𝑛)𝑡 = ((V)𝑡)𝑥𝑦−𝜂1𝑥𝑦−𝜂2 ⋅⋅⋅𝑥𝑦−𝜂𝑛−1 𝑥𝑦−𝜂𝑛= ((V)𝑡)𝑤∗. This shows that 𝑥𝑦−𝜂1𝑥𝑦−𝜂2 ⋅⋅⋅𝑥𝑦−𝜂𝑛−1𝑥𝑦−𝜂𝑛 also fixes a point (V)𝑡. Proof. Let V𝑖, V𝑗, and V𝑘be arbitrary vertices of the circuits (𝑛1, 𝑛2, . . . , 𝑛2𝑘), (𝑚1, 𝑚2, . . . , 𝑚2𝑘), and (𝑚󸀠 1, 𝑚󸀠 2, . . . , 𝑚󸀠 2𝑘), 5 The Scientific World Journal The Scientific World Journal 5 h e Sc e ti c Wo d Jou a 5 Figure 4 1 Figure 5 2 Figure 6 1 Figure 7 u2 Figure 8 u3n2 u3n2−2 u3n2−1 u1 u2 u3 u4 u5 u6 1 2 3 4 5 6 3n1 3n1−2 3n1−1 Figure 9 Theorem 13. If a vertex V is fixed by a word 𝑤in a circuit (𝑛1, 𝑛2), then there exists a vertex V∗in (𝑛1, 𝑛2) such that V∗ is fixed by 𝑤∗. Proof. Consider a simple circuit (𝑛1, 𝑛2). Since ∼is an equivalence relation, hence Figure 6 Let the homomorphic image of the fragment (Figure 10) occur in the coset diagram 𝐷(𝜃, 𝑞). Since 𝐷(𝜃, 𝑞) admits the axis of symmetry, the mirror image of the fragment under the permutation 𝑡will also occur as shown in Figure 11. Proposition 14. If the fragment 𝛾has one vertex V fixed by 𝐹[𝑤𝑖, 𝑤𝑗], then its mirror image 𝛾∗has one vertex fixed by 𝐹[𝑤∗ 𝑖, 𝑤∗ 𝑗]. Proof. Let 𝛾be the fragment with one vertex V fixed by 𝐹[𝑤𝑖, 𝑤𝑗]. Since 𝛾∗is the mirror image of 𝛾, therefore 𝛾∗has Figure 7 The Scientific World Journal 6 in (𝑛1, 𝑛2), with the vertex V2, fixed by (𝑥𝑦)𝑚1/2(𝑥𝑦−1)𝑚2 (𝑥𝑦)𝑚1/2 in (𝑚1, 𝑚2), has the same orientation as that of its mirror image. Figure 10 Figure 10 Proof. (i) Let 𝛾1 be the fragment which has one vertex say 𝑢, fixed by (𝑥𝑦−1) (𝑛1−1)/2(𝑥𝑦) 𝑛2(𝑥𝑦−1) (𝑛1+1)/2, (𝑥𝑦−1) (𝑚1−1)/2(𝑥𝑦)𝑚2(𝑥𝑦−1) (𝑚1+1)/2. (19) (19) Then, by Proposition 14, its mirror image fragment 𝛾∗ 1 con- tains a vertex, say, 𝑢∗, fixed by (𝑥𝑦)(𝑛1−1)/2(𝑥𝑦−1) 𝑛2(𝑥𝑦)(𝑛1+1)/2 and (𝑥𝑦)(𝑚1−1)/2(𝑥𝑦−1) 𝑚2(𝑥𝑦)(𝑚1+1)/2. By Remark 4, the vertex (𝑢∗)𝑦of fragment 𝛾∗ 1 is fixed by Figure 10 one vertex (V)𝑡. Then, by Theorem 12, the vertex (V)𝑡is fixed by 𝐹[𝑤∗ 𝑖, 𝑤∗ 𝑗]. one vertex (V)𝑡. Then, by Theorem 12, the vertex (V)𝑡is fixed by 𝐹[𝑤∗ 𝑖, 𝑤∗ 𝑗]. one vertex (V)𝑡. Then, by Theorem 12, the vertex (V)𝑡is fixed by 𝐹[𝑤∗ 𝑖, 𝑤∗ 𝑗]. 𝑦(𝑥𝑦)(𝑛1−1)/2(𝑥𝑦−1) 𝑛2(𝑥𝑦)(𝑛1+1)/2𝑦−1 = ((𝑥𝑦−1) (𝑛1−1)/2(𝑥𝑦)𝑛2(𝑥𝑦−1) (𝑛1+1)/2) −1 (20) (20) Theorem 15. The polynomials obtained from the fragment 𝛾 and its mirror image 𝛾∗are the same. and 𝑦(𝑥𝑦)(𝑚1−1)/2(𝑥𝑦−1) 𝑚2(𝑥𝑦)(𝑚1+1)/2𝑦−1 = ((𝑥𝑦−1)(𝑚1−1)/2 ( )/ −1h and 𝑦(𝑥𝑦)(𝑚1−1)/2(𝑥𝑦−1) 𝑚2(𝑥𝑦)(𝑚1+1)/2𝑦−1 = ((𝑥𝑦−1)(𝑚1−1)/2 (𝑥𝑦)𝑚2(𝑥𝑦−1)(𝑚1+1)/2) −1. This implies that 𝑢= (𝑢∗)𝑦. Hence fragment 𝛾1 has the same orientation as that of its mirror image 𝛾∗ 1 . Proof. Let 𝑓(𝜃) be the polynomial obtained by choosing a pair of words 𝐹[𝑤𝑖, 𝑤𝑗] from 𝛾and let 𝑔(𝜃) be the polynomial obtained by choosing a pair of words 𝐹[𝑤∗ 𝑖, 𝑤∗ 𝑗] from 𝛾∗. Let 𝑓(𝜃) = 0. Then, by Theorem 2, there is a vertex say V in 𝐷(𝜃, 𝑞) or in PL(𝐹𝑞) which is fixed by 𝐹[𝑤𝑖, 𝑤𝑗]. Then, by Theorem 12, the vertex (V)𝑡is fixed by 𝐹[𝑤∗ 𝑖, 𝑤∗ 𝑗]. So, by Theorem 2, 𝑔(𝜃) = 0. Similarly, it can be proved that if 𝑔(𝜃) = 0, then 𝑓(𝜃) = 0. Hence, 𝑓(𝜃) = 𝑔(𝜃). Proof. Since ∼is an equivalence relation, hence Let 𝑓(𝜃) be the polynomial obtained by choosing a pair of words 𝐹[𝑤𝑖, 𝑤𝑗] from 𝛾and let 𝑔(𝜃) be the polynomial obtained by choosing a pair of words 𝐹[𝑤∗ 𝑖, 𝑤∗ 𝑗] from 𝛾∗. Let 𝑓(𝜃) = 0. Then, by Theorem 2, there is a vertex say V in 𝐷(𝜃, 𝑞) or in PL(𝐹𝑞) which is fixed by 𝐹[𝑤𝑖, 𝑤𝑗]. Then, by Theorem 12, the vertex (V)𝑡is fixed by 𝐹[𝑤∗ 𝑖, 𝑤∗ 𝑗]. So, by Theorem 2, 𝑔(𝜃) = 0. Similarly, it can be proved that if 𝑔(𝜃) = 0, then 𝑓(𝜃) = 0. Hence, 𝑓(𝜃) = 𝑔(𝜃). (𝑥𝑦)𝑚2(𝑥𝑦−1)(𝑚1+1)/2) −1. This implies that 𝑢= (𝑢∗)𝑦. Hence fragment 𝛾1 has the same orientation as that of its mirror image 𝛾∗ 1 . g 𝛾1 (ii) Let 𝛾2 be the fragment which has one vertex, say, V, fixed by (𝑥𝑦−1) 𝑛1/2(𝑥𝑦)𝑛2(𝑥𝑦−1) 𝑛1/2, (𝑥𝑦−1) 𝑚1/2(𝑥𝑦)𝑚2(𝑥𝑦−1) 𝑚1/2. (21) (21) Since a unique polynomial is obtained from a fragment 𝛾 and its mirror image 𝛾∗, hence 𝐹[𝑤𝑖, 𝑤𝑗] ∼𝐹[𝑤∗ 𝑖, 𝑤∗ 𝑗]. Then, its mirror image fragment 𝛾∗ 2 has one vertex, say, V∗, fixed by 𝑗 𝑗 Consider two circuits (𝑛1, 𝑛2) and (𝑚1, 𝑚2). In general, fragment 𝛾and its mirror image 𝛾∗do not have the same orientation. There are certain fragments which have the same orientations as those of their mirror images. These kinds of fragments have vertical symmetry and may have fixed points of 𝑡. The following Theorem shows how these fragments are composed. (𝑥𝑦)𝑛1/2(𝑥𝑦−1) 𝑛2(𝑥𝑦)𝑛1/2, (𝑥𝑦)𝑚1/2(𝑥𝑦−1) 𝑚2(𝑥𝑦)𝑚1/2. (22) (22) The vertex (V∗)𝑥of fragment 𝛾∗ 2 is fixed by The vertex (V∗)𝑥of fragment 𝛾∗ 2 is fixed by 𝑥(𝑥𝑦)𝑛1/2(𝑥𝑦−1) 𝑛2(𝑥𝑦)𝑛1/2𝑥 = ((𝑥𝑦−1) 𝑛1/2(𝑥𝑦)𝑛2(𝑥𝑦−1) 𝑛1/2) −1 (23) Theorem 16. (i) The fragment composed by joining a vertex 𝑢1, fixed by Theorem 16. (i) The fragment composed by joining a vertex 𝑢1, fixed by (23) (𝑥𝑦−1) (𝑛1−1)/2(𝑥𝑦)𝑛2(𝑥𝑦−1) (𝑛1+1)/2 (16) (16) and 𝑥(𝑥𝑦)𝑚1/2(𝑥𝑦)𝑚2(𝑥𝑦)𝑚1/2𝑥 = ((𝑥𝑦−1)𝑚1/2(𝑥𝑦)𝑚2 1h and 𝑥(𝑥𝑦)𝑚1/2(𝑥𝑦)𝑚2(𝑥𝑦)𝑚1/2𝑥 = ((𝑥𝑦−1)𝑚1/2(𝑥𝑦)𝑚2 (𝑥𝑦−1)𝑚1/2) −1. This implies that V = (V∗)𝑥. Hence, 𝛾2 has the same orientation as that of its mirror image 𝛾∗ 2 . in (𝑛1, 𝑛2), with the vertex 𝑢2, fixed by (𝑥𝑦−1)(𝑚1−1)/2(𝑥𝑦)𝑚2 (𝑥𝑦−1)(𝑚1+1)/2 in (𝑚1, 𝑚2), has the same orientation as that of its mirror image.hi (𝑥𝑦−1)𝑚1/2) −1. This implies that V = (V∗)𝑥. Hence, 𝛾2 has the same orientation as that of its mirror image 𝛾∗ 2 . (𝑥𝑦−1)𝑚1/2) −1. This implies that V = (V∗)𝑥. 4. Motivation and Open Problems [4] R. Bowen and C. Series, “Markov maps associated with fuchsian groups,” Publications Math´ematiques de l’Institut des Hautes ´Etudes Scientifiques, vol. 50, no. 1, pp. 153–170, 1979. If we join a pair of circuits at a certain point, we get a fragment and hence a polynomial. Since each such polynomial splits linearly in a suitable Galois [16] and corresponding to each zero, we get a triplet (𝑥, 𝑦, 𝑡) [15] which is a group. This shows that each pair of circuits can be related to a group. A pair of circuits has finitely many points of connections. Two distinct points of connection may or may not give the same fragment. So, it is important to ask the following question. [5] R. Moeckel, “Geodesics on modular surfaces and continued fractions,” Ergodic Theory and Dynamical Systems, vol. 2, no. 1, pp. 69–83, 1982. [6] C. Series, “The modular surfaces and continued fractions,” Journal London Mathematical Society, vol. 2, no. 31, pp. 69–80, 1985. [7] M. G. Humbert, “Sur les fractions continues et les formes quadratiques binaires indefinies,” Comptes Rendus de l’Acad´emie des Sciences, vol. 162, pp. 23–26, 1916. Problem 17. How many fragments (polynomials) are obtained if we join a pair of circuits (𝑛1, 𝑛2) and (𝑚1, 𝑚2) at all points of connection? [8] Q. Mushtaq, “Reduced inde.nite binary quadratic forms and orbits of the modular group,” Radovi Matematicki, vol. 4, pp. 331–336, 1988. In order to give the answer of the above question, we first have to find those pair of words which are equivalent. In other words, we have to find those points of connection, at which we get the same polynomial. This issue is resolved in this paper. We will give the answer of the above question in an other paper. After that, one can establish a connection between a class of groups and a pair of circuits (𝑛1, 𝑛2) and (𝑚1, 𝑚2), which is indeed a great development. [9] Q. Mushtaq and U. Hayat, “Pell Numbers, Pell-Lucas Numbers and Modular Group,” Algebra Colloquium, vol. 14, no. 1, pp. 97– 102, 2007. [10] N. H. Bong and Q. Mushtaq, “Fibonacci and Lucas numbers through the action of the modular group on real quadratic fields,” The Fibonacci Quarterly, vol. 42, no. 1, pp. 20–27, 2004. [11] M. Ashiq, “Action of a two generator group on a real quadratic field,” Southeast Asian Bulletin of Mathematics, vol. 30, no. 3, pp. References 𝑥(𝑥𝑦)𝑛1/2(𝑥𝑦−1) 𝑛2(𝑥𝑦)𝑛1/2𝑥 = ((𝑥𝑦−1) 𝑛1/2(𝑥𝑦)𝑛2(𝑥𝑦−1) 𝑛1/2) −1 (26) [1] M. Akbas, “On suborbital graphs for the modular group,” The Bulletin of the London Mathematical Society, vol. 33, no. 6, pp. 647–652, 2001. (26) [2] E. Fujikawa, “Modular groups acting on infinite dimensional Teichmuller spaces,” Contemporary Mathematics, vol. 355, pp. 239–253, 2004. and 𝑥(𝑥𝑦−1)𝑚1/2(𝑥𝑦)𝑚2(𝑥𝑦−1)𝑚1/2𝑥 = ((𝑥𝑦)𝑚1/2(𝑥𝑦−1)𝑚2 (𝑥𝑦)𝑚1/2) −1. This implies that V = (V∗)𝑥. Hence, 𝛾3 has the same orientation as that of its mirror image 𝛾∗ 3 . and 𝑥(𝑥𝑦−1)𝑚1/2(𝑥𝑦)𝑚2(𝑥𝑦−1)𝑚1/2𝑥 = ((𝑥𝑦)𝑚1/2(𝑥𝑦−1)𝑚2 (𝑥𝑦)𝑚1/2) −1. This implies that V = (V∗)𝑥. Hence, 𝛾3 has the same orientation as that of its mirror image 𝛾∗ 3 . [3] O. Koruo˘glu, “The determination of parabolic points in mod- ular and extended modular groups by continued fractions,” Bulletin of the Malaysian Mathematical Sciences Society, vol. 33, no. 3, pp. 439–445, 2010. 4. Motivation and Open Problems 399–404, 2006. Conflict of Interests [12] B. Everitt, “Alternating quotients of the (3, 𝑞, 𝑟) triangle groups,” Communications in Algebra, vol. 25, no. 6, pp. 1817–1832, 1997. The authors declare that there is no conflict of interests regarding the publication of this paper. [13] G. Higman and Q. Mushtaq, “Generators and relations for PSL(2, Z),” The Arab Gulf Journal of Scientific Research, vol. 1, no. 1, pp. 159–164, 1983. Since ∼is an equivalence relation, hence Hence, 𝛾2 has the same orientation as that of its mirror image 𝛾∗ 2 . g 𝛾2 (iii) Let 𝛾3 be the fragment which has one vertex, say, V, fixed by g (ii) The fragment composed by joining a vertex 𝑢1, fixed by (𝑥𝑦−1) 𝑛1/2(𝑥𝑦) 𝑛2(𝑥𝑦−1) 𝑛1/2 (17) (𝑥𝑦−1) 𝑛1/2(𝑥𝑦)𝑛2(𝑥𝑦−1) 𝑛1/2, (𝑥𝑦)𝑚1/2(𝑥𝑦−1) 𝑚2(𝑥𝑦)𝑚1/2. (24) (17) (24) (24) in (𝑛1, 𝑛2), with the vertex 𝑢2, fixed by (𝑥𝑦−1)𝑚1/2(𝑥𝑦)𝑚2 (𝑥𝑦−1)𝑚1/2 in (𝑚1, 𝑚2), has the same orientation as that of its mirror image.hi Then, its mirror image fragment 𝛾∗ 3 has one vertex, say, V∗, fixed by Then, its mirror image fragment 𝛾∗ 3 has one vertex, say, V∗, fixed by (iii) The fragment composed by joining a vertex V1, fixed by (𝑥𝑦)𝑛1/2(𝑥𝑦−1) 𝑛2(𝑥𝑦)𝑛1/2, (𝑥𝑦−1) 𝑚1/2(𝑥𝑦)𝑚2(𝑥𝑦−1) 𝑚1/2. (25) (𝑥𝑦−1) 𝑛1/2(𝑥𝑦) 𝑛2(𝑥𝑦−1) 𝑛1/2 (𝑥𝑦−1) 𝑛1/2(𝑥𝑦) 𝑛2(𝑥𝑦−1) 𝑛1/2 (18) (18) The Scientific World Journal 7 ∗ Figure 11 Figure 11 The vertex (V∗)𝑥of fragment 𝛾∗ 3 is fixed by R The vertex (V∗)𝑥of fragment 𝛾∗ 3 is fixed by Acknowledgment [14] A. Torstensson, “Coset diagrams in the study of .nitely pre- sented groups with an application to quotients of the modular group,” Journal of Commutative Algebra, vol. 2, no. 4, pp. 501– 514, 2010. The authors would like to thank the referee for his very helpful comments, which have significantly improved the presentation of this paper. The Scientific World Journal 8 [15] Q. Mushtaq, “Parameterization of all homomorphisms from PGL(2, Z) into PSL(2, q),” Communications in Algebra, vol. 4, no. 20, pp. 1023–1040, 1992. [16] Q. Mushtaq, “A condition for the existence of a fragment of a coset diagram,” The Quarterly Journal of Mathematics, vol. 39, no. 153, pp. 81–95, 1988.
https://openalex.org/W4295357648	https://www.emerald.com/insight/content/doi/10.1108/CAFR-06-2022-0068/full/pdf?title=government-subsidization-and-corporate-product-strategies-evidence-from-chinese-exporters	English	null	Government subsidization and corporate product strategies: evidence from Chinese exporters	Zhongguo Kuaiji yu caiwu yanjiu/China accounting and finance review	2,022	cc-by	13,053	The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/1029-807X.htm The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/1029-807X.htm Abstract Purpose – This study aims to take advantage of exporters’ product codes and examine the effects of government subsidization on corporate product strategies by focusing on the dimension of product differentiation. Design/methodology/approach – This study uses harmonized system (HS) product codes to construct a novel measure of product differentiation among a sample of Chinese exporters during 2000–2012. It uses propensity score matching to construct a comparable sample of control firms for exporters receiving government subsidies, and then a difference-in-differences (DID) analysis is conducted. Findings – This study finds that product differentiation decreases immediately upon receiving a government subsidy. This finding suggests that in an emerging market, firms use their subsidy to imitate competitors rather than increase innovation. Further analyses show that this effect is concentrated among wholly foreign- owned enterprises and firms that focus on general trade rather than processing trade. In addition, the authors find some evidence that government subsidization leads to an increase in the number of product lines and decreases in domestic value added and export product quality. Originality/value – This study constructs a novel measure of product differentiation for a large sample of Chinese exporters and provides insights that government subsidization can affect corporate product strategies. Keywords Government subsidy, Product differentiation, Product strategy, Product market competition Paper type Research paper Paper type Research paper 293 Received 21 June 2022 Revised 28 July 2022 14 August 2022 Accepted 19 August 2022 Janus Jian Zhang Hong Kong Baptist University, Kowloon, Hong Kong JEL Classification — G30, M11 © Xiaodong Lu, Jingjun Liu and Janus Jian Zhang. Published in China Accounting and Finance Review. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and no commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at http:// creativecommons.org/licences/by/4.0/legalcode The authors thank the editor and anonymous reviewers for their helpful comments and suggestions. Xiaodong Lu acknowledges the financial support from the National Natural Science Foundation of China (Grant Number 71873150). The authors thank the editor and anonymous reviewers for their helpful comments and suggestions. Xiaodong Lu acknowledges the financial support from the National Natural Science Foundation of China (Grant Number 71873150). JEL Classification — G30, M11 © Xiaodong Lu, Jingjun Liu and Janus Jian Zhang. Published in China Accounting and Finance Review. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and no commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at http:// creativecommons.org/licences/by/4.0/legalcode Th h h k h di d i f h i h l f l d i Government subsidization and corporate product strategies: evidence from Chinese exporters Xiaodong Lu and Jingjun Liu Lingnan College, Sun Yat-Sen University, Guangzhou, China, and Corporate product strategies 1. Introduction C ’ China’s rapid growth in international trade has spurred great interest in its supporting policies. China and other developing countries rely heavily on subsidies to promote exports (e.g. Haley & Haley, 2008; Defever & Ria~no, 2017). After China joined the World Trade Organization in 2001, its subsidy policy became a sensitive and controversial issue (Hwang & Mai, 2007; Bown & Hillman, 2019). A large body of literature on this issue focuses on the quantitative aspect of international trade (i.e. export volume) (e.g. Hoffmaister, 1992; Chen, Mai, & Yu, 2006; Eckaus, 2006; G€org, Henry, & Strobl, 2008; Girma, Gong, G€org, & Yu, 2009). In particular, Girma et al. (2009) document that China’s subsidies encourage existing exporters to export more (intensive margin) but do not effectively encourage nonexporters to start exporting (extensive margin). However, to the best of our knowledge, much less JEL Classification — G30, M11 China Accounting and Finance Review Vol. 25 No. 3, 2023 pp. 293-312 Emerald Publishing Limited e-ISSN: 2307-3055 p-ISSN: 1029-807X DOI 10.1108/CAFR-06-2022-0068 The authors thank the editor and anonymous reviewers for their helpful comments and suggestions. Xiaodong Lu acknowledges the financial support from the National Natural Science Foundation of China (Grant Number 71873150). literature on government subsidies delves into the qualitative aspect of international trade, such as export product structure and product quality. Our study adds to the literature by investigating the effects of government subsidization on exporters’ product strategies, specifically the dimension of product differentiation [1]. p y p [ ] The effects of government subsidization on exporters’ product strategies depend on how these subsidized exporters leverage governmental financial supports. On the one hand, firms may invest more in research and development (R&D) to promote product innovation, which in turn enables them to better differentiate their products from those of competitors. Product differentiation is an important strategy for firms to maintain sustainable competitive advantage (e.g. Smith, 1956; Murray, Kotabe, & Zhou, 2005; Cho & Tsang, 2020). On the other hand, with the help of government support, firms may invest more in mature product lines to imitate their competitors and follow the market trends, which decreases product differentiation. However, this strategy would inevitably increase product market competition unless the market demand exceeds the total supply. A large body of literature examines the effectiveness of government subsidies in promoting industrial innovation, but the empirical evidence is inconclusive (Dimos & Pugh, 2016). 1. Introduction C ’ One major reason might be that firms are heterogeneous and thus they may have different preferences in investment and product strategies. In addition, firm managers need to allocate scarce resources efficiently and choose the most suitable strategy (Sorenson, 2000). For example, some may prioritize R&D investments, which are risky and uncertain but also can generate competitive advantages. Others may invest in mature product lines, which are less risky but result in higher level of competition. Therefore, it is ex ante unclear how government subsidies affect exporters’ choice of competition mode and the degree of their product differentiation [2]. 294 exporters choice of competition mode and the degree of their product differentiation [2]. To empirically examine the impact of government subsidies on exporters’ competition strategy, we must address two main challenges. First, a firm’s choice of competition strategy is unobservable and difficult to measure though it can be inferred from the degree of product differentiation (e.g. Smith, 1956; Dickson & Ginter, 1987; Mukherjee, 2014; Hoberg & Phillips, 2016). Using transaction-level international trade data of China exporters, we construct a novel measure of product differentiation to capture the extent to which an exporter’s product space differs from that of its competitors. Second, government subsidies are not randomly assigned; hence, it is challenging to make causal inferences. To address this concern, we use propensity score matching to identify a group of control firms with comparable firm-level characteristics. Based on this matched sample, we then conduct a difference-in-differences (DID) analysis to establish causality between government subsidization and exporters’ product differentiation. y g p p The DID analysis of a large sample of Chinese exporters during 2000–2012 shows that, compared with exporters that do not receive any subsidies, subsidized exporters experience significant decreases in product differentiation in the postsubsidy period. This finding suggests that government subsidization increases the use of imitation, rather than innovation, as a competitive strategy. As previously argued, firms still face fierce competition, but government subsidies and cheap labor costs allow exporters in China to compete via low prices (Haley & Haley, 2008). Our finding also aligns with prior studies demonstrating China’s quantity-driven export growth (e.g. Shi, 2011). Further analyses reveal significant heterogeneous effects of government subsidies. Specifically, we find that the effect of government subsidies on exporters’ product differentiation is concentrated in foreign-owned enterprises and firms focused on general, rather than processing, trade. CAFR 25,3 Corporate product strategies 1. Introduction C ’ To provide more insights, we also examine other potential consequences of government subsidies. As our main finding suggests that exporters in China tend to invest in mature product lines instead of R&D, we expect recipients of a government subsidy to increase the number of products manufactured and to decrease product quality. Consistent with our expectation, we find some evidence that government subsidization leads to a larger number of export products and a lower level of domestic value added and export product quality. Taken together, our study provides novel insights that government subsidization can affect exporters’ choice of competition strategy. In particular, our findings suggest that government subsidies in China lead exporters to engage in price-based competition, which in turn decreases product differentiation and quality. Taken together, our study provides novel insights that government subsidization can affect exporters’ choice of competition strategy. In particular, our findings suggest that government subsidies in China lead exporters to engage in price-based competition, which in turn decreases product differentiation and quality. Our paper makes two key contributions to the literature. First, we introduce a novel measure of product differentiation that is available for a large sample of exporters in China. Prior literature measures product differentiation by classifying export products into homogeneous and heterogeneous categories (e.g. Rauch, 1999; Hu & Tan, 2016) or using survey data (e.g. Boehe & Barin Cruz, 2010). Recently, Hoberg and Phillips (2016) use textual analysis to collect product names disclosed in financial reports, but this approach offers limited accuracy. By using harmonized system (HS) codes, we can accurately identify the product space of each exporter. As such, our measures should be useful for future studies on China’s role in international economics. 295 295 Second, our study adds to the literature by providing evidence on the effects of government subsidies on exporters’ mode of competition, product differentiation and product quality. Prior literature on the intersection of government subsidization and international trade mainly focus on quantity (e.g. Hoffmaister, 1992; Eckaus, 2006; G€org et al., 2008; Girma et al., 2009). Our paper complements this literature by investigating the impact of subsidization on product differentiation and export quality. Our paper also enriches the literature on corporate product strategy (e.g. Smith, 1956; Cooper & Kleinschmidt, 1985) and firms’ response to product market competition (e.g. Mayer, Melitz, & Ottaviano, 2014; Ryou, Tsang, & Wang, 2022). Our findings should be of interest to policymakers, especially those in developing countries. The rest of this paper is organized as follows. Section 2 describes the empirical methodology, including data sources, sample selection, variables and DID model. Section 3 introduces the propensity score matching procedure and presents our main empirical results. In section 4, we explore the heterogeneous effects of government subsidization. We examine other potential outcomes of government subsidization in section 5 and conclude in section 6. 2. Empirical methodology 2.1 Data sources Following Hoberg and Phillips (2016), we calculate the product cosine similarity for each pair of exporters year by year. As the first step, we use the following vector with N dimensions to represent the product space of a given firm i: 296 (1) Pi ¼ ðpi1; pi2; pi3; . . . ; pik; . . . ; piNÞ (1) Pi ¼ ðpi1; pi2; pi3; . . . ; pik; . . . ; piNÞ In vector Pi, the element pik is defined as the ratio of export value of product k to the total export value of firm i’s major products. That is, pik equals 0 if firm i does not export product k. In this way, we weight each product according to its contribution to firm i’s annual export value. N refers to the total number of unique products exported by all Chinese firms in a given year. In our sample, N is 6,041 in 2000 and 7,168 in 2012. We then normalize the vector Pi to have unit length as follows: Vi ¼ Pi ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Pi$Pi p (2) (2) We repeat this procedures for any other firm j in the same year to obtain its normalized vector Vj. The product cosine similarity between firm i and firm j in each year is calculated as follows: We repeat this procedures for any other firm j in the same year to obtain its normalized vector Vj. The product cosine similarity between firm i and firm j in each year is calculated as follows: Similarityij ¼ ðVi$VjÞ (3) (3) Because both vectors Vi and Vj are normalized to have unit length, the above similarity score can be any real number bounded by 0 and 1. Intuitively, the similarity score increases when both firms export more of the same products. The weight also matters, as we weight each product according to its export value. For example, the similarity score equals 1 for a pair of firms exporting the same set of products with the same weights. Suppose there are two firms, both export products A and B but one firm primarily exports product A and the other primarily exports product B. In this case, the similarity score is positive but close to 0 even though both firms share the same scope of products. 2. Empirical methodology 2.1 Data sources Our study uses export and financial data from Chinese exporters. To measure exporters’ product differentiation, we rely on detailed export data compiled by the General Administration of Customs of the People’s Republic of China. The export data covers 2000 to 2012 and provides detailed information on each trade, including exporter name, nature (e.g. general trade and processing trade), eight-digit HS code of exported product, volume and value. To construct government subsidization and other control variables, we extract financial data from the Chinese industrial enterprises database maintained by the National Bureau of Statistics of China. The bureau conducts an annual survey of all state-owned enterprises, regardless of firm size, and of large non-state-owned enterprises with an output value of 5 million or more Chinese Yuan. These surveyed firms account for 98% of total exports from China (Brandt, Van Biesebroeck, & Zhang, 2012). Because the survey data cover a longer period than the export data, we limit our sample to the 13 years in which they overlap, yielding a final sample that covers 2000 to 2012. The export and survey databases use different firm identifiers, so we cannot directly merge the financial data with the export data. However, we can reliably match firms in both databases using information such as firm name, name of legal representative and executives, postal code and telephone number. 2.2 Product differentiation As previously mentioned, we rely on export data to measure product differentiation. To construct the measure, we analyze the whole dataset at the firm-year-product level, where a ff As previously mentioned, we rely on export data to measure product differentiation. To construct the measure, we analyze the whole dataset at the firm-year-product level, where a product is identified by its unique eight-digit HS code [3]. To limit our focus to major products only, we exclude products that contribute to less than 1% of the company’s annual export value [4]. This exclusion leaves over ten million firm-year-product observations across our sample period of 13 years, covering 489,183 unique exporters and 9,538 unique products. product is identified by its unique eight-digit HS code [3]. To limit our focus to major products only, we exclude products that contribute to less than 1% of the company’s annual export value [4]. This exclusion leaves over ten million firm-year-product observations across our sample period of 13 years, covering 489,183 unique exporters and 9,538 unique products. CAFR 25,3 2.3 Government subsidization Government subsidy data are available from the Chinese industrial enterprises database. To ensure that the subsidy received by the exporter is substantial for its operation, we select treatment firms based on two conditions: 1) the firm’s annual subsidy exceeds 500,000 RMB or 2) its subsidy ratio (SUBRATIO), calculated as subsidy amount divided by annual sales, exceeds 1% and the amount exceeds 100,000 RMB [6]. Throughout our sample period, some treatment firms may receive multiple subsidies. Our main DID analysis focuses on treatment firms receiving their first subsidy. To implement a DID analysis, we create an indicator of treatment firms (TREAT), which takes a value of 1 for treatment firms and 0 for control firms. Control firms are those that never receive a subsidy during our sample period. As detailed in section 3.1, we use propensity score matching to identify a control firm for each treatment firm. For each pair of matched firms, we include in our regression sample six-year observations from t3 to tþ2 (i.e. three years before and after receiving a government subsidy in year t). The dummy variable POST equals 1 in the postsubsidy period (from year t to tþ2) and 0 otherwise. 2.4 Model specification To examine the effects of government subsidization on product differentiation, we estimate the following DID model: PDIFFi;t ¼ αi þ mt þ β1TREATi;t 3 POSTi;t þ β2POSTi;t þ γXi;t−1 þ εi;t (6) 2.4 Model specification To examine the effects of government subsidization on product differentiation, we estimate the following DID model: PDIFFi;t ¼ αi þ mt þ β1TREATi;t 3 POSTi;t þ β2POSTi;t þ γXi;t−1 þ εi;t (6) 2.4 Model specification To examine the effects of government subsidization on product differentiation, we estimate the following DID model: PDIFFi;t ¼ αi þ mt þ β1TREATi;t 3 POSTi;t þ β2POSTi;t þ γXi;t−1 þ εi;t (6) Model specification examine the effects of government subsidization on product differentiation, we estimate following DID model: To examine the effects of government subsidization on product differentiation, we estimate the following DID model: PDIFFi;t ¼ αi þ mt þ β1TREATi;t 3 POSTi;t þ β2POSTi;t þ γXi;t−1 þ εi;t (6) PDIFFi;t ¼ αi þ mt þ β1TREATi;t 3 POSTi;t þ β2POSTi;t þ γXi;t−1 þ εi;t (6) (6) In this model, the dependent variable is one of our product differentiation measures, as constructed in section 2.2. The subscripts i and t represent firm and year, respectively. Corporate product strategies 297 297 2. Empirical methodology 2.1 Data sources Accordingly, the similarity score also would be 0 for a pair of firms that do not share any common products. Next, we aggregate firms’ pairwise similarity scores to the firm-year level as our measure of product differentiation. Following Hombert and Matray (2018), we calculate the pairwise product differentiation as 1 minus the pairwise similarity score: Differentiationij ¼ 1 ðVi$VjÞ (4) (4) Likewise, the pairwise product differentiation can take any value between 0 and 1. In each year, we take the average of pairwise product differentiation among the firm’s competitors as our firm-year measure of product differentiation: Likewise, the pairwise product differentiation can take any value between 0 and 1. In each year, we take the average of pairwise product differentiation among the firm’s competitors as our firm-year measure of product differentiation: PDIFFi ¼ 1 Ci X Ci j¼1 ð1 ðVi$VjÞÞ ¼ 1 1 Ci X Ci j¼1 ðVi$VjÞ (5) (5) In Equation (5), Ci is the number of competitors for firm i in a specific year. Instead of using the traditional approach based on industry classification, we take advantage of the export product network to identify firm i’s competitors. Specifically, firm i and firm j are deemed competitors if Vi$Vj > 0. The rationale for this identification is that firms sharing at least one common product compete to some extent. During our sample period, direct sales from manufacturing firms contribute to about 80% of Chinese exports. Traders, which purchase products from domestic suppliers to resell overseas, contribute to about 20% of total exports [5]. We follow Ahn, Khandelwal and Wei’s (2011)approach to identify trading companies as those whose translated Chinese names include the words “trading,” “importer” or “exporter.” Accordingly, we construct two measures of product differentiation: PDIFF1 is based on exports of both manufacturing firms and trading companies and PDIFF2 is based on exports made by manufacturers only. 3. Propensity score matching and DID results 3. Propensity score matching and DID results 3.1 Propensity score matching In our DID specification, we compare subsidized exporters’ actual level of product differentiation with the counterfactual level if they did not receive a government subsidy. The main challenge is that the counterfactual level of product differentiation is unobservable. Following prior literature (e.g. Rosenbaum & Rubin, 1985; Heckman, Ichimura, & Todd, 1998; Dehejia & Wahba, 2002; Shipman, Swanquist, & Whited, 2017; Yang, He, Zhu, & Li, 2018; Brucal, Javorcik, & Love, 2019), we use propensity score matching to identify a reasonable counterfactual for each subsidized exporter [9]. Specifically, we use the following probit model to estimate the propensity score: 298 TREATi;t ¼ β0 þ β1ΔPDIFF2i;t−123 þ γXi;t−123 þ Ownership F:E: þ Province F:E: þ Industry F:E: þ YearF:E: þ ε (7) In Equation (7), we use the average value of each independent variable in the past three years (i.e. from year t3 to t1) to predict whether a firm will receive a government subsidy in year t. The dependent variable is the treatment dummy, which equals 1 for firms receiving subsidy and 0 otherwise. In the prediction model, we include all firm-level control variables, as defined in Equation (6), as well as ownership fixed effects, province fixed effects, industry fixed effects and year fixed effects. In addition, we also control for the average change of product differentiation in the past three years to enhance the parallel trend of our outcome variable in the pre-subsidy period [10]. In Equation (7), we use the average value of each independent variable in the past three years (i.e. from year t3 to t1) to predict whether a firm will receive a government subsidy in year t. The dependent variable is the treatment dummy, which equals 1 for firms receiving subsidy and 0 otherwise. In the prediction model, we include all firm-level control variables, as defined in Equation (6), as well as ownership fixed effects, province fixed effects, industry fixed effects and year fixed effects. In addition, we also control for the average change of product differentiation in the past three years to enhance the parallel trend of our outcome variable in the pre-subsidy period [10]. p y p [ ] We run this prediction model on a sample consisting of both treatment firms and potential control firms. Treatment firms receive substantial government subsidization, and control firms never receive any subsidy during our sample period. 2.3 Government subsidization The regression includes firm fixed effects, αi, which captures the time-invariant characteristics of each sample firm [7]. We also include year fixed effects (μt) to account for time-variant macroeconomic factors. Our focus is the coefficient on the interaction term between the treatment dummy (TREATi,t) and postsubsidy dummy (POSTi,t) defined in section 2.3. A significantly positive (negative) β1 would suggest that exporters increase (decrease) their product differentiation after receiving a government subsidy. In Equation (6), we also control for a series of firm-level control variables (Xi,t1). Specifically, we control for the firm’s total factor productivity (TFP), estimated using Olley and Pakes’ (1996) methodology; firm size (SIZE), defined as the natural logarithm of total assets; return on assets (ROA), calculated as net income scaled by lagged total assets; financial leverage (LEV), calculated as total liability divided by total assets; and firm age (AGE), the log of 1 plus the number of years since establishment. We also control for product diversity, captured by the number of export products (NP) because as firms increase their product diversity, it becomes more difficult to differentiate their products from those of their competitors. All control variables are lagged by one year. Continuous variables in our regression model are winsorized at the 1st and 99th percentiles, and standard errors are clustered at firm level [8]. 3. Propensity score matching and DID results 3.1 Propensity score matching In our DID specification, we compare subsidized exporters’ actual level of product differentiation with the counterfactual level if they did not receive a government subsidy. The main challenge is that the counterfactual level of product differentiation is unobservable. Following prior literature (e.g. Rosenbaum & Rubin, 1985; Heckman, Ichimura, & Todd, 1998; Dehejia & Wahba, 2002; Shipman, Swanquist, & Whited, 2017; Yang, He, Zhu, & Li, 2018; Brucal, Javorcik, & Love, 2019), we use propensity score matching to identify a reasonable counterfactual for each subsidized exporter [9]. Specifically, we use the following probit model to estimate the propensity score: FR 8 3. Propensity score matching and DID results To calculate the average value of each variable, we drop observations with missing variables in the previous three years. We keep all available observations of each potential control firm to increase the chance of successful matching, but we keep only the year t observation of each treatment firm (i.e. the year in which the firm receives subsidy). As we already take the average of each variable from year t3 to t1, this treatment observation captures characteristics of the treatment firm before receiving subsidization. In short, we intend to find a control firm for each treatment firm based on firm characteristics in the presubsidy period. For the prediction model, the final sample comprises 68,132 firm-year observations, including 54,738 observations of control firms (TREAT 5 0) and 13,394 observations of treatment firms (TREAT 5 1). ( ) ( ) Table 1 presents the implementation of propensity score matching. In Column (1) of Panel A, we present the results of predicting government subsidization. Consistent with our intuition, the Chinese government is more likely to subsidize larger firms and firms with poor performance. Based on the estimation in Column (1), we can derive each observation’s propensity score (i.e. the likelihood of receiving a subsidy). We then do a one-by-one match without replacement. Specifically, for each treatment firm, we try to find a control firm with a close propensity score within the same ownership type, location province, industry and year. To ensure similarity between matched firm pairs, we further require the difference between their propensity scores to be less than 10%. We successfully match 2,615 pairs of firms, which we use for our main analyses [11]. To check the matching performance, we rerun the prediction model on the matched sample. As shown in Column (2) of Panel A, the pseudo R2 is nearly 0, and the p-value of the Wald χ2 test is 1, suggesting that our matching procedure significantly reduces the prediction power of firm-level characteristics. In Panel B, we compare the sample means of the variables used in our matching procedure between the treatment group and the control (1) before matching (2) after matching Dep. var. 3. Propensity score matching and DID results 5 TREAT TREAT Panel A: estimation of propensity scores TFP 0.0052 (0.51) 0.0037 (0.13) SIZE 0.5404* (54.17) 0.0026 (0.14) ROA 0.1021 (2.43) 0.1457* (1.78) LEV 0.1852* (11.79) 0.0770* (2.64) AGE 0.0768* (4.24) 0.1518* (4.34) NP 0.0912*** (6.46) 0.0020 (0.07) ΔPDIFF2 0.1586* (1.67) 0.0450 (0.20) Ownership fixed effects Yes Yes Province fixed effects Yes Yes Industry fixed effects Yes Yes Year fixed effects Yes Yes N 68,132 5,230 Pseudo R2 0.3590 0.0045 p-value of Wald χ2 test 0.0000 1.0000 Before matching After matching Variable Control group N 5 54,738 Treatment group N 5 13,394 Difference in mean Control group N 5 2,615 Treatment group N 5 2,615 Difference in mean Panel B: checking matching performance TFP 2.7139 1.7683 0.9456* 1.8667 1.8510 0.0157 SIZE 9.8873 11.3560 1.4686* 10.4743 10.4958 0.0215 ROA 0.1481 0.1238 0.0243* 0.1324 0.1362 0.0038 LEV 0.5899 0.7387 0.1488* 0.7690 0.7125 0.0565 AGE 2.0234 2.1011 0.0777* 1.9949 2.0621 0.0672* NP 0.9818 1.0026 0.0208 1.0364 1.0377 0.0013 ΔPDIFF2 0.0012 0.0034 0.0022 0.0035 0.0030 0.0005 Propensity Score 0.1215 0.5046 0.3831* 0.2958 0.2937 0.0021 Note(s): This table presents our propensity score matching. Panel A uses a probit model to estimate propensity scores, and Panel B checks the performance of our matching procedure. We do a one-by-one match without replacement. Specifically, for each treatment firm, we try to find the control firm with the closest propensity score within the same ownership type, location province, industry and year. *, and * represent statistical significance at the 1, 5 and 10% levels, respectively Table 1. Propensity score matching Corporate product strategies 299 Note(s): This table presents our propensity score matching. Panel A uses a probit model to estimate propensity scores, and Panel B checks the performance of our matching procedure. We do a one-by-one match without replacement. Specifically, for each treatment firm, we try to find the control firm with the closest propensity score within the same ownership type, location province, industry and year. *, and * represent statistical significance at the 1, 5 and 10% levels, respectively Table 1. Propensity score matching group. Though we observe significant differences between treatment and control groups before matching, most of these differences disappear after matching. We also find that the overall propensity scores of matched control firms are essentially the same as that of matched treatment firms, although these control firms are slightly younger and have significantly higher financial leverage. 3. Propensity score matching and DID results Taken together, our propensity score matching performs reasonably well in identifying control firms. Treatment and control firms in our matched sample are comparable in terms of firm characteristics in the presubsidy period. Based on these matched pairs of firms, we construct a panel sample to conduct our main DID test. For these matched firms, we include in our regression sample six-year observations from t3 to tþ2 (i.e. three years before and three years after receiving a government subsidy in year t). After dropping observations with missing regression variables, we drop matched firm pairs in which a treatment or control firm does not have any available observations in either the pre- or post-subsidy period. Our matched final sample comprises 17,218 firm-year observations. Table 2 presents the descriptive statistics for this matched sample. Our two Mean SD P25 Median P75 PDIFF1 0.5183 0.1470 0.4310 0.5359 0.6264 PDIFF2 0.4469 0.1596 0.3468 0.4632 0.5645 TREAT 0.4971 0.5000 0.0000 0.0000 1.0000 POST 0.5378 0.4986 0.0000 1.0000 1.0000 TFP 2.4781 1.8302 0.0000 3.1787 3.9972 SIZE 10.5680 1.1268 9.7848 10.5101 11.2583 ROA 0.1230 0.1785 0.0212 0.0611 0.1467 LEV 0.5486 0.2563 0.3600 0.5564 0.7434 AGE 2.1195 0.5651 1.7918 2.1972 2.4849 NP 1.0587 0.7571 0.6931 1.0986 1.6094 Note(s): This table presents the descriptive statistics of the regression variables in our matched sample. We present the mean, standard deviation (SD), 25th percentile (P25), median and 75th percentile (P75) of each regression variable. All continuous variables are winsorized at the 1st and 99th percentiles Table 2. Descriptive statistics of matched sample (N 5 17,218) CAFR 25,3 300 Note(s): This table presents the descriptive statistics of the regression variables in our matched sample. We present the mean, standard deviation (SD), 25th percentile (P25), median and 75th percentile (P75) of each regression variable. All continuous variables are winsorized at the 1st and 99th percentiles Table 2. Descriptive statistics of matched sample N 5 17,218) measures of product differentiation, PDIFF1 and PDIFF2, have mean values of 0.5183 and 0.4469, respectively. Because this is a matched sample, the treatment dummy (TREAT) and postsubsidy dummy (POST) both have means close to 50%. measures of product differentiation, PDIFF1 and PDIFF2, have mean values of 0.5183 and 0.4469, respectively. Because this is a matched sample, the treatment dummy (TREAT) and postsubsidy dummy (POST) both have means close to 50%. 3.2 DID results T i h 5 (1) (2) (3) (4) PDIFF1 PDIFF2 PDIFF1 PDIFF2 TREAT 3 POST 0.0090* (2.76) 0.0100* (2.74) 0.0082* (2.58) 0.0091 (2.57) POST 0.0006 (0.23) 0.0007 (0.24) 0.0018 (0.67) 0.0020 (0.65) TFP 0.0003 (0.21) 0.0006 (0.37) SIZE 0.0063* (3.09) 0.0061* (2.66) ROA 0.0063 (1.07) 0.0050 (0.77) LEV 0.0041 (0.80) 0.0033 (0.57) AGE 0.0038 (0.89) 0.0036 (0.77) NP 0.0239* (10.06) 0.0269* (10.12) Year fixed effects Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes N 17,218 17,218 17,218 17,218 Adjusted R2 0.709 0.689 0.713 0.693 Note(s): This table presents our main results by estimating the baseline DID model on the matched sample. The dependent variables are one of our measures of product differentiation. The dummy variable TREAT takes a value of 1 for treatment firms and 0 for control firms. The dummy variable POST equals 1 in the postsubsidy period (from year t to tþ2) and 0 otherwise. All continuous variables are winsorized at the 1st and 99th percentiles. The model includes firm and year fixed effects. Presented in the parentheses below each coefficient is the t-value based on standard errors clustered by firm. Constant terms are estimated but omitted for presentation. *, and * represent statistical significance at the 1, 5 and 10% levels, respectively Table 3. Main results Corporate product strategies 301 Dep. var. 5 (1) (2) (3) (4) PDIFF1 PDIFF2 PDIFF1 PDIFF2 TREAT 3 POST 0.0090* (2.76) 0.0100* (2.74) 0.0082* (2.58) 0.0091 (2.57) POST 0.0006 (0.23) 0.0007 (0.24) 0.0018 (0.67) 0.0020 (0.65) TFP 0.0003 (0.21) 0.0006 (0.37) SIZE 0.0063* (3.09) 0.0061* (2.66) ROA 0.0063 (1.07) 0.0050 (0.77) LEV 0.0041 (0.80) 0.0033 (0.57) AGE 0.0038 (0.89) 0.0036 (0.77) NP 0.0239* (10.06) 0.0269* (10.12) Year fixed effects Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes N 17,218 17,218 17,218 17,218 Adjusted R2 0.709 0.689 0.713 0.693 Note(s): This table presents our main results by estimating the baseline DID model on the matched sample. The dependent variables are one of our measures of product differentiation. The dummy variable TREAT takes a value of 1 for treatment firms and 0 for control firms. The dummy variable POST equals 1 in the postsubsidy period (from year t to tþ2) and 0 otherwise. All continuous variables are winsorized at the 1st and 99th percentiles. The model includes firm and year fixed effects. Presented in the parentheses below each coefficient is the t-value based on standard errors clustered by firm. Table 3. Main results 3.2 DID results T i h To examine the effect of government subsidy on exporters’ product differentiation, we estimate the DID model specified in Equation (6) on the matched final sample of 17,218 firm- year observations. Table 3 presents the DID results. In Columns (1) and (3), we focus on PDIFF1, a measure of product differentiation based on the combined product space of two types of exporters (i.e. manufacturing firms and trading companies). In Columns (2) and (4), we focus on PDIFF2, an alternative measure of product differentiation constructed solely on the product space of manufacturing exporters. We start our analyses with a simplified DID model in which we do not control for firm-level control variables. As shown in the first two columns, we find significantly negative coefficients on the interaction terms of TREAT 3 POST. After controlling for a series of firm-level characteristics, we continue to find similar results in the last two columns. For instance, in Column (4), the coefficient on TREAT 3 POST is 0.0091 and significant at the 5% level (t-value 5 2.57) [12]. These results show that, compared with that of control firms, treatment firms’ product differentiation decreases after receiving government subsidies. This finding suggests that Chinese exporters tend to leverage governmental financial supports to invest in mature product lines to imitate their competitors and follow the market trends, instead of investing in innovation projects. This finding is consistent with prior literature documenting that Chinese exporters tend to compete via low prices (Haley & Haley, 2008) and government subsidies induce firms’ overinvestment behavior (Zhang, An, & Zhong, 2019). ( g, , g, ) In terms of control variables, most are insignificant, which is expected because our analyses are based on matched samples. In the last two columns, we find that larger firm size (SIZE) is associated with lower product differentiation, perhaps because larger firms face more competition and hence may find it harder to differentiate their products from competitors. We also find that product diversity (NP) is negatively associated with product differentiation, which is consistent with our expectation that increased product diversity makes it harder for firms to differentiate their products from those of their competitors. An effective DID model requires treatment and control groups to maintain similar trends in the preevent period. We check this parallel trend assumption and examine the dynamic effects of government subsidization in Table 4. We replace the dummy of POST in Dep. var. 3.2 DID results T i h Constant terms are estimated but omitted for presentation. *, and * represent statistical significance at the 1, 5 and 10% levels, respectively Corporate product strategies 301 Table 3. Main results Dep. var. 5 (1) (2) (3) (4) PDIFF1 PDIFF2 PDIFF1 PDIFF2 TREAT 3 Period(2) 0.0019 (0.43) 0.0011 (0.21) 0.0034 (0.74) 0.0026 (0.52) TREAT 3 Period(1) 0.0067 (1.31) 0.0057 (1.00) 0.0073 (1.43) 0.0064 (1.13) TREAT 3 Period(0) 0.0095* (1.84) 0.0091 (1.59) 0.0100** (1.99) 0.0098* (1.73) TREAT 3 Period(1) 0.0131** (2.16) 0.0124* (1.84) 0.0128** (2.15) 0.0122* (1.84) TREAT 3 Period(2) 0.0172* (2.59) 0.0190 (2.57) 0.0161 (2.49) 0.0178 (2.48) Firm-level controls No No Yes Yes Period indicators Yes Yes Yes Yes Year fixed effects Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes N 17,218 17,218 17,218 17,218 Adjusted R2 0.709 0.689 0.713 0.694 Note(s): This table tests the parallel trend assumption and dynamic effects of government subsidy. The dependent variables represent one of our measures of product differentiation. The dummy variable TREAT equals 1 for treatment firms and 0 for control firms. The period indicator Period(x) equals 1 in year t þ x and 0 otherwise, where year t is the year of receiving a government subsidy. The model includes firm and year fixed effects and firm-level control variables. All continuous variables are winsorized at the 1st and 99th percentiles. Presented in the parentheses below each coefficient is the t-value based on standard errors clustered by firm. Constant terms are estimated but omitted for presentation. *, and * represent statistical significance at the 1, 5 and 10% levels, respectively Table 4. Parallel trend and dynamic effects CAFR 25,3 302 Equation (6) with a series of relative period dummies. In our matched sample, we focus on an event window covering three years before and after the treatment firms receive a subsidy. We use year t3 as the benchmark period and include five period indicators from t2 to tþ2 and their interaction terms with the treatment dummy (TREAT). Insignificant coefficients on TREAT 3 Period(2) and TREAT 3 Period(1) suggest that treatment and control firms maintain similar trends in product differentiation in the pre-subsidy period. Indeed, we find that these coefficients are statistically insignificant across four columns: whereas the first two columns do not control for firm-level characteristics, the last two columns do. 3.2 DID results T i h These results validate the parallel trend assumption of our main DID model, and validation tests hold for both measures of product differentiation. Therefore, our main DID results are less likely to be driven by the violation of the parallel trend assumption. Equation (6) with a series of relative period dummies. In our matched sample, we focus on an event window covering three years before and after the treatment firms receive a subsidy. We use year t3 as the benchmark period and include five period indicators from t2 to tþ2 and their interaction terms with the treatment dummy (TREAT). Insignificant coefficients on TREAT 3 Period(2) and TREAT 3 Period(1) suggest that treatment and control firms maintain similar trends in product differentiation in the pre-subsidy period. Indeed, we find that these coefficients are statistically insignificant across four columns: whereas the first two columns do not control for firm-level characteristics, the last two columns do. These results validate the parallel trend assumption of our main DID model, and validation tests hold for both measures of product differentiation. Therefore, our main DID results are less likely to be driven by the violation of the parallel trend assumption. y p p As for the dynamic effects of government subsidization, we find that product differentiation of treatment firms starts to decline in the same year they receive a subsidy, as evidenced by significantly negative coefficients on TREAT 3 Period(0). More importantly, we also find that the coefficients on TREAT 3 Period(1) and TREAT 3 Period(2) are significantly negative, suggesting that the negative effect of government subsidization on product differentiation lasts for three years or longer. In addition, we observe an increasing trend in coefficient magnitudes from year t to tþ2 (e.g. in Column (4), the coefficients of TREAT 3 Period(0), TREAT 3 Period(1), and TREAT 3 Period(2) are 0.0098, 0.0122 and 0.0178, respectively. Taken together, these results highlight the importance of considering the long-term effects of government subsidization. 3.3 Robustness checks In Table 5, we conduct a series of robustness checks for our main results. In our main DID analyses, we focus on treatment firms receiving a substantial government subsidy (i.e. the subsidy exceeds 500,000 RMB, or the subsidy exceeds 100,000 RMB and exceeds 1% of the firm’s annual sales). One might argue that this selection criterion is somewhat arbitrary. 3.2 DID results T i h To address this issue, we first use various alternative cut-offs to identify a treatment sample of Threshold T 5 0.50 T 5 1.00 T 5 2.00 Dep. var. 5 PDIFF1 (1) PDIFF2 (2) PDIFF1 (3) PDIFF2 (4) PDIFF1 (5) PDIFF2 (6) Panel A: requiring the government subsidy amount to exceed T% of annual sales and exceed 100,000 RMB TREAT 3 POST 0.0097* (2.68) 0.0108* (2.68) 0.0133* (3.09) 0.0155* (3.23) 0.0169* (2.76) 0.0193* (2.81) POST 0.0027 (0.87) 0.0022 (0.64) 0.0008 (0.21) 0.0011 (0.26) 0.0009 (0.17) 0.0024 (0.40) Firm-level controls Yes Yes Yes Yes Yes Yes Year fixed effects Yes Yes Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes Yes Yes N 13,036 13,036 8,456 8,456 4,345 4,345 Adjusted R2 0.710 0.691 0.728 0.711 0.734 0.717 Threshold T 5 300 T 5 500 T 5 1,000 Dep. var. 5 PDIFF1 (1) PDIFF2 (2) PDIFF1 (3) PDIFF2 (4) PDIFF1 (5) PDIFF2 (6) Panel B: requiring the amount of government subsidy to exceed T thousand RMB TREAT 3 POST 0.0084 (2.55) 0.0092 (2.49) 0.0077* (1.80) 0.0084* (1.76) 0.0122** (2.11) 0.0126* (1.93) POST 0.0017 (0.61) 0.0018 (0.57) 0.0001 (0.02) 0.0006 (0.14) 0.0005 (0.10) 0.0002 (0.03) Firm-level controls Yes Yes Yes Yes Yes Yes Year fixed effects Yes Yes Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes Yes Yes N 15,953 15,953 9,921 9,921 5,144 5,144 Adjusted R2 0.712 0.691 0.714 0.692 0.724 0.702 Dep. var. 5 PDIFF1 (1) PDIFF2 (2) PDIFF1 (3) PDIFF2 (4) Panel C: matching all government subsidies regardless of the amount SUBRATIO 3 POST 0.1779 (2.45) 0.1970 (2.40) 0.1773 (2.49) 0.1961 (2.43) POST 0.0003 (0.23) 0.0000 (0.02) 0.0013 (0.95) 0.0011 (0.67) Firm-level controls No No Yes Yes Year fixed effects Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes N 42,221 42,221 42,221 42,221 Adjusted R2 0.712 0.694 0.717 0.699 Note(s): This table presents various robustness checks for our main results. In Panels A and B, we use different threshold to select the government subsidy amount and redo the propensity score matching and DID analysis. In Panel C, we match all government subsidies regardless of the amount and replace the treatment dummy (TREAT) with the subsidy ratio (SUBRATIO), calculated as subsidy amount divided by annual sales. The model includes firm and year fixed effects and firm-level control variables. All continuous variables are winsorized at the 1st and 99th percentiles. Table 5. Robustness checks 3.2 DID results T i h Because we include all subsidy events regardless of the amount, we use the subsidy ratio (SUBRATIO) to capture the economic magnitude of government subsidization and modify Equation (6) as follows: PDIFFi;t ¼ αi þ mt þ β1SUBRATIOi;t 3 POSTi;t þ β2POSTi;t þ γXi;t−1 þ εi;t (8) PDIFFi;t ¼ αi þ mt þ β1SUBRATIOi;t 3 POSTi;t þ β2POSTi;t þ γXi;t−1 þ εi;t (8) (8) In Equation (8), SUBRATIO is calculated as the subsidy amount divided by the exporter’s annual sales. We assign the positive subsidy ratio to all observations in the event window for each treatment exporter that receives any government subsidy. By contrast, observations of control exporters always have a subsidy ratio equal to 0. In this way, the defined variable of SUBRATIO not only captures the intensity of government subsidization but also differentiates between treatment and control exporters. A more intensive subsidy should have a stronger impact on exporters’ product differentiation. We run Equation (8) on the newly constructed testing sample. Consistent with our expectation, we find significantly negative coefficients on SUBRATIO 3 POST, suggesting that treatment firms experience larger decreases in product differentiation after receiving a higher subsidy. The results from the alternative model continue to support our main findings. Given that this alternative model specification complements our baseline DID model, we set forth to report both results for our later analyses. In Equation (8), SUBRATIO is calculated as the subsidy amount divided by the exporter’s annual sales. We assign the positive subsidy ratio to all observations in the event window for each treatment exporter that receives any government subsidy. By contrast, observations of control exporters always have a subsidy ratio equal to 0. In this way, the defined variable of SUBRATIO not only captures the intensity of government subsidization but also differentiates between treatment and control exporters. A more intensive subsidy should have a stronger impact on exporters’ product differentiation. We run Equation (8) on the newly constructed testing sample. Consistent with our expectation, we find significantly negative coefficients on SUBRATIO 3 POST, suggesting that treatment firms experience larger decreases in product differentiation after receiving a higher subsidy. The results from the alternative model continue to support our main findings. Given that this alternative model specification complements our baseline DID model, we set forth to report both results for our later analyses. 3.2 DID results T i h Presented in the parentheses below each coefficient is the t-value based on standard errors clustered by firm. Constant terms are estimated but omitted for presentation. *, and * represent statistical significance at the 1, 5 and 10% levels, respectively Table 5 Robustness checks Corporate product strategies 303 303 subsidized exporters. In Panel A, we use the subsidy ratio (i.e. subsidy amount divided by annual sales). Specifically, we require the subsidy amount to exceed 100,000 RMB and the subsidy ratio to be larger than 0.5, 1 or 2%. For each cut-off ratio, we identify a sample of government-subsidized firms and implement propensity score matching, as introduced in subsection 3.1, to construct a matched sample. For example, in the first two columns in Panel A, we start with a sample of firms receiving a government subsidy exceeding 0.5% of their annual sales. We then match these companies with exporters receiving no subsidy to get a matched sample of 13,036 firm-year observations during the event window of [t3, tþ2]. The choice of different cut-offs can dramatically affect our final sample size, yet we continue to find significantly negative coefficients on the interaction terms of TREAT 3 POST. 304 g y g In Panel B, we use the subsidy amount to construct alternative testing samples. Specifically, we require the subsidy amount to exceed 300,000, 500,000 or 1,000,000 RMB. The lower amounts result in larger sample sizes, but we get qualitatively similar results across all three alternative samples: compared with control firms, treatment firms’ product differentiation decreases after receiving government subsidies. Taken together, the results in Panels A and B show that changing the selection criteria of government subsidization does not change our inferences. g In Panel C, weuse an alternativeapproach to further address the concern of sampleselection. Instead of imposing restrictions on the economic magnitude of government subsidization, we include all exporters’ with a first-time subsidy events during our sample period. Again, we implement propensity score matching to find matching control firms, yielding 42,221 firm-year observations during the event window [t3, tþ2]. CAFR 25,3 4. Heterogeneous effects of government subsidization In this section, we investigate the heterogeneous effects of government subsidization. We first focus on the role of equity ownership by exploring if the relation between government subsidization and product differentiation varies across different types of ownership. Then, we compare the effects of government subsidization between general trade exporters and processing trade exporters. Corporate product strategies In terms of ownership, our sample covers four types of exporters: joint ventures of local firms and foreign companies, wholly foreign-owned enterprises, state-owned enterprises and others. These different types of exporters may have different incentives that affect the relation between government subsidization and product differentiation. To explore this conjecture, we divide our sample into four subsamples and rerun the regressions on each subsample. Table 6 presents the results [13]. In Panel A, we divide our baseline sample into four subsamples according to exporters’ equity ownership type. Results show that the regression coefficient on TREAT 3 POST is significantly negative only for wholly foreign- owned enterprises (Coeff. 5 0.0149, t-value 5 2.70) subsamples. In Panel B, the results from matching all government subsidies, regardless of the amount, show the same pattern. The concentration of depressive effects among wholly foreign-owned exporters suggests that using government subsidies to attract foreign investments may reduce product differentiation. 305 China has made great efforts to attract foreign capital since its economic reform. During our sample period, which covers China’s admittance to the World Trade Organization, several government policies at the central and local levels intend to offer incentives and preferential treatment to foreign investors. While these supporting polices may facilitate long-term innovation and thus promote product differentiation, it is also plausible that firms Dep. var. 5 PDIFF2 JVs (1) WFOEs (2) SOEs (3) Non-SOEs (4) Panel A: results based on our baseline sample TREAT 3 POST 0.0007 (0.09) 0.0149*** (2.70) 0.0103 (0.57) 0.0083 (1.40) POST 0.0039 (0.62) 0.0067 (1.44) 0.0093 (0.51) 0.0012 (0.22) Firm-level controls Yes Yes Yes Yes Year fixed effects Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes N 4,098 6,331 603 6,186 Adjusted R2 0.695 0.732 0.682 0.644 Panel B: matching all government subsidies regardless of the amount SUBRATIO 3 POST 0.2645 (1.27) 0.2054* (1.87) 0.1015 (0.27) 0.1399 (1.00) POST 0.0070 (2.24) 0.0006 (0.26) 0.0052 (0.47) 0.0021 (0.70) Firm-level controls Yes Yes Yes Yes Year fixed effects Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes N 10,823 17,203 1,185 13,010 Adjusted R2 0.706 0.730 0.678 0.647 Note(s): This table presents subsample analyses for each type of firm ownership. We divide our matched sample into four subsamples: joint ventures of local firms and foreign companies (JVs), wholly foreign-owned enterprises (WFOEs), state-owned enterprises (SOEs) and others (labeled as Non-SOEs). Table 6. Heterogeneous effects: type of firm ownership Corporate product strategies 4.1 The role of ownership type 4.1 The role of ownership type China government provides a lot of financial support to firms in private sector and plays a pivotal role in shaping firm behavior (e.g. Fang, Lerner, Wu, & Zhang, 2018; Gong, Shan, & Yu, 2022; Pan, Zhang, & Zhang, 2022). Given that government policies (e.g. subsidy and taxation) often vary for firms with different ownership types, firm ownership is an important dimension of firm-level characteristics in China studies (e.g. Hu, 2001; Qian, Gao, & Tsang, 2015; Fang et al., 2018; Han, He, Pan, & Shi, 2018). We therefore explore if the effect of government subsidization is heterogeneous for firms with different ownership. Corporate product strategies In Panel A, we present the results based on our baseline sample. In Panel B, we match all government subsidies regardless of the amount and replace the treatment dummy (TREAT) with the subsidy ratio (SUBRATIO), calculated as the subsidy amount divided by annual sales. The model includes firm and year fixed effects and firm-level control variables. All continuous variables are winsorized at the 1st and 99th percentiles. Presented in the parentheses below each coefficient is the t-value based on standard errors clustered by firm. Constant terms are estimated but omitted for presentation. , * and * represent statistical significance at the 1, 5 and 10% levels, respectively Table 6. Heterogeneous effects: type of firm ownership Dep. var. 5 PDIFF2 JVs (1) WFOEs (2) SOEs (3) Non-SOEs (4) Panel A: results based on our baseline sample TREAT 3 POST 0.0007 (0.09) 0.0149*** (2.70) 0.0103 (0.57) 0.0083 (1.40) POST 0.0039 (0.62) 0.0067 (1.44) 0.0093 (0.51) 0.0012 (0.22) Firm-level controls Yes Yes Yes Yes Year fixed effects Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes N 4,098 6,331 603 6,186 Adjusted R2 0.695 0.732 0.682 0.644 Panel B: matching all government subsidies regardless of the amount SUBRATIO 3 POST 0.2645 (1.27) 0.2054* (1.87) 0.1015 (0.27) 0.1399 (1.00) POST 0.0070 (2.24) 0.0006 (0.26) 0.0052 (0.47) 0.0021 (0.70) Firm-level controls Yes Yes Yes Yes Year fixed effects Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes N 10,823 17,203 1,185 13,010 Adjusted R2 0.706 0.730 0.678 0.647 N ( ) Thi bl b l l f h f fi hi W di id h d Dep. var. 5 PDIFF2 JVs (1) WFOEs (2) SOEs (3) Non-SOEs (4) Note(s): This table presents subsample analyses for each type of firm ownership. We divide our matched sample into four subsamples: joint ventures of local firms and foreign companies (JVs), wholly foreign-owned enterprises (WFOEs), state-owned enterprises (SOEs) and others (labeled as Non-SOEs). In Panel A, we present the results based on our baseline sample. In Panel B, we match all government subsidies regardless of the amount and replace the treatment dummy (TREAT) with the subsidy ratio (SUBRATIO), calculated as the subsidy amount divided by annual sales. The model includes firm and year fixed effects and firm-level control variables. All continuous variables are winsorized at the 1st and 99th percentiles. Corporate product strategies Presented in the parentheses below each coefficient is the t-value based on standard errors clustered by firm. Constant terms are estimated but omitted for presentation. , * and * represent statistical significance at the 1, 5 and 10% levels, respectively Note(s): This table presents subsample analyses for each type of firm ownership. We divide our matched sample into four subsamples: joint ventures of local firms and foreign companies (JVs), wholly foreign-owned enterprises (WFOEs), state-owned enterprises (SOEs) and others (labeled as Non-SOEs). In Panel A, we present the results based on our baseline sample. In Panel B, we match all government subsidies regardless of the amount and replace the treatment dummy (TREAT) with the subsidy ratio (SUBRATIO), calculated as the subsidy amount divided by annual sales. The model includes firm and year fixed effects and firm-level control variables. All continuous variables are winsorized at the 1st and 99th percentiles. Presented in the parentheses below each coefficient is the t-value based on standard errors clustered by firm. Constant terms are estimated but omitted for presentation. *, and * represent statistical significance at the 1, 5 and 10% levels, respectively benefiting from these policies may expand product lines to exploit short-term profitability, resulting in lower product differentiation. Our main finding supports the latter argument that government subsidization induces homogenous competition among exporters in China because exporters tend to use subsidy funding to expand product lines rather than invest in innovation. Our subsample analyses suggest that this is especially true for wholly foreign- owned enterprises. benefiting from these policies may expand product lines to exploit short-term profitability, resulting in lower product differentiation. Our main finding supports the latter argument that government subsidization induces homogenous competition among exporters in China because exporters tend to use subsidy funding to expand product lines rather than invest in innovation. Our subsample analyses suggest that this is especially true for wholly foreign- owned enterprises. p Several features of wholly foreign-owned exporters might help explain why the depressive effects of government subsidization on product differentiation concentrate in these exporters. First, parent companies’ intention of establishing facilities in China might be building a profit center as appose to an innovation center. These exporters are typically subsidiaries of foreign companies from developed countries where can better nurture innovation. As an emerging country, China’s relative advantage is the cheap labor costs, not technology or innovation. Corporate product strategies Second, because of their foreign background, these exporters gain better understanding of the international market. Better knowing the market enables them to identify and follow the trends. Third, these exporters probably perceive higher uncertainty in operational environment that may discourage long-term investments such as innovation projects. For instance, the perceived uncertainty may result from the lack of political connection and a poor understanding of Chinese culture. Taken together, compared with other exporters in our sample, it is more likely to be true for these wholly foreign-owned exporters that making use of government subsidies in expanding mature product lines instead of developing new products is an optimal product strategy. 306 4.2 General trade versus processing trade CAFR 25,3 4.2 General trade versus processing trade p g To promote exportation, the Chinese government has focused on processing trade since the 1980s. In our sample period (2000–2012), the percentage of processing trade gradually decreased and general trade began to dominate exports. Given the noticeable differences in these two types of trades (e.g. Dai, Maitra, & Yu, 2016), one might expect government subsidization to affect each firm type differently. To explore this conjecture, we split our sample into two subsamples: firms focusing solely on general trade and firms engaging in some degree of processing trade. Table 7 presents the results. In Panel A, results show that the regression coefficients on TREAT 3 POST are negative for both subsamples but significant only for general trade exporters (Coeff. 5 0.0100, t-value 5 2.08). In Panel B, the results from matching all government subsidies regardless of the amount also show that the coefficient on SUBRATIO 3 POST is significantly negative only for the general trade subsample. Our results suggest that the depressive effects of government subsidization on product differentiation is concentrated among general-trade exporters, which is consistent with the notion that processing-trade exporters are mainly responsible for product manufacture, whereas general-trade exporters can choose what to design, produce and sell. 5. Additional analyses on the effects of government subsidization In prior sections, we document a significantly negative effect of government subsidization on product differentiation among exporters in China. This finding is consistent with our argument that subsidization induces homogenous competition. That is, companies tend to use subsidy funding to expand existing product lines that are similar to those of competitors. Accordingly, subsidized exporters should have more diversified product lines, compared to unsubsidized ones. In addition, because our results suggest that subsidy funding is not invested in innovation, it seems unlikely that it can positively affect exporters’ product value and quality. In this section, we shift our focus from studying product differentiation to Dep. var. 4.2 General trade versus processing trade 5 PDIFF2 General trade (1) Processing trade (2) Panel A: results based on our baseline sample TREAT 3POST 0.0100 (2.08) 0.0069 (1.32) POST 0.0018 (0.44) 0.0001 (0.03) Firm-level controls Yes Yes Year fixed effects Yes Yes Firm fixed effects Yes Yes N 9,921 7,297 Adjusted R2 0.668 0.723 Panel B: matching all government subsidies regardless of the amount SUBRATIO 3 POST 0.3042 (2.57) 0.0607 (0.58) POST 0.0008 (0.37) 0.0027 (1.11) Firm-level controls Yes Yes Year fixed effects Yes Yes Firm fixed effects Yes Yes N 25,231 16,990 Adjusted R2 0.676 0.728 Note(s): This table presents subsample analyses by dividing our matched sample into firms focusing on general trade and those focusing on processing trade. In Panel A, we present results based on our baseline sample. In Panel B, we match all government subsidies regardless of the amount and replace the treatment dummy (TREAT) with the subsidy ratio (SUBRATIO), calculated as the subsidy amount divided by annual sales. The model includes firm and year fixed effects and firm-level control variables. All continuous variables are winsorized at the 1st and 99th percentiles. Presented in the parentheses below each coefficient is the t-value based on standard errors clustered by firm. Constant terms are estimated but omitted for presentation. *, and * represent statistical significance at the 1, 5 and 10% levels, respectively Table 7. Heterogeneous effects: General versus processing trade Corporate product strategies 307 Dep. var. 5 PDIFF2 General trade (1) Processing trade (2) Panel A: results based on our baseline sample TREAT 3POST 0.0100 (2.08) 0.0069 (1.32) POST 0.0018 (0.44) 0.0001 (0.03) Firm-level controls Yes Yes Year fixed effects Yes Yes Firm fixed effects Yes Yes N 9,921 7,297 Adjusted R2 0.668 0.723 Panel B: matching all government subsidies regardless of the amount SUBRATIO 3 POST 0.3042 (2.57) 0.0607 (0.58) POST 0.0008 (0.37) 0.0027 (1.11) Firm-level controls Yes Yes Year fixed effects Yes Yes Firm fixed effects Yes Yes N 25,231 16,990 Adjusted R2 0.676 0.728 Corporate product strategies 307 Note(s): This table presents subsample analyses by dividing our matched sample into firms focusing on general trade and those focusing on processing trade. In Panel A, we present results based on our baseline sample. In Panel B, we match all government subsidies regardless of the amount and replace the treatment dummy (TREAT) with the subsidy ratio (SUBRATIO), calculated as the subsidy amount divided by annual sales. 4.2 General trade versus processing trade The model includes firm and year fixed effects and firm-level control variables. All continuous variables are winsorized at the 1st and 99th percentiles. Presented in the parentheses below each coefficient is the t-value based on standard errors clustered by firm. Constant terms are estimated but omitted for presentation. *, and * represent statistical significance at the 1, 5 and 10% levels, respectively Table 7 Heterogeneous effects General versu processing trad examining potential outcomes of government subsidization on product diversity, value added and quality. To empirically test these potential outcomes, we use the number of products (NP) to capture exporters’ product diversity based on eight-digit HS codes. To capture domestic value added, we follow Kee and Tang (2016) to calculate the ratio of domestic value added for exports to gross exports (DVAR). Finally, we follow prior literature to construct two measures of product quality: Khandelwal’s (2010) product quality measure (QK) and Fan, Li, and Yeaple’s (2015) estimated export product quality (QF). Table 8 presents the results. Panel A shows the results based on our baseline sample, and Panel B shows the results from matching all government subsidies regardless of amount. In Column (1), NP is the dependent variable. Consistent with our expectation, we find that subsidized exporters have more export products than unsubsidized ones. In Column (2), we focus on domestic value added [14]. We find that the value added in products of subsidized exporters is significantly lower in the post-subsidy period, compared with that of unsubsidized exporters. In Columns (3)–(4), we use two measures of export product quality as independent variables, and we find negative coefficients on TREAT 3 POST. Although the coefficients in Panel A are insignificant, they are statistically significant in Panel B. Taken together, we find some evidence that government subsidization results in exporters’ expanding their product lines but decreasing their domestic value added and export product quality. These findings corroborate our main finding, suggesting that government subsidization induces homogenous competition among exporters in China. 6. Conclusion I hi In this paper, we investigate the effect of government subsidization on exporters’ product strategy. Exploiting comprehensive product-level export data on exporters in China during Dep. var. 5 NP (1) DVAR (2) QK (3) QF (4) Panel A: results based on our baseline sample TREAT 3POST 0.0420* (3.10) 0.0389* (2.62) 0.0760 (0.60) 0.0321 (0.49) POST 0.0231* (1.90) 0.0088 (0.56) 0.1564 (1.50) 0.0807 (1.45) Firm-level controls Yes Yes Yes Yes Year fixed effects Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes N 17,218 6,807 13,873 13,873 Adjusted R2 0.776 0.659 0.710 0.740 Panel B: matching all government subsidies regardless of the amount SUBRATIO 3POST 0.6299* (1.86) 1.0159* (3.62) 9.0183 (2.45) 4.8141* (2.71) POST 0.0022 (0.34) 0.0068 (0.90) 0.0099 (0.18) 0.0014 (0.05) Firm-level controls Yes Yes Yes Yes Year fixed effects Yes Yes Yes Yes Firm fixed effects Yes Yes Yes Yes N 42,221 17,347 32,642 32,642 Adjusted R2 0.782 0.703 0.688 0.722 Note(s): This table presents the effects of government subsidization on firms’ other outcomes. Specifically, we focus on the total number of export products (NP), Kee and Tang’s (2016) ratio of domestic value added in exports to gross exports (DVAR), Khandelwal’s (2010) product quality measure (QK) and Fan et al.’s (2015) estimated export product quality (QF). In Panel A, we present results based on our baseline sample. In Panel B, we match all government subsidies regardless of the amount and replace the treatment dummy (TREAT) with the subsidy ratio (SUBRATIO), calculated as the subsidy amount divided by annual sales. The model includes firm and year fixed effects and firm-level control variables. All continuous variables are winsorized at the 1st and 99th percentiles. Presented in the parentheses below each coefficient is the t-value based on standard errors clustered by firm. Constant terms are estimated but omitted for presentation. , * and * represent statistical significance at the 1, 5 and 10% levels, respectively Table 8. Other outcomes of government subsidization CAFR 25,3 308 2000–2012, we introduce a novel measure of product differentiation that captures the extent to which an exporter can differentiate its product space from that of competitors. We use a propensity score matching procedure to construct a sample of comparable treatment and control firms, and our DID analyses provide evidence that the product differentiation of treatment exporters decreases in the postsubsidy period. 6. Conclusion I hi We further explore potential heterogeneous effects of government subsidization and find that the depressive effects of subsidization on product differentiation are concentrated mostly among wholly foreign- owned exporters and general-trade exporters. In addition, we find some evidence that subsidization leads to increases in product lines and decreases in domestic value added and overall product quality. Overall, our results suggest that government subsidization induces homogenous competition among exporters in China. Relying on a new measure of product differentiation for a large sample of these exporters, our study provides insights that government subsidization can affect exporters’ mode of competition. Nevertheless,afewcaveatsneedtobeconsidered.First,duetothedatalimitation,weusefirm-level data of general production-related subsidies rather than export-specific subsidies. The central and local governments have different incentives to grant subsidies, and granted subsidies may have differentpurposes(Girmaet al.,2009; Lee, Walker, &Zeng,2014). Therefore,ourpaper onlyspeaks to the average effect of general subsidies, and without knowing the specific policy objective, it is difficult to tell if it is an intended or unintended consequence. Second, we acknowledge that the association between government subsidization and product differentiation may not be causal because government do not randomly select firms for subsidization and the selection process is largely unobservable. For instance, government may tend to provide financial support to firms facing higher level of product market competition for some unknown reasons. We try to establish causality by using a DID design combined with propensity score matching, but the propensity score matching can only deal with differences in observed characteristics between treatment and controlgroup.Toenhancecausalinferences,futurestudymayconsiderbettersettings,ifavailable, e.g. natural experiments. score matching can only deal with differences in observed characteristics between treatment and controlgroup.Toenhancecausalinferences,futurestudymayconsiderbettersettings,ifavailable, e.g. natural experiments. Notes 1. Note that government subsidies in our study refer to general production-related subsidies to exporters. Due to the same data limitation as in Girma et al. (2009), we have firm-level data of general production-related subsidies but not export-specific subsidies. According to our own calculation, the total subsidies received by exporters in China from 1998 to 2013 grew annually by around 15%, from 6.8 bn to 56 bn Chinese yuan. 309 2. It is also possible that government subsidies could have no effects on exporters’ product strategies because changing long-term strategies can be costly and the subsidies may not be sufficient to cover the costs. 3. The harmonized system (HS) is a global product classification system, and the HS codes are commonly used in international trade. More details about HS codes can be found at https://www. trade.gov/harmonized-system-hs-codes 4. Given that we conduct matrix operations with a large dataset, this exclusion also considerably reduces the computational burden. 4. Given that we conduct matrix operations with a large dataset, this exclusion also considerably reduces the computational burden. 5. These trading companies serve as an intermediary in international trade. As documented in Ahn et al. (2011), 22% of Chinese exports were handled by trading companies in 2005. 5. These trading companies serve as an intermediary in international trade. As documented in Ahn et al. (2011), 22% of Chinese exports were handled by trading companies in 2005. 6. In the robustness checks (see Table 5), changing the requirements for treatment firms does not change our inferences. 6. In the robustness checks (see Table 5), changing the requirements for treatment firms does not change our inferences. 7. Because firm fixed effects absorb the dummy of treatment firms (TREAT), we do not include TREAT in the regression model. 7. Because firm fixed effects absorb the dummy of treatment firms (TREAT), we do not includ TREAT in the regression model. 7. Because firm fixed effects absorb the dummy of treatment firms (TREAT), we do not include TREAT in the regression model. 8. In all regression analyses, we report the t-value, which is calculated based on standard errors clustered by firm and shown in parentheses below each coefficient. Constant terms are estimated but omitted for presentation. 8. In all regression analyses, we report the t-value, which is calculated based on standard errors clustered by firm and shown in parentheses below each coefficient. Notes Constant terms are estimated but omitted for presentation. 9. We acknowledge that the propensity score matching has its own limitations (e.g. Cram, Karan, & Stuart, 2009; Shipman et al., 2017). Propensity score matching cannot deal with unobservable differences between treatment and control groups and we can only match between treatment and control firms based on observable characteristics (Shipman et al., 2017). In addition, there could be some characteristics that cannot be perfectly matched and accordingly, we follow Cram et al.’s (2009) suggestion and control for these factors in the analyses of matched sample. 10. The parallel trend assumption is pivotal to DID estimation. However, the parallel trend requires only the relative change, rather than the absolute level, to be similar in the preevent period. Therefore, we control for the average value of annual changes in PDIFF2 in the previous three years. Our results are similar if we control for the average change in PDIFF1 or control for neither of them. 11. We find suitable control firm matches for about 20% (52615/13,394) of our treatment firms. The rest lack a suitable match based on type of ownership, location province, industry and year. 11. We find suitable control firm matches for about 20% (52615/13,394) of our treatment firms. The rest lack a suitable match based on type of ownership, location province, industry and year. 12. This magnitude is moderate but can be economically meaningful: compared with the mean value of the dependent variable (PDIFF2) in Column (4), the coefficient on TREAT3POST represents a 2% (50.0091/0.4469) decrease in product differentiation. 12. This magnitude is moderate but can be economically meaningful: compared with the mean value of the dependent variable (PDIFF2) in Column (4), the coefficient on TREAT3POST represents a 2% (50.0091/0.4469) decrease in product differentiation. 13. Results are essentially the same when we alternatively use the PDIFF1 as dependent variable. 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https://openalex.org/W4395037433	https://link.springer.com/content/pdf/10.1007/s12304-024-09562-1.pdf	English	null	Metaphysics of the Organic Whole: Ehrenfels, Uexküll, and Merleau-Ponty	Biosemiotics. Journal/Biosemiotics	2,024	cc-by	12,904	Biosemiotics (2024) 17:341–360 https://doi.org/10.1007/s12304-024-09562-1 Biosemiotics (2024) 17:341–360 https://doi.org/10.1007/s12304-024-09562-1 https://doi.org/10.1007/s12304-024-09562-1 RESEARCH RESEARCH Abstract The aim of this paper is to compare the theory of Gestalt qualities, introduced by the Austrian philosopher Christian von Ehrenfels (1859–1932), with the concept of Umwelt, proposed by Jakob von Uexküll (1864–1944). The primary basis for the comparison will be the phenomenology of Maurice Merleau-Ponty (1908–1961), who extensively discusses the two concepts in his work. In the Uexküll–Ehrenfel sian context, we focus on analysing the similarities and differences of their argu mentation and model approaches to understanding the living and non-living natural entities, their mutual communication, development, and ontological grounding. We also consider the role of individual experience with the environment: in that con text, the metaphysical frameworks within which the two thinkers operate in their efforts to explain natural phenomena are central to our comparative reflections. Keywords Gestalt · Umwelt · Phenomenology · Darwinism · Teleology · Complexity · Experience Keywords Gestalt · Umwelt · Phenomenology · Darwinism · Teleology · Complexity · Experience Keywords Gestalt · Umwelt · Phenomenology · Darwinism · Teleology · Complexity · Experience Lenka Ovčáčková lenka.ovcackova@natur.cuni.cz Metaphysics of the Organic Whole: Ehrenfels, Uexküll, and Merleau-Ponty Lenka Ovčáčková1 · Jana Švorcová1 Received: 15 December 2023 / Accepted: 14 April 2024 / Published online: 23 April 2024 © The Author(s) 2024 1 Department of Philosophy and History of Science, Faculty of Science, Charles University, Prague, Czech Republic @ 1 Department of Philosophy and History of Science, Faculty of Science, Charles University, Prague, Czech Republic Introduction In the following, we focus primarily on the metaphysical background of the analysed approaches to the living: Ehrenfels’s metaphysics of the organic world, Uexküll’s counterpoint teleology, and Merleau-Ponty’s particular metaphys ics, which moves on the interface of the finite and infinite. After a brief biographical introduction of the proponents, we will point out pos sible analogies between Ehrenfels’s theory of Gestalt and Uexküll’s Umwelt. In the following section, we discuss the ambiguous conceptions of teleology of both Uexküll and Ehrenfels and, in the fourth section, we highlight their ambiguous rela tionship to Darwinism. The last section which deals with Merleau-Ponty’s phenom enological perception of nature also outlines a synthesis of Ehrenfels’s and Uexküll’s views. While Merleau-Ponty’s direct references to Gestalt and Umwelt run through all preceding sections, they are discussed in detail in the sixth part of the paper. We conclude with a reflection on how the reconstructed conceptual patterns could be applicable to current theoretical biology. Introduction This paper presents a comparative analysis of Christian von Ehrenfels’s (1859–1932) and Jakob von Uexküll’s (1864–1944) scientific and philosophical reflections upon natural phenomena, with a focus on Ehrenfels’s concept of the theory of Gestalt qualities and Uexküll’s concept of Umwelt. Although the two thinkers were contem poraries, we found no instances of mutual referencing in their work, and the same applies to the reception of their scientific work. Nevertheless, the philosophical and scientific reflections of the French phenomenologist Maurice Merleau-Ponty (1908– 1 3 3 L. Ovčáčková, J. Švorcová 342 1961), published under the title Nature. Course Notes from the Collège de France, do provide a sort of link between the richness of their thought and therefore also an inspiring framework for our comparison. Merleau-Ponty discusses Uexküll’s theo retical-biological reflections of organismal perception in detail in the second series of lectures (Animality, the Human Body, and the Passage to Culture, 1957–1958, subchapter ‘Animality: The Study of Animal Behavior’) and repeatedly returns to Uexküll’s notion of Umwelt also in the third series of lectures (Nature and Logos: The Human Body, 1959–1960). He also repeatedly references the concept of Gestalt in his phenomenological reflections, both directly in relation to Uexküll’s Umwelt and within broader phenomenological considerations upon the relation of parts and whole, subject and object, and the visible and invisible. When discussing Gestalt, however, he does not speak directly about Christian von Ehrenfels, the ‘father of the theory of Gestalt qualities’. For the most part, he draws on the work of thinkers who further developed Ehrenfels’s theory, such as Koffka, Köhler, and Wertheimer (see below). We will, however, work primarily with Ehrenfels’s interpretations of the theory of Gestalt qualities, which is rooted in the cosmogonic dualistic dynamic prin ciple of juxtaposition of the shaping force (gestaltende Kraft) and chaos. Although Ehrenfels’s primary academic background was in philosophy, he also reflected upon the origin and development of living beings, on the relationality of animate and inani mate nature, and on the physical and psychic dimensions of reality, while consider ing both pro- and anti-Darwinian positions. Although we did not find any analysis related to perception or subjectivity in Ehrenfels’s work in comparison to Uexküll, Ehrenfels’s broader insights into ways of understanding natural phenomena are no less important. Short Scientific Biographies The Austrian philosopher Christian von Ehrenfels (1859–1932) was a versatile thinker active in the late 19th and early 20th century. His most influential teach ers were Franz Brentano in Vienna and Alexius Meinong in Graz, but his academic 1 3 Metaphysics of the Organic Whole: Ehrenfels, Uexküll, and… 343 career was strongly linked to the academic environment of Prague.1 Ehrenfels’s aca demic work focused on value ethics Über Fühlen und Wollen, 1888 [On Feeling and Willing], System der Werttheorie, 1897–1898 [System of Value Theory] but he also made important contributions to several subjects in psychology, aesthetics, logic, social biology, Darwinism, vitalism, and eugenics. He lectured, among other things, on cosmic morphology and prime numbers. Surprisingly, the topic of the theory of Gestalt qualities was not widely represented in his lectures (except for the years 1918, 1923, and 1926 at the University of Prague; Ovčáčková, 2022).2 Ehrenfels proposed the concept of Gestalt qualities in a seminal paper from 1890 entitled Über Gestaltqualitäten [On Gestalt qualities], whose basic message was that the whole is not only made up of its part: it is something more (see further below). Ehrenfels unfortunately did not explicitly return to this subject in writing for another 32 years, when he critically reassessed and further developed his original Gestalt con cept in his work on prime numbers (Das Primzahlengesetz, 1922 [The law of prime numbers]. At this occasion, he not only republished his paper Über Gestaltqualitäten but also added an accompanying text entitled Weiterführende Bemerkungen [Explan atory Notes], which built on his book Kosmogonie, 1916 [Cosmogony], where the cosmogonic Gestalt principles play an important role.3 Jakob von Uexküll (1864–1944), a German biologist of Estonian origin, exempli fies a vibrant discourse between a mechanistic and a non-mechanistic worldview. Since his student years, Uexküll was influenced by the philosophy of Immanuel Kant, which led him to reflections upon the subjective world: ‘He realized that the transcen dental analysis that Kant directed to the minds of human beings could be extended to other animal species too’ (Brentari 2015: 23). Uexküll rejected the idea that ani mals are reflective machines adapted to random physical and chemical environments. Instead, he argued that animal behaviour can only be explained by taking into con sideration processes in their subjective Umwelt (Kriszat, 1956b: 167). 1 In 1896, Ehrenfels became an extraordinary and in 1899 a full professor of philosophy at the German part of the Charles-Ferdinand University (renamed the German University in 1920) in Prague. He retired in 1929 but worked at the university until his death in 1932 (cf. Ovčáčková, 2022). 2 In the overview of lectures, Ehrenfels’s courses at the Department of Philosophy in Prague were described as intended for students of natural sciences and Ehrenfels was also instrumental in founding, the Department of Natural Philosophy (Lehrstuhl für Naturphilosophie) at the Faculty of Science of the German University in 1929 (cf. Ovčáčková 1922). After Ehrenfels’s death, the department was headed by the philosopher and mathematician Rudolf Carnap until 1936. 3 Gestalt qualities, with an overlap to biology, are also the subject of Ehrenfels’s scientific reform religion. Besides his book Cosmogony, see also his essays Gedanken über die Religion der Zukunft (Reflections on the Religion of the Future, 1922) and Die Religion der Zukunft (The Religion of the Future, 1929). 3 Gestalt qualities, with an overlap to biology, are also the subject of Ehrenfels’s scientific reform religion. Besides his book Cosmogony, see also his essays Gedanken über die Religion der Zukunft (Reflections on the Religion of the Future, 1922) and Die Religion der Zukunft (The Religion of the Future, 1929). 1 In 1896, Ehrenfels became an extraordinary and in 1899 a full professor of philosophy at the German part of the Charles-Ferdinand University (renamed the German University in 1920) in Prague. He retired in 1929 but worked at the university until his death in 1932 (cf. Ovčáčková, 2022). 2 In the overview of lectures, Ehrenfels’s courses at the Department of Philosophy in Prague we described as intended for students of natural sciences and Ehrenfels was also instrumental in foundin the Department of Natural Philosophy (Lehrstuhl für Naturphilosophie) at the Faculty of Science of th German University in 1929 (cf. Ovčáčková 1922). After Ehrenfels’s death, the department was heade by the philosopher and mathematician Rudolf Carnap until 1936. Short Scientific Biographies Uexküll’s modified Kantianism also included a teleological and holistic metaphysics of Nature (Jaroš & Brentari, 2022), where Nature is understood as the goal-directed natural fac tor arranging the Umwelten of all living beings so that they can interact effectively. Uexküll studied biology at the University of Dorpat, now Tartu. Initially enthusi astic about Darwinism, he soon became its fierce opponent, as evidenced by his fre quent anti-Darwinist statements and a tendency to contrast the absolute and relative worlds (see below). Uexküll then worked for many years at the Institute of Physiol ogy at the University of Heidelberg. While there, he undertook a number of research 1 1 3 3 L. Ovčáčková, J. Švorcová 344 stays at the Stazione Zoologica in Naples, where he met, Hans Driesch, among oth ers (Brentari 2015: 26). The highlight of his career in theoretical biology was the founding of the Institute for Umwelt Research at the University of Hamburg. Uexküll introduced the concept of Umwelt in his book Umwelt und Innenwelt der Tiere, 1909 [Umwelt and Inner World of Animals] and developed it further in his much later publications from 1934, such as Streifzüge durch die Umwelten von Tieren und Men schen [A Foray into the Worlds of Animals and Humans] and Die Bedeutungslehre [A Theory of Meaning]. In the following, we also draw on the essays Uexküll published from 1910 to 1913 in the journal Die neue Rundschau. The French phenomenologist Maurice Merleau-Ponty (1908–1961) was strongly inspired by Edmund Husserl and Martin Heidegger but developed their thought fur ther, with a focus on the significance of subjective experience within a broad inter disciplinary framework, e.g., Nature, but also Le Visible et l’Invisible, 1964 [The Visible and The Invisible] and Phénoménologie de la perception, 1945 [Phenomenol ogy of Perception]. He summarised his basic approach as follows: ‘There is nature wherever there is a life that has meaning, but where, however, there is not thought…’ (Merleau-Ponty, 2003: 3). In the introduction to the first course in Nature, Merleau- Ponty points to the undercurrent of the relation between Nature and Physis (φύσις), that is, the plant world whose ‘primordial and lexical sense’ is related to phyo, i.e. the birth and growth of plants. The related reference to ‘autoproduction of mean ing’ clearly indicates Merleau-Ponty’s phenomenological position where nature is ‘unconstructed’ and characterised by permanence and eternity. 4 The roots of Ehrenfels’s dualism of entelechy and chaos go explicitly back to the thoughts of Anaxi mander and Anaxagoras. For both Ehrenfels and Uexküll, the richness of Kant’s and Goethe’s thought is an important source of inspiration. Short Scientific Biographies Nature is the nour ishing soil that ‘carries us’ (Merleau-Ponty, 2003: 3–4). These ideas anticipate the overall framework of his reflections on nature, closely associated with the concepts of Gestalt and Umwelt. Ehrenfels’s Concept of Gestalt In line with their holistic approach to the living, both Uexküll and Ehrenfels work with the analogy of melody. Ehrenfels’s seminal article Über Gestaltqualitäten (1890)7 was significantly influenced by his deep appreciation of music. The example of melody and musical harmony allowed him to express his thoughts on Gestalt qual ities with emphasis on the relationship between the parts and the whole. The whole, he notes, is not merely the sum of parts or elements or a reference to the relationships between them; the whole becomes a higher reality than its parts with the emergence of a new ‘quality’: If the memory images of the successive tones are present as a simultaneous complex of consciousness, then the idea of a new category can emerge in con sciousness, namely a unified idea which is connected in a subtle way with the idea of the tone complex in question. The imagination of this whole belongs to a new category for which the name “fundierte Inhalte” has become common. Not all fundierte Inhalte are of a descriptive nature and related to the concept of melody. (Ehrenfels, 1932, in Weinhandl, 1960: 61)8 In connection with the above, it should be added that every grounded content (fundi erter Inhalt) requires a certain foundation (Fundament). This does not mean, however, 5 As illustrated below, Ehrenfels dismisses chance as the sole principle of formation. He regards chance (identified with chaos) as one of the dualistic principles that contrasts with the second principle, namely, the shaping force. 6 As shown below, Ehrenfels, in contrast to Uexküll, rejects a teleological interpretation of natural devel opment. His concept of the shaping force, nonetheless, embodies a formation principle that enhances complexity, emphasizing not the origin of specific species, but rather the diversity of life itself. Thus, we employ the term ‘specific teleology’ within this context. 7 For Ehrenfels, the key inspiration for this essay was Ernst Mach’s Beiträge zur Analyse der Empfindun gen (1886), where the essence of a melody is identified with the sum of the perceptions of the individual notes. According to Ehrenfels, however, these perceptions were not sufficiently clarified (cf. Ehrenfels, 1890, 1932). Ehrenfels’s theory of Gestalt qualities is nowadays relatively little known, and the histori cal reception of the Gestalt theory is associated mainly with members of the Berlin/Frankfurt school of Gestalt psychology such as the aforementioned Max Wertheimer, Wolfgang Köhler, and Kurt Koffka. Ehrenfels’s Gestalt and Uexküll’s Umwelt A comparison between Uexküll’s concept of Umwelt and Ehrenfels’s Gestalt theory may at first sight seem difficult. Uexküll’s concept of Umwelt is based on significa tion, that is, it works with the notion of animals finding their bearings in the world by means of their sensory organs and in accordance with their Bauplan. Umwelt is thus a concept placed primarily within a framework of physiology. Ehrenfels, on the other hand, in his theory of the Gestalt focuses on morphology, especially metaphysical morphology, within the context of theoretical biology.4 Nevertheless, one can also find a number of substantive similarities between these two concepts: they both aim at holistic explanations of phenomena in nature, both react to Darwinism, both reject 1 3 Metaphysics of the Organic Whole: Ehrenfels, Uexküll, and… 345 chance as a primary factor determining the character of living nature5, and they both work with a specific teleology.6 chance as a primary factor determining the character of living nature5, and they both work with a specific teleology.6 6 As shown below, Ehrenfels, in contrast to Uexküll, rejects a teleological interpretation of natural devel opment. His concept of the shaping force, nonetheless, embodies a formation principle that enhances complexity, emphasizing not the origin of specific species, but rather the diversity of life itself. Thus, we employ the term ‘specific teleology’ within this context. 5 As illustrated below, Ehrenfels dismisses chance as the sole principle of formation. He regards chance (identified with chaos) as one of the dualistic principles that contrasts with the second principle, namely, the shaping force. 7 For Ehrenfels, the key inspiration for this essay was Ernst Mach’s Beiträge zur Analyse der Empfindun gen (1886), where the essence of a melody is identified with the sum of the perceptions of the individual notes. According to Ehrenfels, however, these perceptions were not sufficiently clarified (cf. Ehrenfels, 1890, 1932). Ehrenfels’s theory of Gestalt qualities is nowadays relatively little known, and the histori cal reception of the Gestalt theory is associated mainly with members of the Berlin/Frankfurt school of Gestalt psychology such as the aforementioned Max Wertheimer, Wolfgang Köhler, and Kurt Koffka. Aside from them, Alexius Meinong’s school of thought in Graz also played an important role. For a historical overview with a detailed account of the post-Ehrenfels development of the Gestalt theory and attempts to link holistic thinking and the natural sciences, see Ash (1995). According to Merleau-Ponty, elementary functions likewise do not form a whole by their sum and the parts of a thing are not linked merely by external association (Merleau-Ponty, 2002). 5 As illustrated below, Ehrenfels dismisses chance as the sole principle of formation. He regards chance (identified with chaos) as one of the dualistic principles that contrasts with the second principle, namely, the shaping force. 6 As shown below, Ehrenfels, in contrast to Uexküll, rejects a teleological interpretation of natural devel opment. His concept of the shaping force, nonetheless, embodies a formation principle that enhances complexity, emphasizing not the origin of specific species, but rather the diversity of life itself. Thus, we employ the term ‘specific teleology’ within this context. 7 For Ehrenfels, the key inspiration for this essay was Ernst Mach’s Beiträge zur Analyse der Empfindun gen (1886), where the essence of a melody is identified with the sum of the perceptions of the individual notes. According to Ehrenfels, however, these perceptions were not sufficiently clarified (cf. Ehrenfels, 1890, 1932). Ehrenfels’s theory of Gestalt qualities is nowadays relatively little known, and the histori cal reception of the Gestalt theory is associated mainly with members of the Berlin/Frankfurt school of Gestalt psychology such as the aforementioned Max Wertheimer, Wolfgang Köhler, and Kurt Koffka. Aside from them, Alexius Meinong’s school of thought in Graz also played an important role. For a historical overview with a detailed account of the post-Ehrenfels development of the Gestalt theory and attempts to link holistic thinking and the natural sciences, see Ash (1995). According to Merleau-Ponty, elementary functions likewise do not form a whole by their sum and the parts of a thing are not linked merely by external association (Merleau-Ponty, 2002). 8 ‘Wenn die Erinnerungsbilder der aufeinanderfolgenden Töne als ein gleichzeitiger Bewusstseinskom plex vorliegen, so kann im Bewusstsein die Vorstellung einer neuen Kategorie auftauchen, und zwar eine einheitliche Vorstellung, welche auf eine eigentümliche Weise mit der Vorstellung des betreffenden Tonkomplexes verbunden ist. Die Vorstellung dieses Ganzen gehört einer neuen Kategorie an, für welche der Name „fundierte Inhalte“ üblich wurde. Nicht alle fundierten Inhalte sind anschaulicher Natur und der Melodievorstellung verwandt’ (translation of German quotes by Ovčáčková). Ehrenfels’s Concept of Gestalt Aside from them, Alexius Meinong’s school of thought in Graz also played an important role. For a historical overview with a detailed account of the post-Ehrenfels development of the Gestalt theory and attempts to link holistic thinking and the natural sciences, see Ash (1995). According to Merleau-Ponty, elementary functions likewise do not form a whole by their sum and the parts of a thing are not linked merely by external association (Merleau-Ponty, 2002). 8 ‘Wenn die Erinnerungsbilder der aufeinanderfolgenden Töne als ein gleichzeitiger Bewusstseinskom plex vorliegen, so kann im Bewusstsein die Vorstellung einer neuen Kategorie auftauchen, und zwar eine einheitliche Vorstellung, welche auf eine eigentümliche Weise mit der Vorstellung des betreffenden Tonkomplexes verbunden ist. Die Vorstellung dieses Ganzen gehört einer neuen Kategorie an, für welche der Name „fundierte Inhalte“ üblich wurde. Nicht alle fundierten Inhalte sind anschaulicher Natur und der Melodievorstellung verwandt’ (translation of German quotes by Ovčáčková). 3 3 L. Ovčáčková, J. Švorcová 346 that every foundation will also acquire a fundierter Inhalt.9 The connection between Gestalt quality and memory is likewise significant.10 For Ehrenfels, memory images (Erinnerungsbilder) become conscious through the unified idea (einheitliche Vorstel lung). Individual elements without a Gestalt quality are thus much more difficult to remember than melodies, which are temporal Gestalt qualities that reflect a process, or harmonies, which are non-temporal Gestalt qualities that reflect a momentary state (Ehrenfels, 1932, in Weinhandl, 1960: 62).11 ( ) The notion of the ‘height of the Gestalt’ (Höhe der Gestalt), which ‘grows with the product of its constituents, its uniformity and the diversity of its parts’ (Ehrenfels, 1922, in Weinhandl, 1960: 50)12 is intrinsically connected to a gradation of Gestalt. This can extend infinitely – unlike the purity of Gestalt, which reaches a maximum threshold that cannot be surpassed. For instance, pure Gestalten may include flawless mathematical solids that achieve maximum purity at a relatively lower Gestalt level. Ehrenfels explores the prospect of building an entire aesthetics based on the prin ciples of the Gestalt theory, especially in relation to the beauty of natural phenomena. Within this framework, the beauty of a Gestalt would increase proportionally with its complexity. A higher Gestalt is, for example, a rose or a swallow compared to a pile of rocks or a lump of dirt. Low Gestalten are ugly because they are disharmoni ous. 9 Some of his pupils disagreed with Ehrenfels on this point. Some considered the interdependence between the Fundament and the fundierter Inhalt to be absolutely necessary (e.g. Meinong and Benusi), for others (Wertheimer, Köhler) the conception of melody was not about the production of the fundierter Inhalt but about perceiving (Bemerken) (Ehrenfels, 1932, in Weinhandl, 1960: 61). 13 The genealogical tree represents both quantitative and qualitative development: ‘From a few and rela tively little differentiated Ur-beginnings, the diversity of shapes grows up towards us in a fan-like manner’ (Ehrenfels, 1916: 11). The target is life itself, the shaping of innumerable diverse forms as the Gestalt series and thus also the complexity increase. Essential for Ehrenfels is the interconnectedness of the ‘Dar winian idea of selection with the assumption of an immaterial, psychoid – but without a sense of purpose – organising principle’ (Ehrenfels, 1916: 84). 9 Some of his pupils disagreed with Ehrenfels on this point. Some considered the interdependence between the Fundament and the fundierter Inhalt to be absolutely necessary (e.g. Meinong and Benusi), for others (Wertheimer, Köhler) the conception of melody was not about the production of the fundierter Inhalt but about perceiving (Bemerken) (Ehrenfels, 1932, in Weinhandl, 1960: 61). 10 This dimension of the Gestalt approach was further developed by the Berlin/Frankfurt School; cf., e.g., Goldmeier, 1982. 11 According to Ehrenfels, mnemonic devices are also based on Gestalt qualities. 12 ‘…wächst mit dem Produkt ihrer Konstituenten, ihrer Einheitlichkeit und der Mannigfaltigkeit ihrer Teile.’ 13 The genealogical tree represents both quantitative and qualitative development: ‘From a few and rela tively little differentiated Ur-beginnings, the diversity of shapes grows up towards us in a fan-like manner’ (Ehrenfels, 1916: 11). The target is life itself, the shaping of innumerable diverse forms as the Gestalt series and thus also the complexity increase. Essential for Ehrenfels is the interconnectedness of the ‘Dar winian idea of selection with the assumption of an immaterial, psychoid – but without a sense of purpose – organising principle’ (Ehrenfels, 1916: 84). 10 This dimension of the Gestalt approach was further developed by the Berlin/Frankfurt School; cf., e.g., Goldmeier, 1982. g q 12 ‘…wächst mit dem Produkt ihrer Konstituenten, ihrer Einheitlichkeit und der Mannigfaltigkeit ihr Teile.’ 11 According to Ehrenfels, mnemonic devices are also based on Gestalt qualities. 12 ‘…wächst mit dem Produkt ihrer Konstituenten, ihrer Einheitlichkeit und der Mannigfaltigkeit ihrer Teile.’ 13 14 ‘Immer deutlicher kommt es unserer Generation zum Bewusstsein […] dass wir dem aufbauenden schöpferischen Prinzip in der Natur, dem Wesen der Welt am nächsten kommen, wenn wir es uns als ein in seiner innersten Natur Gestalten treibendes zur Vorstellung zu bringen suchen, – als ein Prinzip, welches nichts will und nichts anstrebt, als Gestalten, und immer neue und höhere Gestalten hervorzubringen – gleichgültig was sonst daraus noch entstehen oder erfolgen mag, – oder besser noch als ein Prinzip, welches auch dieses nicht “will” (in irgendeiner anthropomorphistischen Deutung des Ausdruckes) – sondern es tut – seinem innersten Wesen nach tun muss, ohne daran gebunden zu sein, es vorher oder während des Tuns auch zu wollen oder gewollt zu haben.’ Ehrenfels’s Concept of Gestalt (Ehrenfels, 1922, in Weinhandl, 1960: 52–53)14 Our generation is realising more and more clearly [… ] that we come closest to the constructive creative principle in nature, to the essence of the world, when we try to visualise it as a principle that drives Gestalten in its innermost nature, – as a principle that wants nothing and strives for nothing but Gestalten, and to bring forth ever new and higher Gestalten– no matter what else may emerge or follow thereof, – or even better as a principle which does not “will” this either Our generation is realising more and more clearly [… ] that we come closest to the constructive creative principle in nature, to the essence of the world, when we try to visualise it as a principle that drives Gestalten in its innermost nature, – as a principle that wants nothing and strives for nothing but Gestalten, and to bring forth ever new and higher Gestalten– no matter what else may emerge or follow thereof, – or even better as a principle which does not “will” this either (in some anthropomorphic interpretation of the expression) - but does it – must do it according to its innermost nature, without being bound to willing or hav ing willed before or during the doing. (Ehrenfels, 1922, in Weinhandl, 1960: 52–53)14 Ehrenfels’s Concept of Gestalt In such Gestalten, there is a competition between the formative elements, that is, between elements where ‘which each represents only that part of a Gestalt which demands completion to a unity, – but in a direction incompatible with that of the other element(s)’, such as a long torso on short legs (Ehrenfels, 1922, in Weinhandl, 1960: 50–51). Ehrenfels was convinced that the doctrine of evolution (see below) is essential for contemplating the multitude of Gestalten. Higher Gestalten emerge during develop ment driven by the shaping principle. The phylogenetic developmental series from the lowest organisms to humans therefore represents an increase in the height of Gestal tung13, as is the case in ontogenetic development, where, however, this increase may be difficult to observe. Beyond aesthetics and the natural sciences, the Gestalt theory could, according to Ehrenfels, be effectively applied not only in psychology or epistemology (where it is widely recognised) but also in cosmogony, which is a crucial basis for our article 1 3 Metaphysics of the Organic Whole: Ehrenfels, Uexküll, and… 347 and comparison. Ehrenfels considered the cosmogenic framework to be especially significant: Our generation is realising more and more clearly [… ] that we come closest to the constructive creative principle in nature, to the essence of the world, when we try to visualise it as a principle that drives Gestalten in its innermost nature, – as a principle that wants nothing and strives for nothing but Gestalten, and to bring forth ever new and higher Gestalten– no matter what else may emerge or follow thereof, – or even better as a principle which does not “will” this either (in some anthropomorphic interpretation of the expression) - but does it – must do it according to its innermost nature, without being bound to willing or hav ing willed before or during the doing. Uexküll’s Concept of Umwelt In his early reflections on Umwelt, Uexküll focused on how animals view the world and which parts of the world are accessible to them (Uexküll, 1910: 638–639). Every organism has its own set of sensory perceptions, motor abilities, and subjective expe riences that shape its Umwelt. Uexküll emphasises that one cannot consider a single Umwelt in isolation. Rather, one must take into account countless Umwelten that cannot be reduced to each other; our human Umwelt is just one among many – it does not have a privileged significance. In contrast to the absolute world, which would exist even without any relations to the sensory organs (Uexküll, 1910: 639), Uexküll constructs a relative world of Umwelt that is based on organismal functional circles (Funktionskreis, i.e., the circu lar relation between sensory and operating organs) and the makeup of the whole body (Bauplan). By perception and transmission of action, sensory organs are connected to the nervous system and the brain. In the brain, there can thus emerge uniform impressions (einheitliche Eindrücke) which correspond to objects of the Umwelt. Uexküll then calls the purposeful cohesion of relations, where the makeup of the brain is adapted to the makeup of the sensory organs and vice versa, the Bauplan of the animal. This Bauplan, which connects the organs in the animal body, becomes a closed whole once objects of the Umwelt are drawn into it. In higher animals, the Umwelt of the sensory organs expands, outgrowing the Umwelt of the ‘working’ organs, i.e., organs typically found also in the lower animals. A close relation is estab lished between the sensory organs and objects. Uexküll thus describes a developmen 1 1 3 348 L. Ovčáčková, J. Švorcová tal series from simple animals with simple Umwelten to differentiated animal bodies with rich and varied Umwelten (Uexküll, 1910: 640–641).f Melody and counterpoint play a central role in efforts to understand Uexküll’s concept. The analogy from music theory emphasises the importance of sensory organs and their relation to individual cells, which form like a living carillon whose individual cellular bells chime in different self-tones: In order to understand this, one must recall that the body of each living being is built from living cells that together form a living carillon. The living cell possesses a specific energy that makes it possible for it to respond to any effect which approaches it from outside with a ‘self-tone’. Uexküll’s Concept of Umwelt Self-tones can be combined with one another into melodies and do not require the mechanical interrelation of their cell bodies in order to have an effect on each other. (Uexküll, 2010: 201) The doctrine of counterpoint enables Uexküll to interpret relationships among living beings analogously to the rules governing the interplay of various tones expressed by different musical instruments in a composition: ‘Like every instrument, every animal harbors a certain number of tones, which enter into contrapuntal relationships to the tones of other animals’ (Uexküll, 2010: 187). Uexküll develops this analogy from simple examples (the flower and the bee being composed in counterpoint to each other) to a general view of the relationships and development of living beings within a broader ontological framework: Only the knowledge that everything in Nature is created according to its mean ing and that all environments [Umwelten] are composed into the world-score opens up a path leading out of the confines of one’s own environment [Umwelt]. (Uexküll, 2010: 200) Possible Bridging between Gestalt and Umwelt In relation to the concept of Gestalt quality, let us now look at Uexküll’s explana tion of a meaning carrier (Bedeutungsträger) within his theory of Umwelt. Both in the context of cosmogony and when considering the development of nature, natural entities, or culture, Ehrenfels remarks that it is important to note that a Gestalt quality does not emerge necessarily, because its emergence depends on our shaping ability or the gestaltende Kraft itself (though, importantly, this power is without purpose). For Ehrenfels, Gestalt theory is the only possible conceptual instrument that can grasp the non-teleological shaping principles in nature. The long-term development of these new, potentially continuously linked Gestalt qualities cannot be guaranteed: it can lead to a dead end. The emergence of a new Gestalt quality, which – like Uexküll’s Umwelt – is not a boundless but restricted phenomenon, is accompanied by resistance that may be difficult to overcome; examples include the emergence of new qualities 3 Metaphysics of the Organic Whole: Ehrenfels, Uexküll, and… 349 of social systems, ethical ideals, but also new biological species (cf. Ovčáčková, 2022). In this context, Uexküll’s concept of Bedeutungsträger in an Umwelt shares some aspects with Ehrenfels’s concept: of social systems, ethical ideals, but also new biological species (cf. Ovčáčková, 2022). In this context, Uexküll’s concept of Bedeutungsträger in an Umwelt shares some aspects with Ehrenfels’s concept: Anything and everything that comes under the spell of an environment [Umwelt] is either redirected and re-formed until it becomes a useful carrier of meaning or it is completely neglected. Thereby, the original components are often crudely torn apart without the slightest consideration for the structural plan which controlled them to that point. (Uexküll, 2010: 144) A comparison between the broader context of Gestalt and Umwelt indicates that Ehrenfels’s and Uexküll’s argumentation takes place within different conceptual frameworks, i.e. different core approaches to explaining living nature. Ehrenfels imagines the formation of nature as a synthesis of random chaotic diversity and shaping unity, while Uexküll is fully grounded in a purposive understanding of the development of living beings. Nevertheless, a comparison between the ‘height of the Gestalt’ and the ‘height of the Umwelt’ reveals their shared emphasis on the significance of development, specifically the increase in complexity, which encom passes both diversity and unity. The concepts of melody and counterpoint also sug gest a similar understanding of development. 15 Interestingly enough, Uexküll at this point uses in the original German edition the term ‘Gestalt’ and not the term ‘Form’. He does the same also in the following paragraph when writing about ‘a glance into the many-membered form [Gestalt] of the performed artwork’ (Uexküll 1956: 142). In this context, a com parison with Ehrenfels’s reflections on the Gestalt convergence of human artefacts is clearly at hand (cf. Ehrenfels, 1916: 193). However, the notions of ‘Gestalt’ and ‘Gestaltung’ are also found in other passages of Uexküll’s Theory of Meaning, for instance in connection with ‘Gestaltbildung’ (form development) or in the following case, where he states: ‘Thanks to its taking on foreign motifs, the body of any and every subject is formed [gestaltet sich] into a recipient of meaning from those carriers of meaning whose forma tive melodies have taken shape [Gestalt] in its body as motifs’ (Uexküll, 2010: 198). Possible Bridging between Gestalt and Umwelt Interestingly, Uexküll speaks of higher Gestalten in the context of the symphony, giving the example of a young man intently following the score during a concert: Each voice of a person or instrument is a being for itself, but one which melts into a higher form [Gestalt]15 through point and counterpoint with other voices, which form then grows further, gaining richness and beauty in order to bring forward to us the composer’s soul. (Uexküll, 2010: 185–186) 18 ‘… Gestaltungsströmen immer die weitaus größte Überzahl den nagenden Einflüssen der zufälligen Widerstände erliegen, ‚blind‛, das heißt ohne Nachkommenschaft endigen, – sich im chaotischen Sande verlaufen. – Aber unerschöpflich drängt gestaltlicher Auftrieb nach auf allen Bahnen. Und wenn hundert Zweigströme versiegen, – der hundertunterste ringt sich durch, zerteilt sich in hundert und mehr als hun dert Arme, findet neue Wege, treibt neue Gestaltungen hervor, so dass – in der weitaus größten Zahl seiner Kämpfe immer besiegt – der Strom des Lebens als Ganzes doch immer anschwillt, Neues gebiert, sich in Vielfältiges zerspellt, ins Unermessliche erweitert.’ Ehrenfels’s Dynamic relationship of Chaos and Gestaltende Kraft To understand Ehrenfels’s argumentation regarding the formation and development of the Gestalt, we need to take a closer look at his cosmogonic framework, which is anchored in a dynamic dualistic metaphysics of contrasting but indispensable prin ciples of chaos and the shaping order. Everything arises through the gestaltende Kraft (or entelechy): it is the primal source of all lawfulness, order, and Gestalt. But this unified divine principle does not create out of chaos but through the resistance offered 1 3 L. Ovčáčková, J. Švorcová 350 by chaos. The first Gestalt arose by accidental miracle out of the bottomless chaos that existed in eternity before the world came into being. This event became the basis of further development driven by mutual cooperation or destruction of one of the principles, that is, by never-ending emergence and extinction (Ehrenfels, 1916: 198–199).16 The development of the organic world is of the same nature. Ehrenfels criticises the mechanistic approach and exclusive reliance on chance, but a teleological approach is equally unacceptable to him. Although an organism seems to be the means (Mittel) and as such purposefully ordered, it is not clear what purpose the organism, as the means, is supposed to serve. According to Ehrenfels, its purpose can be stated only with reference to the organism itself: ‘Self-preservation and the preservation of the species – the preservation of that shape of Gestaltung, which is definitely and in every respect a “means”, is also its only plausible “pur pose”. Means and purpose seem to coincide in the organic’ (Ehrenfels, 1916: 81).17 Ehrenfels’s contemplations here unmistakably display Kantian influences (Ehrenfels, 1916: 150–153). Ehrenfels’s gestaltende Kraft enables the shaping of the whole in the course of life processes and increases the likelihood of viable variations in the evolutionary development compared to mere chance. For Ehrenfels, Gestalt development in the organic world (which is not in conflict with his dualistic approach) moreover forms a genealogical tree: … the vast majority of formative streams always succumb to the gnawing influ ences of random resistance, end ‘blindly’, that is, without offspring, – get lost in chaotic sand. – But shaping impetus pushes inexhaustibly along all paths. 17 ‘Die Selbst- und Arterhaltung, – die Erhaltung jener Gestaltungsform, welche durchaus und in jed Beziehung „Mittel“ ist, ist auch ihr einzig plausibler „Zweck“. Mittel uns Zweck scheinen im Organische zusammenfallen.’ 16 See also Arnheim, 1971 for this context. 16 See also Arnheim, 1971 for this context. 17 ‘Die Selbst- und Arterhaltung, – die Erhaltung jener Gestaltungsform, welche durchaus und in jeder Beziehung „Mittel“ ist, ist auch ihr einzig plausibler „Zweck“. Mittel uns Zweck scheinen im Organischen zusammenfallen.’ 18 ‘… Gestaltungsströmen immer die weitaus größte Überzahl den nagenden Einflüssen der zufälligen Widerstände erliegen, ‚blind‛, das heißt ohne Nachkommenschaft endigen, – sich im chaotischen Sande verlaufen. – Aber unerschöpflich drängt gestaltlicher Auftrieb nach auf allen Bahnen. Und wenn hundert Zweigströme versiegen, – der hundertunterste ringt sich durch, zerteilt sich in hundert und mehr als hun dert Arme, findet neue Wege, treibt neue Gestaltungen hervor, so dass – in der weitaus größten Zahl seiner Kämpfe immer besiegt – der Strom des Lebens als Ganzes doch immer anschwillt, Neues gebiert, sich in Vielfältiges zerspellt, ins Unermessliche erweitert.’ Uexküll’s Planfulness Ehrenfels’s dynamic account of the origin and development of formation in nature stands in a opposition to Uexküll’s purposive understanding of similar phenomena. In adult animals, Uexküll even speaks of a double purposiveness: ‘firstly, the organism is purpose-built and secondly, the organism is purpose-fitted into its environment’ (Uexküll, 1912: 100).19 Uexküll often uses the term ‘purposiveness’ (Zweckmäßig keit) but also suggests using the term ‘planfulness’ (Planmäßigkeit) instead. By ‘plan fulness’, he means ‘that the parts are arranged according to a ground plan or a plan in such a way that together they form a uniformly functioning whole’ (Uexküll, 1912: 100).20 This approach to teleology hints at the specific causality of living beings, a notion that can be traced back to Kant (2000 [1790]). Also for Uexküll, the Darwinian term adaptation or Anpassung falls short of fully capturing the relationship between organism and the environment, because organisms do not merely adjust to the physi cally existing world – they respond to the world of signs. Uexküll prefers the term Einpassung (Uexküll, 1927) and this term encompasses both the matching of organs and their parts and the matching of organisms and the environment as noted above, but also the matching of Umwelten to each other (Uexküll, 1927: 696, Kull, 2004)i Significantly, compared to Ehrenfels’s ‘mysterious force’ (geheimnisvolle Kraft), Uexküll speaks of a ‘mysterious plan’ (geheimnisvoller Plan) in his reflections upon the evolution of humans and other organic beings: ‘The development of the human individual takes place in the same way as that of animals. The same protoplasm forms the basis of our body and in the same way the genes mould our structure according to a mysterious plan’ (Uexküll, 1913: 1080).21 This ‘mysterious plan’ seems to be identical to Uexküll’s Bauplan, which raises the question of how flexible and open to change this Bauplan is. Ehrenfels’s unified gestaltende Kraft could, after all, represent a kind of flexible Bauplan, although one that lacks intentionality because it is in constant contact with chance and therefore chaos. According to Ehrenfels, God’s objective is not the life ‘of certain forms of Gestaltung – but life in general’, which means ‘to shape as much and as many and as rich as possible’. Life shapes itself, ‘as the unpurposed success of this shaping, in that the ongoing shaping always offers the next starting conditions for new shaping’ (Ehrenfels, 1916: 82). 19 ‘…einmal ist der Organismus zweckmäßig gebaut und zweitens ist der Organismus zweckmäßig in seine Umgebung eingepasst.’ 20 ‘…dass die Teile entsprechend einem Grundrisse oder einem Plane derart angeordnet sind, dass sie gemeinsam ein einheitlich funktionierendes Ganzes bilden.’ 21 ‘Die Entwicklung des menschlichen Individuums läuft in gleicher Weise ab, wie die der Tiere. Das gleiche Protoplasma bildet die Grundlage unseres Körpers und in gleicher Weise formen die Gene unsere Struktur nach einem geheimnisvollen Plan.’ Ehrenfels’s Dynamic relationship of Chaos and Gestaltende Kraft And if a hundred branching streams dry up, – the hundred and hundredth one struggles through, divides itself into a hundred and more than a hundred arms, finds new paths, drives forth new shapes, so that – always defeated in by far the greatest number of its battles – the stream of life as a whole nevertheless always swells, gives birth to something new, splits up into manifold forms, expands into the immeasurable. (Ehrenfels, 1922, in Fabian 1990: 249)18 1 3 Metaphysics of the Organic Whole: Ehrenfels, Uexküll, and… 351 Uexküll’s Planfulness In most cases, Uexküll speaks of a fixed Bauplan to whose command the cells of organs and entire organs are subjected. They do, nevertheless, have their own meaning tone (Bedeutungston), which is important. The ‘life tone’ of an entire animal thus consists of its ‘organ tones’. A living animal is thus not merely a physical mechanism made up of cells and organs in accordance with the order of development (Uexküll, 2010: 157). On the other hand, Uexküll’s understanding of 1 3 L. Ovčáčková, J. Švorcová 352 melody is rather narrow, although, like Ehrenfels’s, it has the tendency to leave the realm of mere elements: melody is rather narrow, although, like Ehrenfels’s, it has the tendency to leave the realm of mere elements: The meaning tone starts up abruptly and activates the form development order in the self-tones of the previously homogeneous cell elements, which then sort themselves out in different tones attuned to each other and allow the form development to proceed according to a previously established melody. (Uexküll, 2010: 156) But what gives the semantic tone the impulse to give the order to shape the form in self- or other tones? Uexküll believes that behind all development in living nature, there is a teleological principle, a plan; this is then also reflected in his terminol ogy (cf. Bauplan, Entstehungsplan, Planmäßigkeit, God–nature, or the ‘mysterious plan’). Relevant in this context are Merleau-Ponty’s reflections on what may be ‘the Umwelt of Umwelten’; this refers to Uexküll’s statement that all Umwelten are car ried by one (perhaps God–nature), which nevertheless remains inaccessible to all Umwelten (Merleau-Ponty, 2003: 177). In Uexküll’s works, we find no hints to inter preting the interconnectedness of Umwelten by reference to some higher anchoring of content, as is the case with Ehrenfels’s fundierter Inhalt. Ehrenfels’s Gestalt shap ing and the Gestalt itself are difficult to grasp, as is – due to its processual nature – the determination of boundaries between Gestalten. 1 3 Communication between Gestalten and Umwelten Both Ehrenfels’s and Uexküll’s approach have a connection to the distinctive Kantian teleology of the living. In Critique of the Power of Judgment (2000 [1790]) Kant emphasised the complexity of ‘natural purposes’ and argued that our minds cannot fully comprehend them. For Kant, a natural purpose is anchored in the interrelation ships between parts that make up the whole and in the interaction between the whole and its parts, where all parts are, reciprocally, both the end and the means, because organisms are both organised and self-organising (sich selbst organisirendes Wesen, Kant 2000: 245; AA 5 374, also Kanócz forthcoming). The problem with grasping natural teleology is not due to its claim that the parts cause the whole, as mechanistic explanations of nature tend to assume, nor is it linked to the relations between the parts themselves. Rather, the difficulty with grasping natural teleology is in how the parts are caused by the whole. Still, the telos is internal, not external, to the whole. This distinction characterises the divide between the realms of the animate and inanimate and captures the internal process of self-creation that is inherently self- directed. Living organisms emerge from within themselves, emerge from their wholeness, thereby becoming the cause of their own existence. This wholeness is irreducible. Brier gives an apt example: If we talk about experience, we cannot say that my brain has experience. We can only say that I, as a feeling and thinking sub ject, have experience (Brier, 2015). Likewise, if we accept that even cells have some rudimentary experience (albeit not conscious), then the cell as a whole cannot be reduced to its parts (ribosomes, etc.). Even Uexküll attributed the functional circles and subjectivity to the cells (Uexküll, 1931). Also, the Uexküllian Plan represents the 1 3 Metaphysics of the Organic Whole: Ehrenfels, Uexküll, and… 353 spatial and temporal whole (Kull, 2004). This teleological causality where the whole both constraints and generates the parts (whose purpose is to realise the whole) makes organisms both self-generating and self-causing (Švorcová forthcoming).22 Such a whole can be understood as a Gestalt, and only with such a Gestalt there emerges an irreducible quality of the whole, represented either by a cell or a more complex organism, which enables them, i.e., the cells or organisms, to attribute meaning, a relation of self-reference, and thus Umwelt. 22 The notion of the whole outlined in this paper expands upon Humberto Maturana and Francisco Varela (1980) and is related to their theory of autopoiesis. Their perspective underscores the independence of living systems that is evident in their ability to self-organise and sustain themselves by regulating their composition and maintaining their boundaries. It also draws upon the organicist tradition, which originated with thinkers such as Kant and Bernard but its roots can be traced back to Schelling and even Aristotle (Švorcová forthcoming in 2024). But Maturana and Varela’s contribution goes beyond this by emphasis ing the concept of embodied information. Embodied information transcends mere representation of the external environment; rather, it embodies its collaborative aspect, suggesting an ongoing and reciprocal causal relationship between organisms and their surroundings. According to Maturana and Varela, the physical world lacks meaning in itself. It acquires significance through the organisms’ active engagement in converting environmental stimuli into functional information. Maturana and Varela endorse a semantic theory of information where information is viewed not as an inherent property of objects but as emerging from interactions between them, akin to the principles found in semiotics. Communication between Gestalten and Umwelten This, we believe, is another point that links the thinking of Uexküll and Ehrenfels. Although Umwelt and Gestalt are not quite the same, Umwelt cannot exist without Gestalt. Teleological thinking, especially as based on the Uexküllian external plan for the whole nature, may appear irrelevant to contemporary biology. But some trends in the philosophy of biology, such as biosemiotics or agency theory, recognize living beings themselves as end-directed, having a certain internal teleology (Švorcová forthcom ing in 2024). According to the classical theory of Darwinism, organisms only appear to be purposeful systems because of natural selection made it seem purposeful, but they are not purposeful systems per se. This is where Uexküll clearly departs from Darwin’s views. 23 ‘…einer geradezu grotesken Überschätzung der tatsächlichen Wahrscheinlichkeitschancen, welche für das zufällige Zustandekommen eines innerlich folgerichtig aufgebauten Ganzen gegeben wird.’ Searching for Similarities through the Relationship to Darwinism Ehrenfels’s dualistic metaphysical concept is strongly rooted in Darwinism, but that does not mean that Ehrenfels accepts it fully. In his view, Darwinism is in explaining organic evolution subject to ‘an almost grotesque overestimation of the actual chances of probability that are given for the chance occurrence of an internally logically con structed whole’ (Ehrenfels, 1922, in Fabian 1990: 240).23 Ehrenfels’s rejection of chance as the principle exclusively responsible for the emergence and development of natural entities is closely related to his identification of chance and chaos. In a world of blind chance, nothing graspable or continuous can emerge. Ehrenfels seeks to find the missing link that would allow us to consider the emergence and develop ment both on the basis of chance and based on a unified shaping principle. Ehren fels’s contribution to Darwinian considerations thus consists of extending the notion 23 ‘…einer geradezu grotesken Überschätzung der tatsächlichen Wahrscheinlichkeitschancen, welche für das zufällige Zustandekommen eines innerlich folgerichtig aufgebauten Ganzen gegeben wird.’ 3 3 L. Ovčáčková, J. Švorcová 354 of chance-based creation to the idea of the emergence of unified Gestalten that not only stand in opposition to chaos but also originate from it.24 Random stimuli give rise to uniform Gestalten. If new Gestalten or Gestalt series have a more permanent character, they attract more intensely other shaping forces of the unified principle. These again give rise to new formations, but always with the aforementioned lack of focus.25 That mysterious force, ‘which begets life and brings forth Gestalten, shows […] an inexhaustible imagination, but no trace, neither of deliberation and foresight, nor of pity and compassion for its creatures’ (Ehrenfels, 1922, in Fabian 1990: 244).26i Uexküll was, in comparison with Ehrenfels, a fierce opponent of Darwinism. His attitude to the evolutionary theory is clearly summed up by Merleau-Ponty: ‘For Darwin, life is endlessly menaced by death; for Uexküll, there is a solidity of super structures, a shuffling of life’ (2003: 171). Uexküll sharply criticised both the materi alistic and mechanical character of Darwinism and the scientific monistic worldview represented by the German zoologist and physician Ernst Haeckel.27 In opposition to Darwinism, Uexküll calls for biology to be governed by meaning and not by a causal order that remains hidden from the ‘great connections’ (Uexküll, 2010: 160). 24 Ehrenfels finds even in inorganic nature a number of remarkable analogies with the world of living organisms (e.g. the Gestalt individuality of crystals). Inorganic and organic nature share the asymmetry of emergence and extinction and, in both cases, it is possible that there will be a rapid ‘Gestalt fall’ into formlessness. Nevertheless, according to Ehrenfels, the inorganic world lacks the constantly overflowing and compulsive power to shape (cf. Ovčáčková, 2022). 25 The dynamic delineation of opposing forces leads to humans who, through their own creativity, have become part of the inner life of God and thus his collaborators. Only at this point does Ehrenfels contem plate divine purpose and purposive consciousness (Ehrenfels, 1916: 207). According to Ehrenfels, the cos mogonic development thus consists of three important events: the beginning of the world, the emergence of life and its psychic dimension, and finally the emergence of the purposive will in humans. 26 ‘…welche Leben zeugt und Gestalten hervortreibt, zeigt […] eine unerschöpfliche Phantasie, aber keine Spur, weder von Überlegung und Voraussicht, noch auch von Mitleid und Erbarmen für ihre Geschöpfe.’ 27 Haeckel formulated his naturphilosophical and Darwinism-anchored worldview in his Generelle Morphologie der Organismen (1866). It culminated with the founding of the German Monistic League (Deutscher Monistenbund) in 1906 (Ovčáčková, 2018). Ehrenfels also turned against Haeckel’s monism. Unlike Uexküll, however, he primarily criticises Haeckel’s monistic cosmogenesis, which is in opposition to his dualistic understanding of the origin and development of the world (cf. Ehrenfels, 1916: 36–46). 25 The dynamic delineation of opposing forces leads to humans who, through their own creativity, have become part of the inner life of God and thus his collaborators. Only at this point does Ehrenfels contem plate divine purpose and purposive consciousness (Ehrenfels, 1916: 207). According to Ehrenfels, the cos mogonic development thus consists of three important events: the beginning of the world, the emergence of life and its psychic dimension, and finally the emergence of the purposive will in humans.l 24 Ehrenfels finds even in inorganic nature a number of remarkable analogies with the world of living organisms (e.g. the Gestalt individuality of crystals). Inorganic and organic nature share the asymmetry of emergence and extinction and, in both cases, it is possible that there will be a rapid ‘Gestalt fall’ into formlessness. Nevertheless, according to Ehrenfels, the inorganic world lacks the constantly overflowing and compulsive power to shape (cf. Ovčáčková, 2022). 25 The dynamic delineation of opposing forces leads to humans who, through their own creativity, have become part of the inner life of God and thus his collaborators. Only at this point does Ehrenfels contem plate divine purpose and purposive consciousness (Ehrenfels, 1916: 207). According to Ehrenfels, the cos mogonic development thus consists of three important events: the beginning of the world, the emergence of life and its psychic dimension, and finally the emergence of the purposive will in humans. 26 ‘…welche Leben zeugt und Gestalten hervortreibt, zeigt […] eine unerschöpfliche Phantasie, aber keine Spur, weder von Überlegung und Voraussicht, noch auch von Mitleid und Erbarmen für ihre Geschöpfe.’ 27 Haeckel formulated his naturphilosophical and Darwinism-anchored worldview in his Generelle Morphologie der Organismen (1866). It culminated with the founding of the German Monistic League (Deutscher Monistenbund) in 1906 (Ovčáčková, 2018). Ehrenfels also turned against Haeckel’s monism. Unlike Uexküll, however, he primarily criticises Haeckel’s monistic cosmogenesis, which is in opposition to his dualistic understanding of the origin and development of the world (cf. Ehrenfels, 1916: 36–46). Merleau-Ponty’s Reflection upon Natural Phenomena within Umwelt and Gestalt Our main link between Uexküll and Ehrenfels in this context is the thought of Mer leau-Ponty. Similar to Uexküll’s approach to animals, Merleau-Ponty challenges the longstanding Cartesian subject–object tradition, where subjects supposedly use their mind or consciousness to perceive external objects in the objective world. According to Merleau-Ponty, our existence in the world is fundamentally perceptual, whereby perception is more a lived experience rather than a purely cognitive one. Uexküll attributes a central role to perception also in animals: it is species-specific and thus grounds the existence of the various Umwelten, the subjective, experiential realms of signs. Consequently, organisms undergo transformation through semiotic causation (Hoffmeyer, 2008) guided by interpretation of environmental signs. Merleau-Ponty also interprets (at least) animals as subjects (Tønnessen, 2009) and his philosophical interpretation of Umwelt through the concept of Gestalt can help us take the connec tion between these two thinkers further: The notion of Umwelt is destined to join what we usually separate: the activ ity that creates the organs and the activity of behavior, lower as well as higher. From animal-machines to animal-consciousness, there is everywhere an unfurl ing of an Umwelt. What is unfurled, and from what? (Merleau-Ponty, 2003: 173) 173) Ehrenfels’s metaphysics of the organic world might offer one answer to the ques tion posed above, but Uexküll’s teleological framework would have to be shifted. This possible direction is supported by Merleau-Ponty’s further statement about ‘the unfurling of an Umwelt as a melody that is singing itself’ (Merleau-Ponty, 2003: 173). It is a fundamental departure from Uexküll’s concept of counterpoint, because the melody is not based on an external but an internal impulse. Thus our arguments are in line with Merleau-Ponty’s (see Sect. 3.4.). The respective quotation, as well as the one just below, allude rather to Ehrenfels’s Gestalt. Merleau-Ponty understands melody as a Platonic idea that cannot be viewed in isolation: Ehrenfels’s metaphysics of the organic world might offer one answer to the ques tion posed above, but Uexküll’s teleological framework would have to be shifted. This possible direction is supported by Merleau-Ponty’s further statement about ‘the unfurling of an Umwelt as a melody that is singing itself’ (Merleau-Ponty, 2003: 173). It is a fundamental departure from Uexküll’s concept of counterpoint, because the melody is not based on an external but an internal impulse. Thus our arguments are in line with Merleau-Ponty’s (see Sect. 3.4.). Searching for Similarities through the Relationship to Darwinism For him, a meaningful form (bedeutungsvolle Form) is always the work of the subject and not of an object that acts unplanned; even plants and animals owe their bodily form to the fact that they are evaluators of meaning factors that come to them from outside (Uexküll, 2010: 151). Uexküll is aware that while we cannot know how animals observe the world (that would be a matter of psychological speculation), we can investigate and find out which parts of the world are accessible to animals through their sensory organs (Uexküll, 1910: 638). The lower organisms form a closed unity with their world in which they do not struggle for their existence. They live in an Umwelt in which they are adapted to dangerous situations. Yet, the Umwelten of humans and animals are irreducible to each other: ‘We can therefore only speak of countless “Umwelten”, among which the world around us is only an individual case but must by no means be 1 3 Metaphysics of the Organic Whole: Ehrenfels, Uexküll, and… 355 regarded as standardising’ (Uexküll, 1910: 639).28 This also implies that animals do not passively adapt to a shared world (see Sect. 3.3. on Einpassung).il Uexküll identifies in the excessive influence of materialism a great danger of his time, noting that the world has become ‘desolate’. Interestingly, Uexküll encourages – quasi in line with Merleau-Ponty – to look from the invisible to the visible: ‘It really is time to shift our gaze from the invisible to the assessable if we want to experience something of the essence of the visible’ (Uexküll, 1910: 646)29. 29 ‘Da ist es wahrhaftig an der Zeit den Blick vom Unsichtbaren auf das Übersichtbare zu lenken, wen man etwas vom Wesen des Sichtbaren erfahren will.’ 28 ‘Wir dürfen daher nur von zahllosen “Umwelten” reden, unter denen die uns umgebende Welt nur einen Einzelfall bildet, aber keineswegs als normgebend angesehen werden darf.’ 28 ‘Wir dürfen daher nur von zahllosen “Umwelten” reden, unter denen die uns umgebende Welt nur einen Einzelfall bildet, aber keineswegs als normgebend angesehen werden darf.’ 29 ‘Da ist es wahrhaftig an der Zeit den Blick vom Unsichtbaren auf das Übersichtbare zu lenken, wenn man etwas vom Wesen des Sichtbaren erfahren will.’ Merleau-Ponty’s Reflection upon Natural Phenomena within Umwelt and Gestalt The respective quotation, as well as the one just below, allude rather to Ehrenfels’s Gestalt. Merleau-Ponty understands melody as a Platonic idea that cannot be viewed in isolation: 1 3 356 L. Ovčáčková, J. Švorcová It is impossible to distinguish the means and the end, the essence and the exis tence in it. From a center of physical matter surges an ensemble of principles of discernment at a given moment, which means that in this region of the world, there will be a vital event. (Merleau-Ponty, 2003: 173) Ehrenfels’s and Uexküll’s reflections upon the formation of the natural world, the processes involved in the development of living beings, the dynamic metaphysi cal framework, and interpretation of meaning in orientation in the world from both immediate and distant perspectives can now be appreciated within Merleau-Ponty’s broader phenomenological framework. This is presented by Merleau-Ponty in his two books The Visible and The Invisible and Phenomenology of Perception. Although Merleau-Ponty’s understanding of Gestalt was shaped mainly by the Gestalt theory as presented by Ehrenfels’s followers (Koffka, Köhler and Wertheimer), it may still help us understand more deeply the Uexküllian perspective. For Merleau-Ponty, the world and life stand in a relation of the visible and the invisible, whereby the invisible is ‘Verborgenheit by principle, i.e. invisible of the visible, Offenheit of the Umwelt and not Unendlichkeit’ (Merleau-Ponty, 1968: 251). In this connection, he also defines his understanding of metaphysics: ‘I am against finitude in the empirical sense, a factual existence that has limits, and that is why I am for metaphysics. But it lies no more in infinity than in the factual finitude’ (Merleau- Ponty, 1968: 251). A similar type of metaphysics can also be found in the works of Uexküll and Ehrenfels. In the fifth chapter of Die Bedeutungslehre, which deals with the regularity of formation of shapes of bodies and the regularity of meaning, Uexküll realizes that his metaphysical ideas will not be easily acceptable to biologists (Uexküll 1956: 123). Ehrenfels, on the other hand, explicitly speaks of a metaphys ics of the organic world (Ehrenfels, 1922, in Fabian 1990: 237–246) and, as we have already shown, the metaphysical dimension is crucial to his understanding of Dar winism and to an elaboration of his neo-vitalist position. Therefore Merleau-Ponty’s discussion of Driesch’s concept of entelechy (a notion to which Uexküll and in part Ehrenfels30 also reacted) cannot be omitted. 30 Ehrenfels defines himself against Driesch by contrasting entelechy with the second metaphysical prin ciple of chaos (Ehrenfels, 1922, in Fabian 1990: 258). Merleau-Ponty’s quotation from the final reflections in the fourth sketch of the third series of lectures from 1959 to 1960, Nature and Logos: The Human Body, is also Merleau-Ponty’s Reflection upon Natural Phenomena within Umwelt and Gestalt Merleau-Ponty’s phenomenological reflections on the ontology of subject and object and the visible and invisible within scientific knowledge seem to circle around a problematic understanding of wholeness that goes hand in hand with not only the concept of entelechy but also, in parallel, with the concept of Gestalt: But we must follow Driesch in this “philosophical” effort because he remains aware of the difficulties that had led him to totality. And moreover, totality is not a key: we must think it itself as a gestalt. And Driesch´s attempt, of course, teaches the difficulties of transcendental totality, sketches the totality of emer gence. (Merleau-Ponty, 2003: 235) Merleau-Ponty’s quotation from the final reflections in the fourth sketch of the third series of lectures from 1959 to 1960, Nature and Logos: The Human Body, is also 1 3 Metaphysics of the Organic Whole: Ehrenfels, Uexküll, and… 357 significant. Following Uexküll and his concept of melody, he returns to a rehabilita tion of the sensory world: ‘This being-there by difference and not by identity we think only by the rehabilitation of the sensible world, not as a “psychological fact” to reconstruct in positive terms, but as the visibility of the invisible’ (Merleau-Ponty, 2003: 238–239). In doing so, Merleau-Ponty emphasises a departure from the anthro pological view: In nature there is no preestablished field […] In any case a new field is realized. Thus, the perceived form is not an anthropomorphic illusion in relation to the nature in-itself behind it, but to nature englobed in living nature, that we must strip of human clothing (= science): we find then a center [foyer] of phenomena, a lateral encroachment of microphenomena on each other, a cohesion around invisible being even der jure, that they envelop, around which they fold up, crystallize the Gestalthafte. (Merleau-Ponty, 2003: 239) This quote clearly reveals a direct connection to Ehrenfels’s cosmogonic Gestalt thinking characterised by a dualistic dynamic framework that contemplates Gestalt while rejecting the idea of a preestablished world. This quote clearly reveals a direct connection to Ehrenfels’s cosmogonic Gestalt thinking characterised by a dualistic dynamic framework that contemplates Gestalt while rejecting the idea of a preestablished world. From the above, becomes obvious that Ehrenfels’s and Uexküll’s perspectives on relating to the natural world have different starting points. Conclusion The concept of Gestalt has held a distinct position in the historical trajectory of biol ogy and numerous thinkers have engaged with it, be it in its original sense put forth by Ehrenfels or in a later, slightly modified interpretation represented by Wertheimer, Köhler and Koffka.31 For example, Portmann also introduced the idea of the whole being more than its parts and in doing so, he explicitly referred to Gestalt. This is dis cussed by Jaroš and Brentari (2022), who emphasise that Portmann understands the whole not as a structure binding parts together but as a distinct quality that empowers the organism to harness lower-order natural processes. Therefore, Gestalt is not just the physical form of an animal but also the totality of appearances. As such, it tran scends the Darwinian focus on a functional interpretation of organismal structures. The influence of the Ehrenfelsian concept is evident. This line of thought could be extended so as to encompass even simpler organ isms, including unicellular ones. It can be argued that closure, topologically repre sented on the basic level by a cellular membrane since the origin of life, is a necessary precondition for agency, organisation, and semiosis (Švorcová & Markoš, 2023). Beyond these first-order closures, there also exist second-order closures character ised by dynamic and often ad hoc formations, such as cell communities or holobionts (higher Gestalten). We posit that first-order cellular closures structurally enable the whole and yet the additional quality emphasised by Ehrenfels and Portmann is irre ducible to any structure of the whole or any of its parts. Only the whole can relate to the world through experience and semiosis (i.e., via Umwelt). However, these ideas are not entirely new. The Czech philosopher Zdeněk Neu bauer, who was inspired among others also by the work of Portmann, emphasised the difference between the bodily forms of the living (or better Appearance, eidos, Gestalt), which are the shape of the Self (being itself and emerging from itself), and the qualitatively quite different objects, which are scientifically studied. When observing the elusive flowering of the hibiscus, Neubauer defined the foundations of his eidetic biology (Neubauer, 2017, cf. Merleau-Ponty’s idea of Physis). The dynamic nature of the living does not emerge from temporal slices, like when a film captures a chain of objective, immobile states; rather, it represents a continuous emer gence and occurrence. 31 Wertheimer, Köhler, and Koffka sought – in line with an empirical philosophical worldview – a ‘radical reconstruction of psychological thinking’ (Ash, 1995: 103). The differences between their and Ehrenfels’s Gestalt approach mainly had to do with the idea of the whole, i.e., the ‘fundierter Inhalt’ (grounded con tent), which for Ehrenfels is necessarily linked to the ‘Fundament’ (foundation). According to Ehrenfels, however, the ‘fundierter Inhalt’ can be missing. For Wertheimer and Köhler, foundation is always linked to founded content. It is thus not its ‘Produktion’ (product) but only its ‘Bemerken’ (notion) (Ehrenfels, 1932, in Weinhandl, 1960: 62). These approaches are also reflected in the conception of the whole as something other (though not necessarily more) than the sum of its parts (cf. Rausch, 1960). Ehrenfels’s approach is thus clearly incompatible with an atomistic and mechanistic understanding of Gestalt shaping (cf. Weinhandl, 1960: 5). Merleau-Ponty’s Reflection upon Natural Phenomena within Umwelt and Gestalt Although both thinkers viewed their approach as metaphysical, Uexküll’s interpretation of natural phenom ena remains primarily in the realm of the sensory world of meaning, i.e., within the world of the visible, whereas Ehrenfels’s position based on his grounding in the cos mogonic perception of the world tends to move towards the world of the invisible. We have shown that these perspectives can not only intermingle, but also comple ment each other. Meaningful reflection on this interface is central to Merleau-Ponty’s phenomenological emphasis on understanding the nature of both Gestalt and Umwelt also in relation to the body and corporeality. Merleau-Ponty’s reflections on Gestalt are most clearly present in his working notes (named Gestalt) from September 1959. This text clearly spells out Merleau-Ponty’s intention to understand Gestalt not as an idea or meaning but as a body: My body is a Gestalt and it is co-present in every Gestalt. It is a Gestalt; it also, and eminently, is a heavy signification, it is flesh; the system it constitutes is ordered about a central hinge or a pivot which is openness to… (Merleau- Ponty, 1968: 205). Such ‘openness’, free of focus, is also characteristic of Ehrenfels’s conception of Gestalt. Gestalt itself, i.e. the Gestalt quality, is also carried on the physical level by gestaltende Kraft that does not have a predetermined goal. Its aim is to shape not only with respect to the whole, but also in reaction to new situations. Such situations may call for an interpretation determined not by an external framework but rather by an inner ability to grasp and perceive the meaning of the inorganic and organic game being played. 1 3 L. Ovčáčková, J. Švorcová 358 Competing Interests The authors declare no competing interests. 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/. Conclusion The living gives itself to the world as a Gestalt/Appearance and only as such it can be perceived and enter into a relationship of counterpoint with other living beings. 1 3 359 Metaphysics of the Organic Whole: Ehrenfels, Uexküll, and… Acknowledgements We would like to thank Anna Pilátová, Ph.D., for her thorough revisions and comments. Author Contributions L.O. wrote the text concerning Ehrenfels, Uexküll and Merleau-Ponty. J.S. added parts of the text on Uexküll and Merleau-Ponty and on specific aspects of current theoretical biology. Funding Open access publishing supported by the National Technical Library in Prague. Lenka Ovčáčková’s research was not supported by any funding. The research of Jana Švorcová was funded by the Czech Science Foundation (GACR) 20–16633 S. ( ) Open access publishing supported by the National Technical Library in Prague. Data Availability No datasets were generated or analysed during the current study. References Arnheim, R. (1971). 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https://openalex.org/W3198374781	https://europepmc.org/articles/pmc8423783?pdf=render	English	null	Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing	Communications biology	2,021	cc-by	12,367	1 Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, Evry, France. 2 CIRAD, UMR AGAP Institut, Montpellier, France. 3 UMR AGAP Institut, Univ Montpellier, CIRAD, INRAE, Institut Agro, Montpellier, France. 4 Commissariat à l’Energie Atomique (CEA), Institut François Jacob, Genoscope, Evry, France. 5 Institute of Experimental Botany of the Czech Academy of Sciences, Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc, Czech Republic. 6These authors contributed equally: Caroline Belser, Franc-Christophe Baurens. ✉email: jmaury@genoscope.cns.fr https://doi.org/10.1038/s42003-021-02559-3 OPEN Results Highly contiguous genome assembly of the banana genome. The efﬁciency of long-reads sequencing depends on the quality of the DNA extraction. Here, DNA was extracted following a plant- dedicated protocol provided by Oxford Nanopore Technologies (“High molecular weight gDNA extraction from plant leaves”). This protocol was particularly effective and allowed us to obtain long DNA fragments (> 50 kbp). Residual short fragments were ﬁltered out using the Short Read Eliminator (SRE) XL kit (Cir- culomics, MD, USA). Almost 93 Gb of nanopore sequences were obtained with a single PromethION ﬂowcell R9.4.1. The 5.2 M reads had a N50 of 31.6 kbp and the genome was covered at 17X with reads longer than 75 kbp. This high-quality set of long reads was assembled using several bioinformatics tools and the assembly obtained with NECAT11 was retained. Indeed the NECAT assembly of this haploid cultivar was the most con- tiguous (contig N50) and had a larger cumulative size. This assembly was polished ﬁrst using long reads with Racon12 and Medaka13 and then using Illumina short-reads with Hapo-G14. This assembly, based on nanopore long reads and without long- range information, was composed of 124 contigs (larger than 50 kbp) and had a cumulative size of 485 Mbp. Half of the assembly size was composed of contigs larger than 32 Mbp and only 16 contigs covered 90% of the total length (Table 1, contig N50 and contig L90). More importantly, the seven largest contigs had a size compatible with complete chromosomes (ranging from 47.7 We selected the banana genome, a medium-size genome in the plant lineage (~500 Mbp), and hypothesized that recent improvement of the ONT technology, coupled with dedicated DNA extraction protocol and efﬁcient software enables the reconstruction of gapless and Telomere-to-Telomere chromo- some sequences. q Banana species are monocotyledonous plants and part of the Zingiberales order and of the Musaceae family. Bananas are mostly cultivated in tropical and subtropical countries, and their fruits are the basis of the diet of several hundred million people and are massively exported to industrialized countries. Four genetic groups have been predicted to be involved in the origins of cultivars, mainly through inter(sub)speciﬁc hybridization and with different extents of contribution: Musa acuminata including various subspecies (A-genome), Musa balbisiana (B-genome), Musa schizocarpa (S-genome) and species of the Australimusa section (T-genome). Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing Caroline Belser 1,6, Franc-Christophe Baurens2,3,6, Benjamin Noel 1, Guillaume Martin 2,3, Corinne Cruaud4, Benjamin Istace 1, Nabila Yahiaoui2,3, Karine Labadie 4, Eva Hřibová5, Jaroslav Doležel 5, Arnaud Lemainque4, Patrick Wincker 1, Angélique D’Hont2,3 & Jean-Marc Aury 1✉ Long-read technologies hold the promise to obtain more complete genome assemblies and to make them easier. Coupled with long-range technologies, they can reveal the architecture of complex regions, like centromeres or rDNA clusters. These technologies also make it possible to know the complete organization of chromosomes, which remained complicated before even when using genetic maps. However, generating a gapless and telomere-to- telomere assembly is still not trivial, and requires a combination of several technologies and the choice of suitable software. Here, we report a chromosome-scale assembly of a banana genome (Musa acuminata) generated using Oxford Nanopore long-reads. We generated a genome coverage of 177X from a single PromethION ﬂowcell with near 17X with reads longer than 75 kbp. From the 11 chromosomes, 5 were entirely reconstructed in a single contig from telomere to telomere, revealing for the ﬁrst time the content of complex regions like cen- tromeres or clusters of paralogous genes. 1 Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, Evry, France. 2 CIRAD, UMR AGAP Institut, Montpellier, France. 3 UMR AGAP Institut, Univ Montpellier, CIRAD, INRAE, Institut Agro, Montpellier, France. 4 Commissariat à l’Energie Atomique (CEA), Institut François Jacob, Genoscope, Evry, France. 5 Institute of Experimental Botany of the Czech Academy of Sciences, Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc, Czech Republic. 6These authors contributed equally: Caroline Belser, Franc-Christophe Baurens. ✉email: jmaury@genoscope.cns.fr 1 COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 L L ong-read technologies are now the standard for generating high-quality assemblies, especially for complex genomes such as plant genomes1–4. Although the impact of these technologies is undeniable, they still lack the maturity to recon- struct complete chromosome sequences from telomere to telo- mere. Generally, assemblies based on long-reads are complemented with long-range data, like optical maps or chro- mosomal conformation sequencing. Recently, the Telomere-to- Telomere (T2T) consortium proposed a telomere-to-telomere assembly of the X chromosome sequence of the human genome5. Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing This high-quality assembly of the human genome was based on a combination of several existing technologies: nanopore sequen- cing from Oxford Nanopore Technology (ONT), single-molecule real-time (SMRT) sequencing provided by Paciﬁc Biosciences (PACBIO), linked reads sequencing from 10X Genomics (10X) and optical mapping provided by Bionano Genomics (BNG). Even if the ﬁnal assembly is very contiguous, there are still several gaps, and the complete X chromosome sequence was obtained by manual curation. This huge effort is not possible for all genome projects because it is far too expensive and time-consuming. It is clear that these multilayer assemblies reveal the architecture of complex regions as well as the complete organization of chro- mosomes, which remained complicated before. Long-range technologies make it possible to organize contigs based on long-reads but they are not able to ﬁll the gaps between these contigs. Indeed, usually complex regions like centromeres or telomeres still contain many gaps, depending on their repetitive content. was published in 2016 and added Illumina long-insert sequences, a low-contiguity optical map as well as a more dense genetic map. Martin et al. proposed an assembly of the 11 chromosomes that included 76% of the estimated genome size. Herein we propose to generate a new version (named V4, the third version was an internal assembly not shared with the community) of the DH- Pahang assembly based on nanopore long-reads. COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio Results Two events appeared during banana domestication: the transition from wild to edible diploids and the emergence of triploids from edible diploids6–8. Recent results suggest that edible cultivar origins are more complex than expected, involving multiple hybridization steps, resulting in inter(sub)speciﬁc mosaic genomes. They also revealed that additional genetic pools to the ones expected were involved, for which the wild contributors are still unidentiﬁed6. In addition, large structural variations in form of reciprocal translocations and a few inversions have been characterized in genetic pools involved in cultivar origins and found widespread in cultivated germplasm6. The complexity of these genomes underlies the importance of producing high-quality assemblies of banana genomes to decipher their evolutionary history and to support genetic studies. Table 1 Comparison of Musa acuminata (DH-Pahang) genome assemblies. D’hont et al. 9 V1 Martin et al. 10 V2 This study V4 Number of contigs 29,437 19,312 124 Cumulative size (bp) 390,477,446 405,522,014 484,058,756 N50 (bp) L50 28,319 3,428 43,237 2,363 32,091,396 7 N90 (bp) L90 5,108 16,551 9,026 10,327 6,704,534 16 Longest contig (bp) 306,660 602,020 47,719,527 Number of chromosomes 11 11 11 Cumulative size (bp) 331,812,599 397,008,016 468,821,802 Cumulative size (ACGT only) 286,824,765 363,519,833 468,133,046 N50 (bp) L50 30,470,408 5 37,593,364 5 43,931,232 5 N90 (bp) L90 25,514,024 10 29,070,452 10 34,826,100 10 Longest (bp) 35,439,739 A08 44,889,171 A08 51,314,288 A08 % of N 13.56% 8.44% 0.68% % of estimated genome 63.4% 75.9% 89.6% % of estimated genome (ACGT only) 54.8% 69.5% 89.5% Complete BUSCO (N = 1,614) 92.6% 98.5% 98.8% Table 1 Comparison of Musa acuminata (DH-Pahang) genome assemblies. In this context, two versions of the Musa acuminata ‘DH- Pahang’ genome have already been proposed9,10. The ﬁrst draft version of the genome of this double haploid genotype (V1) was published in 2012 and based on 454, Sanger (fosmids and BAC- ends), and Illumina sequencing. Furthermore, scaffolds were organized using a sparse genetic map, resulting in the anchoring of 63% of the estimated genome size. The second version (V2) 2 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 Fig. 1 Musa genomes architecture comparison. The tracks represent the following elements (from outer to inner): (1) schematic representation of M acuminata (A), M. balbisiana (B) and M. schizocarpa (S) chromosome sequences, (2) contigs colored in green if the chromosome sequence is composed o 1−4 contigs, in red if the chromosome sequence is composed of more than 5 contigs. (3) Density of the centromeric repeats. Results (4) Density of the Gyps elements. (5) Density of the Copia elements. (6) Density of the DNA transposons. (7) Density of genes. (8) Synteny relationships. The red lines show translocations between B01 and A03 and between S10 and A10. The blue lines show inversions between B05 and A05, S04 and A04, S05 and A05, S0 and A09. \| p // g/ / C Fig. 1 Musa genomes architecture comparison. The tracks represent the following elements (from outer to inner): (1) schematic representation of M. acuminata (A), M. balbisiana (B) and M. schizocarpa (S) chromosome sequences, (2) contigs colored in green if the chromosome sequence is composed of 1−4 contigs, in red if the chromosome sequence is composed of more than 5 contigs. (3) Density of the centromeric repeats. (4) Density of the Gypsy elements. (5) Density of the Copia elements. (6) Density of the DNA transposons. (7) Density of genes. (8) Synteny relationships. The red lines show translocations between B01 and A03 and between S10 and A10. The blue lines show inversions between B05 and A05, S04 and A04, S05 and A05, S09 and A09. to 32.1 Mbp). The anchoring of contigs was performed following the methodology described in Martin et al. 10. As expected, the ﬁve largest contigs correspond to complete chromosome sequences and harbor telomeric repeats at both extremities (Fig. 1). The six remaining chromosome sequences were com- posed of a small number of contigs (between four and eight). Interestingly, the remaining gaps are mainly located in rDNA clusters: 5S for chromosomes 1, 3, and 8 and 45S for chromosome 10 or in other tandem and inverted repeats: chromosomes 1 and 5 (Fig. 2a). These rDNA clusters are composed of a large number of tandemly repeated genes and are generally very difﬁcult to assemble. Even if these clusters still contain a few gaps, it is now possible to decipher the architecture of these large and complex regions. In addition, smaller contigs not anchored to the 11 chromosomes correspond to the chloroplastic and mitochondrial genomes (one and 45 contigs, respectively). A total of 37 contigs were ﬁltered out because they were included in larger contigs and contained highly repeated sequences. Validation of telomere-to-telomere chromosome sequences. A kmer analysis and a ﬁrst alignment of the largest contigs with the previous version of the DH-Pahang assembly did not reveal chimeric contigs (Supplementary Figs. 1 and 2). Results In addition, all COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio 3 3 2 Comparison of the V2 and V4 assemblies. a Localization and density of several repeated elements on chromosome sequences of the V4 (li nge) and V2 (white) assemblies (scale in Mbp on the right) with Nanica LINE and CRM chromovirus Gypsy retrotransposon (red), 5S rDNA (blue), 4 NA (violin), tandem repeat cluster CL18 (dark green), tandem repeat cluster CL33 (light green), Maximus Copia retrotransposon (gray) and telom uences (black triangles). Horizontal black lines and black dots correspond to the 15 remaining gaps in the V4 assembly. b Comparison of the A0 omosomes of the V2 and V4 assemblies. Tracks represent the following elements (from outer to inner): (1) density of the centromeric repeats. nsity of genes. (3) Synteny relationships between the V2 chromosome 1 and the V4 chromosome 1. RTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-0255 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 Fig. 2 Comparison of the V2 and V4 assemblies. a Localization and density of several repeated elements on chromosome sequences of the V4 (light orange) and V2 (white) assemblies (scale in Mbp on the right) with Nanica LINE and CRM chromovirus Gypsy retrotransposon (red), 5S rDNA (blue), 45S rDNA (violin), tandem repeat cluster CL18 (dark green), tandem repeat cluster CL33 (light green), Maximus Copia retrotransposon (gray) and telomeric sequences (black triangles). Horizontal black lines and black dots correspond to the 15 remaining gaps in the V4 assembly. b Comparison of the A01 chromosomes of the V2 and V4 assemblies. Tracks represent the following elements (from outer to inner): (1) density of the centromeric repeats. (2) Density of genes. (3) Synteny relationships between the V2 chromosome 1 and the V4 chromosome 1. Fig. 2 Comparison of the V2 and V4 assemblies. a Localization and density of several repeated elements on chromosome sequences of the V4 (light orange) and V2 (white) assemblies (scale in Mbp on the right) with Nanica LINE and CRM chromovirus Gypsy retrotransposon (red), 5S rDNA (blue), 45S rDNA (violin), tandem repeat cluster CL18 (dark green), tandem repeat cluster CL33 (light green), Maximus Copia retrotransposon (gray) and telomeric sequences (black triangles). Horizontal black lines and black dots correspond to the 15 remaining gaps in the V4 assembly. b Comparison of the A01 chromosomes of the V2 and V4 assemblies. Tracks represent the following elements (from outer to inner): (1) density of the centromeric repeats. COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio Results More importantly, the cumulative size is closer to the estimated genome size, suggesting that complex regions are better represented in this new release (Table 1). With a very small number of contigs, anchoring on the eleven chro- mosomes using the genetic map was easier especially in the centromeric regions, which are generally difﬁcult to organize due to their lower density of genetic markers. The size of the DH- Pahang genome was estimated by ﬂow cytometry at 523 Mbp9. The 11 chromosome sequences of our long-read assembly cover almost 90% of this estimated size, while the ﬁrst two versions were largely incomplete (55 and 70% respectively, Table 1). As a consequence, the assembled size of each chromosome sequence has increased, between 7% for chromosome 10−43% for chro- mosome 1 (Fig. 2a and Supplementary Fig. 2). Chromosome sequences of the two versions were aligned and large genomic regions (>100 kbp) absent in the previous assembly were repor- ted. The 11 chromosome sequences totalized 247 new regions that covered 141.4 Mbp, i.e., 29.2% of the assembly (Supple- mentary Fig. 4). The largest region, close to 6 Mbp, is localized on chromosome 1 (Fig. 2b). Unsurprisingly, these blocks are mainly composed of repeated elements (more than 85%), and localized in centromeric regions or rDNA clusters (Supplementary Fig. 5). We annotated 246 Mbp of the genome (52.6%) as transposable elements (TE), compared to 152 Mbp in V2, which illustrates the much better representation and completion of these repetitive elements in the V4 assembly (Supplementary Table 3 and Sup- plementary Fig. 6). Figure 2a shows the distribution of several tandem repeats and TE along the chromosomes, including the Maximus Copia retrotransposons, which are the most abundant TEs in the Pahang genome. Tandemly duplicated genes (TDGs). Gene duplication is an important evolutionary mechanism that contributes to the appearance of novel functions and to adaptation. The events leading to gene duplication have contributed to important plant agronomic traits, such as grain quality, fruit shape, and ﬂowering time20. A special case of gene duplication relates to genomic/ tandem duplication events, which generate, locally, repetitive regions in the genome. These TDGs are generally harder to capture in short-read assemblies, especially in the case of recent multi-copy clusters. We found 1,700 genes that have been annotated only in our long-read assembly. Results Three clusters are located on one arm of chromosome 8 representing in total around 2.2 Mbp and two large clusters of 5S rDNA repeat have been integrated to chro- mosome 1 and 3 centromeric regions, representing around 3.5 and 4 Mbp, respectively. These results are in accordance with previous cytogenetic results17 and the position of the rDNA clusters have been clariﬁed showing that they colocalize with the centromeric Nanica clusters of these chromosomes. Clusters of 5S rDNA are organized in canonical gene/spacer tandem repeat of different lengths due to the insertion in the spacer of various repeated elements such as Nanica or CRM sequences as observed in the 5S cluster of the centromeric regions of chromosomes 1 and 3 (Fig. 3A, B and c). Furthermore, a large cluster of 1.8 Mbp containing around 110 45S rDNA units, consisting in canonical gene/spacer tandem repeat, is localized on chromosome 10 between positions 4.4 and 6.2 Mbp (Fig. 2a). genome assembly contains only ﬁfteen gaps that are concentrated in large highly repetitive regions (Supplementary Table 1). g g y p g pp y As Illumina paired-end were available for the DH-Pahang genome, an assessment of quality and completeness was performed using Merqury16 (Supplementary Table 2). As expected the reported completeness is higher for the long-read assembly (95.7% compared to 98.1%). However, the consensus quality (QV) is lower for the nanopore assembly (38.8 vs 49.2). This lower value can be explained by the fact that ﬁrstly the nanopore assembly contains regions that are not present in the short-read assembly (such as repetitive regions that are more difﬁcult to polish and can generally contain more errors in nanopore assemblies) and that secondly, the error rate of the nanopore technology is still too high, complicating the correction of the consensus using polishing algorithms. As a control, we calculated the QV score only on the regions shared between both assemblies, and observed a decrease in the difference in quality between the two assemblies (45.9 versus 50.1). This difference may be due to the even higher error rate in the consensus of nanopore assemblies compared to short-read assemblies. Comparison of Musa acuminata assemblies. Unsurprisingly, compared to previous versions, the contiguity of our DH-Pahang assembly is greatly improved. The contig N50 goes from a few tens of kbp (28 and 43 kbp for V1 and V2 respectively) to a few tens of Mbp (32 Mbp). Results (2) Density of genes. (3) Synteny relationships between the V2 chromosome 1 and the V4 chromosome 1. eleven chromosome sequences harbor plant-speciﬁc telomeric repeats (T3AG3) at both sides, underlining the complete assem- bly of chromosome ends. 469 and 474 Mbp lengths, respectively, and had a N50 of 35 and 16 Mbp, respectively. We used these two optical maps to ﬁrst validate the contigs, and then order and orient them. As a result, only one contig of 380 kbp, composed of tandem repeated elements, was ﬂagged as conﬂictual with the optical maps and split into two contigs (Supplementary Fig. 3). All other contigs were in accordance with the maps, which strongly validate the accuracy of the NECAT assembler. The 124 contigs were ordered in 96 scaffolds using the Bionano Solve workﬂow and the BiscoT15 software (88 scaffolds correspond each to one contig). In the end, eight of the eleven chromosomes are represented by a single scaffold and the other four remain in two scaffolds. The whole- However, we decided to validate the quality of our assembly using two Bionano optical maps that were generated using the Saphyr instrument commercialized by Bionano Genomics (BNG). High molecular weight DNA was extracted and labeled using two different labeling chemistries independently, the Direct Label chemistry (DLS) and the Nick-Label-Repair and Stain chemistry (NLRS) based on nicking endonucleases. Two optical maps were generated using DLS with the DLE-1 enzyme and NLRS with the BspQI enzyme. The resulting DLE-1 and BspQI optical maps were COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio 4 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 and Supplementary Fig. 6). Several elements of chromovirus CRM clade, a lineage of Ty3/Gypsy retrotransposons, were also found restricted to these centromeric regions. Some members of this plant retroelement have been shown to have the ability to target their insertion almost exclusively to the functional centromeres18,19. The position of two other tandem repeats (CL18 and CL33) previously identiﬁed17 could also be reﬁned between V2 and V4 and the localization of the main clusters on chromosomes 1 and 2 are in accordance with cytogenetic karyotypes17. Regarding the 5S rDNA sequences, in the V2 assembly, they were present in a few numbers in chromosomes 5, 9, and 8 spanning 7,5 kbp of sequences (around 130 gene units) (Supplementary Table 4). In the new assembly, six major loci containing 5S rDNA gene clusters are present accounting for around 7,696 gene units. COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio Results These genes are dis- tributed over the different chromosomes, with chromosomes 1 and 10 having the greatest number of new genes, 13.3 and 14.3% respectively (Supplementary Table 5). Interestingly, a large pro- portion (38.3%) of these genes are TDGs included in a gene cluster and the proportion of TDGs in new genes is higher when compared to the whole gene catalog (38.3% versus 9.9%). p g g By focusing on TDGs and detecting gene clusters in the short and long-read assemblies, we found 31% more clusters in the V4 compared to V2 assembly (1,134 compared to 866 clusters). These blocks of TDGs contain respectively 3,649 and 1,134 genes. The largest in the long-read assembly contains 38 genes on chromosome 7 (between 31.8 and 32.8 Mbp) and was split into two smaller clusters of 11 and 9 genes in the V2. This TDG cluster is located in a region with several gaps in previous versions, and we found three regions (165, 111 and 110 kbp) between positions 31.8 and 32.3 Mbp of the chromosome 7 that are speciﬁc to the V4 assembly. These new regions allow the creation of a complete cluster of TDGs (Fig. 4a) which contain motifs of the terpene synthase family that are responsible for the synthesis of terpenoid compounds playing a role in plant ﬂavor21 and more generally in the interactions between the plant and its environment22,23. This family is known to contain TDGs and is expanded in several plant species24. Architecture of centromeric regions and rDNA clusters. Earlier cytogenetic analysis showed that a long interspersed element (LINE), named Nanica, is present in the centromeric regions of banana9,17. A very few LINE sequences were present in the ﬁrst release of the assembly despite being present in unassembled reads9 and they had a scattered distribution on the pericen- tromeric and centromeric regions of the V2 assembly. In this new assembly, clusters of Nanica tandem repetitions are found grouped in the centromeric regions of all chromosomes (Fig. 2a 5 5 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 Fine structure and density of main (peri)centromeric repeated sequences on chromosome 1. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 genes. Plant disease resistance genes encoding pro- nucleotide binding leucine rich repeat (NLR) domains Fig. 8). They all have a larger size compared to V2, with sizes ranging from 132 up to 227 kbp and additional detected NLR loci parison of tandemly duplicated gene regions in the V2 and V4 assemblies. a Synteny visualization of gene clusters of the terpene synthase cluster is located between 31.89 and 32.82 Mbp on the V4 (chromosome 7). Gene synteny relationships are colored in green, and genes are ording to their orientation (blue if forward and green otherwise). b Comparison of the structure of a NLR cluster on chromosome 3. The LR loci for each version are represented by blue boxes on the x- and y-axis of the dot plots. Red boxes represent regions bearing undetermined Region coordinates are also indicated. ig. 4 Comparison of tandemly duplicated gene regions in the V2 and V4 assemblies. a Synteny visualization of gene clusters of the terpene synth amily. The cluster is located between 31.89 and 32.82 Mbp on the V4 (chromosome 7). Gene synteny relationships are colored in green, and genes olored according to their orientation (blue if forward and green otherwise). b Comparison of the structure of a NLR cluster on chromosome 3. The redicted NLR loci for each version are represented by blue boxes on the x- and y-axis of the dot plots. Red boxes represent regions bearing undetermi Fig. 4 Comparison of tandemly duplicated gene regions in the V2 and V4 assemblies. a Synteny visualization of gene clusters of the terpene synthase family. The cluster is located between 31.89 and 32.82 Mbp on the V4 (chromosome 7). Gene synteny relationships are colored in green, and genes are colored according to their orientation (blue if forward and green otherwise). b Comparison of the structure of a NLR cluster on chromosome 3. The predicted NLR loci for each version are represented by blue boxes on the x- and y-axis of the dot plots. Red boxes represent regions bearing undetermined nucleotides. Region coordinates are also indicated. Resistance genes. Plant disease resistance genes encoding pro- teins with nucleotide-binding leucine-rich repeat (NLR) domains are often clustered in genomes, sometimes forming large, rapidly evolving clusters of highly homologous genes25. The NLR- annotator program26 allows the identiﬁcation of NLR loci i.e., genomic regions likely associated with an NLR gene (or pseu- dogene). COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio Results Nanica LINE (red), CRM chromovirus G ransposon (yellow), 5S rDNA (lilac), tandem repeat cluster CL18 (dark green), Maximus Copia retrotransposon (gray) are represented o chromosome 1: (b) a zoom and a dot-plot alignment of a 1 Mbp segment in the centromeric region containing Nanica, 5S rDNA, and CM zoom and a dot-plot alignment of a 30 kbp segment containing 5S rDNA repeats and a CMR. TICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003 021 Fig. 3 Fine structure and density of main (peri)centromeric repeated sequences on chromosome 1. Nanica LIN Fig. 3 Fine structure and density of main (peri)centromeric repeated sequences on chromosome 1. Nanica LINE (red), CRM chromovirus Gypsy retrotransposon (yellow), 5S rDNA (lilac), tandem repeat cluster CL18 (dark green), Maximus Copia retrotransposon (gray) are represented on: (a) the entire chromosome 1: (b) a zoom and a dot-plot alignment of a 1 Mbp segment in the centromeric region containing Nanica, 5S rDNA, and CMR repeats, (c) a zoom and a dot-plot alignment of a 30 kbp segment containing 5S rDNA repeats and a CMR. Fig. 3 Fine structure and density of main (peri)centromeric repeated sequences on chromosome 1. Nanica LINE (red), CRM chromovirus Gypsy retrotransposon (yellow), 5S rDNA (lilac), tandem repeat cluster CL18 (dark green), Maximus Copia retrotransposon (gray) are represented on: (a) the entire chromosome 1: (b) a zoom and a dot-plot alignment of a 1 Mbp segment in the centromeric region containing Nanica, 5S rDNA, and CMR repeats, (c) a zoom and a dot-plot alignment of a 30 kbp segment containing 5S rDNA repeats and a CMR. COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio 6 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 Interestingly, the two ONT assemblies have a higher contiguity (contig N50 of 32 and 6.5 Mbp compared to 1.8 Mbp) suggesting the usage of longer reads, or the difﬁculty to extract and sequence long DNA fragments with the PACBIO device (Table 3). Indeed, the PACBIO library was size-selected in order to obtain frag- ments around 20 kbp28, which was perhaps an optimal condition for PACBIO sequencing at this time. The B genome was assembled from reads with an N50 of 16.6 kbp whereas A and S genomes were assembled with reads having a N50 of 31.6 and 24.4 kbp respectively. As a consequence, in addition, we noticed that chromosome sequences of the A and S genomes contain fewer gaps. A difference between the PACBIO and ONT sequencing technologies is already mentioned29. The eleven A and S chromosome sequences contain 15 and 166 gaps respec- tively whereas B chromosome sequences contain 683 gaps and no chromosome sequence is gapless (Fig. 1). Centromeric regions, detected with centromeric repeats, are very fractionated in the case of the PACBIO-based assembly (from 24 contigs for the chromosome 7 to 111 contigs for the chromosome 1), underlying the importance of ultra-long reads to resolve these highly repe- titive regions. However, sequencing technologies evolve rapidly and this comparison does not reﬂect the current capability of each technology. In this study, we combined recent development from DNA extraction, sequencing, and genome assembly and showed that plant chromosome sequences can now be assembled in a single contig, gapless, and from telomere to telomere, at least to a certain extent. We chose the genome of Musa acuminata, the ﬁrst monocotyledonous species sequenced outside Poales, because its reference genome, even of low-quality, has been widely used and constitutes an important resource for the scientiﬁc community. To date, three Musa species: Musa acuminata, Musa balbisiana, and Musa schizocarpa are available at the chromosome-scale. These three species are of particular interest because they are involved in the origin of banana cultivars; their high-quality genome assem- blies will thus be a valuable resource to explore the evolutionary history and biology of current banana cultivars. We reported that the PACBIO assembly of M. balbisiana is more fragmented which can be related to the input read size and underline the importance of long reads to resolve highly repetitive regions. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 A total of 128 NLR loci were detected in this assembly compared to 111 loci in the V2 assembly (Table 2, and Supple- mentary Data 1 and 2). Four major clusters of NLR loci were found in this assembly: two in chromosome 3, one in chromo- some 7, one in chromosome 10 (Fig. 4b and Supplementary Fig. 8). They all have a larger size compared to V2, with sizes ranging from 132 up to 227 kbp and additional detected NLR loci. These clusters were improved in sequence quality with a complete absence of undetermined nucleotides within the corresponding genomic regions (Supplementary Table 6). Comparison of A, B and S-genome assemblies. The B and S genomes have already been recently sequenced using a long-read strategy27,28. Taking into account this new version of the M. acuminata genome, three high-quality banana genomes are now available. The A and S genomes were sequenced using ONT while MMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio 7 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 Table 3 Comparison of A (Musa acuminata), B (Musa balbisiana) and S (Musa schizocarpa) genomes assemblies. Musa acuminata Musa balbisiana Musa schizocarpa Number of contigs 124 3,787 379 Cumulative size 484,058,756 491,421,783 517,486,196 N50 (bp) L50 32,091,396 7 1,801,976 59 6,493,909 24 N90 (bp) L90 6,704,534 16 56,360 578 1,047,001 84 Longest contig (bp) 47,719,527 14,987,599 18,138,554 Number of chromosomes 11 11 11 Cumulative size 468,821,802 430,021,147 496,921,565 Cumulative size (ACGT only) 468,133,046 429,290,714 490,105,212 % Anchored sequences 96.8% 87.3% 94.7% N50 (bp) L50 43,931,232 5 42,323,520 5 46,993,692 5 N90 (bp) L90 34,826,100 10 30,518,812 10 36,762,080 10 Longest (bp) 51,314,288 48,736,620 54,858,060 Number of gaps 15 683 166 Estimated genome size 523 Mb 520 Mb 587 Mb % of estimated genome size 89.6% 82.6% 84.6% Number of annotated genes 36,979 35,148 32,809 Complete BUSCO (N = 1,614) 98.8% 96.9% 97.6% Table 2 Comparison of gene prediction statistics. Reference Martin et al. 10 V2 This study V4 # Number of genes 35,276 36,979 #Exons per spliced gene (avg:med) 6.08: 5 6.05: 4 Gene sizes (avg:med) 4,542: 2,824 4,604.68: 2,758 CDS sizes (avg:med) 1,171: 981 1,180: 972 Complete BUSCO (N = 1,614) 98.5% 98.8% NLR loci 111 128 Table 2 Comparison of gene prediction statistics. Table 3 Comparison of A (Musa acuminata), B (Musa balbisiana) and S (Musa schizocarpa) genomes assemblies. the B genome was sequenced using the PACBIO technology. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 Overall, the synteny conservation between the three genomes is high, we detected one inversion between the chromosome B05 of Musa balbisiana and the chromosome A05 of Musa acuminata and a translocation between the chromosome B01 and the chromosome A03 as already reported28 (Fig. 1 and Supplemen- tary Fig. 9). Four inversions between Musa schizocarpa and Musa acuminata were also detected on the chromosomes S10, S04, S05, and S09 and one translocation on chromosome A10 (Fig. 1 and Supplementary Fig. 10). The corresponding regions on chromo- some A10 contain the 45S rDNAS gene cluster. The contigs organization in these ﬁve regions was manually validated in the two genome assemblies using optical maps. g g y p g We generated a highly contiguous assembly of the eleven chromosome sequences of Musa acuminata of which ﬁve were obtained in a single contig. At the same time, optical maps were used to validate the nanopore assembly. It is important to men- tion that only one small contig, essentially composed of repetitive elements, was detected as a potential chimera underlining the high quality of the contigs produced by the NECAT assembler. However, it should be noted that homozygous material was used and this limited complexity may not be representative of the situation in other species. All eleven chromosome sequences, build with the help of a genetic map, contain telomeric repeats at both ends, which is an important element in asserting on the one hand that the reconstruction of the chromosome sequences is of good quality and on the other hand that the still missing part of the genome is contained in the remaining ﬁfteen gaps, although 7 are of unknown length. One of the advantages of optical maps is that the size of the gaps can be estimated if the map is sufﬁciently contiguous (Supplementary Table 1 and Supplementary Fig. 11). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 Comparison of the distribution of repeated sequences (tandem repeat and TE) between V2 and V4 showed that the integration of these elements that were typically difﬁcult to assemble with past technologies are greatly improved in the new assembly and are now very congruent with cytogenetic karyotype. All centromeres are now clearly identiﬁed with large clusters of Nanica LINE tandem repeats and CMR TE, and in addition, for two of them large clusters of 5S rDNA tandem repeats. Such a case of recruitment of 5S rDNA gene array in centromere was also reported in one of the switchgrass chromosomes30. This high- resolution of centromeric regions opens new avenues to study how satellites repeats originate and evolve in the centromeric region and more generally to better understand the organization and functioning of centromeres that are essential chromosomal domains for kinetochore assembly and correct chromosome segregation31,32. In addition, chromosome reciprocal transloca- tions were recently shown to have accompanied subspecies evo- lution in Musa6, and some of them have their breakpoints in centromeric regions. Having access to the sequence of these centromeric regions will permit investigating the mechanism and sequences involved in the origin of these translocations. Finally, comparison with the Musa acuminata V2 assembly highlights a higher proportion of each class of transposable elements, and a large amount of additional sequences in the centromeric regions, like Nanica elements, or large retro-transposon derivatives. ratios) for purity estimation. DNA samples with a fragment size above 50 kbp, a A260/A280 ratio close to 2 and a A260/A230 ratio above 1.5 were kept. ratios) for purity estimation. DNA samples with a fragment size above 50 kbp, a A260/A280 ratio close to 2 and a A260/A230 ratio above 1.5 were kept. p In order to generate long reads on the Oxford Nanopore Technologies devices, high-quality and high-molecular-weight DNA is needed. To that end, DNA was isolated following the protocol provided by Oxford Nanopore Technologies, “High molecular weight gDNA extraction from plant leaves” downloaded from the ONT Community in March, 2019 (CTAB-Genomic-tip). This protocol involves a conventional CTAB extraction followed by puriﬁcation using the commercial Qiagen Genomic tip (QIAGEN, MD, USA), but size selection was performed using Short Read Eliminator XL (Circulomics, MD, USA) instead of AMPure XP beads. Brieﬂy, 1.2 g of leaves were cryoground in liquid nitrogen. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 One 20 µl agarose mini-plug was prepared from each batch of nuclei according to Šimková et al. 38. Miniplugs were washed and solubilized using agarase enzyme (Thermo Fisher Scientiﬁc) to release high molecular weight (HMW) DNA. HMW DNA was further puriﬁed by drop dialysis and was then homogenized a few days prior to the quality control. The concentration and purity of the extracted DNA were evaluated using a Qubit ﬂuorometer (Thermo Fisher Scientiﬁc) and a Nanodrop spectrophotometer (Thermo Fisher Scientiﬁc). DNA integrity was checked by pulsed-ﬁeld gel electrophoresis (Pippin Pulse, Sage Science). DNA molecules were detectable between 50 and 300 kbp in size. Finally, we showed that gapless and telomere-to-telomere assembly of chromosome sequences is now possible thanks to long-read sequencing, at least in the case of homozygous gen- omes. The critical point remains the DNA extraction protocols that generally need adaptation for each species. These closed assemblies will allow new discoveries and will shed new light on these genomes in particular in complex repetitive regions such as centromeres, which have essential biological function but are so far poorly characterized. Illumina PCR-free library preparation and sequencing. DNA (1.5 μg) was sonicated to a 100−1500 bp size range using a Covaris E220 sonicator (Covaris, Woburn, MA, USA). The fragments were end-repaired and 3′-adenylated. Illumina adapters were added using the Kapa Hyper Prep Kit (KapaBiosystems, Wilming- ton, MA, USA). The ligation products were puriﬁed with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA, USA). The libraries were quantiﬁed by qPCR using the KAPA Library Quantiﬁcation Kit for Illumina Libraries (Kapa- Biosystems), and the library proﬁles were assessed using a DNA High Sensitivity LabChip kit on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The libraries were sequenced on an Illumina HiSeq2500 instrument (Illu- mina, San Diego, CA, USA) using 250 base-length read chemistry in paired- end mode. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 One 20 µl agarose mini-plug was prepared from each batch of nuclei according to Šimková et al. 38. Miniplugs were washed and solubilized using agarase enzyme (Thermo Fisher Scientiﬁc) to release high molecular weight (HMW) DNA. HMW DNA was further puriﬁed by drop dialysis and was then homogenized a few days prior to the quality control. Generating optical maps requires high molecular weight (HMW) DNA. Here HMW DNA of M. acuminata DH Pahang was prepared according to Safář et al. 37 with several modiﬁcations. Brieﬂy, 0.5 cm long segments of leaf midribs and young leaf tissues were ﬁxed for 20 min at 4 °C in Tris buffer (10 mM Tris, 10 mM EDTA, 100 mM NaCl, pH 7.5) containing 2% formaldehyde. After three 5 min washes in Tris buffer, the segments were homogenized using chopping by a razor blade in petri dish containing 1 ml of ice-cold IB buffer (15 mM Tris, 10 mM EDTA, It is often mistakenly thought that short-read assemblies are complete at the gene level. This hypothesis is mainly based on the results given by the BUSCO software, which only focuses on single-copy genes. Accordingly, the gene content completeness was already high, in previous M. acuminata assembly versions, according to the BUSCO score. However, here, using ultralong reads, we were able to assemble many additional copies of TDG clusters which contain important gene families like terpene syn- thases or disease resistance genes. Banana crops are currently particularly threatened by diseases including Black leaf streak disease that requires massive use of pesticide33 and by a new strain of Fusarium wilt (Tropical Race 4) that is currently spreading around the world and for which no chemical control is possible34. This new assembly will facilitate the search for resis- tance genes to these devastating diseases35. 130 mM KCl, 20 mM NaCl, 1 mM spermine, 1 mM spermidine and 0.1% Triton X- 100, pH 9.4) and immediately before use, 33 μl of β-mercaptoethanol were added to 10 ml of IB buffer. Nuclei suspension was passed through a 50 μm nylon mesh and stained with DAPI at a ﬁnal concentration of 2 μg/mL. Six batches of 900,000 G1- phase nuclei were sorted into 77 µl of IB buffer with β-mercaptoethanol in 1.5 ml polystyrene tubes using a FACSAria SORP ﬂow cytometer and sorter (Becton Dickinson, San José, CA, United States) equipped with solid-state UV laser. ARTICLE ARTICLE COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 The ﬁne powder was transferred to 20 mL of Carlson buffer (100 mM Tris-HCl pH 9.5, 2% CTAB, 1.4 M NaCl, 1% PEG 8000, 20 mM EDTA, 0.25% b-mercaptoethanol (v/v)) prewarmed to 65 °C. Then 40 µl of RNase A (100 mg/ml) was added before incubation at 65 °C for 1 h (with intermittent agitation). Proteins removal was performed by addition of one volume of chloroform and centrifugation at 5,500×g for 10 min at 4 °C. DNA was then precipitated with 0.7 V of isopropanol and centrifugation at 5,500×g for 30 min at 4 °C. The pellet was then puriﬁed using the Qiagen Genomic-tip 100/G, following the manufacturer’s instruction: DNA pellet was ﬁrst dissolved at 50 °C for 15 min in 9.5 mL of G2 buffer before loading onto the pre-equilibrated Genomic-tip column. Puriﬁed gDNA was ﬁnally precipitated with 0.7 volumes of isopropanol, washed with 2 ml of 70% ethanol, dried, and eluted in 100 µL of TE Buffer. DNA was quantiﬁed by a dsDNA-speciﬁc ﬂuorometric quantitation method using Qubit dsDNA HS Assays (ThermoFisher Scientiﬁc, Waltham, MA). DNA quality was checked on a 2200 TapeStation automated electrophoresis system (Agilent, CA, USA) (Supplementary Fig. 12). g pp y g Generating optical maps requires high molecular weight (HMW) DNA. Here HMW DNA of M. acuminata DH Pahang was prepared according to Safář et al. 37 with several modiﬁcations. Brieﬂy, 0.5 cm long segments of leaf midribs and young leaf tissues were ﬁxed for 20 min at 4 °C in Tris buffer (10 mM Tris, 10 mM EDTA, 100 mM NaCl, pH 7.5) containing 2% formaldehyde. After three 5 min washes in Tris buffer, the segments were homogenized using chopping by a razor blade in petri dish containing 1 ml of ice-cold IB buffer (15 mM Tris, 10 mM EDTA, 130 mM KCl, 20 mM NaCl, 1 mM spermine, 1 mM spermidine and 0.1% Triton X- 100, pH 9.4) and immediately before use, 33 μl of β-mercaptoethanol were added to 10 ml of IB buffer. Nuclei suspension was passed through a 50 μm nylon mesh and stained with DAPI at a ﬁnal concentration of 2 μg/mL. Six batches of 900,000 G1- phase nuclei were sorted into 77 µl of IB buffer with β-mercaptoethanol in 1.5 ml polystyrene tubes using a FACSAria SORP ﬂow cytometer and sorter (Becton Dickinson, San José, CA, United States) equipped with solid-state UV laser. Discussion d Long read sequencing technology emergence has paved the way for high-quality genome assemblies. The rapid evolution of the DNA extraction protocols now allows the community to sequence very long DNA sequences. Coupled with the evolution of the bioinformatic tools, the generation of high-quality assem- blies has been greatly simpliﬁed. The latest improvements of the ONT technology, especially the base-calling efﬁciency, result in a decrease of the error rate. Several assembly tools were specially developed around long reads and are able to manage noisy reads and have their speciﬁcity, as well as a speciﬁc margin of progress. We think that it is still important to use the latest release of several assemblers and choose the most efﬁcient for each genome assembly project. COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio 8 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 UK) were then ligated using the NEBNext Quick Ligation Module (NEB). After puriﬁcation with AMPure XP beads (Beckmann Coulter, Brea, CA, USA), half of the library was mixed with the sequencing buffer (ONT) and the loading bead (ONT) and loaded on a PromethION R9.4.1 ﬂow cell. The second half of the library was loaded on the ﬂow cell after a Nuclease Flush using the Flow Cell Wash Kit EXP-WSH003 (ONT) according to the Oxford Nanopore protocol. Reads were basecalled using Guppy version 4.0.1. The nanopore long reads were not cleaned and raw reads were used for genome assembly. most of them corresponding to rDNA sequences. Because of their strong homology to scaffolds included in the chromosome sequences these scaffolds were discarded from the assembly. UK) were then ligated using the NEBNext Quick Ligation Module (NEB). After puriﬁcation with AMPure XP beads (Beckmann Coulter, Brea, CA, USA), half of the library was mixed with the sequencing buffer (ONT) and the loading bead (ONT) and loaded on a PromethION R9.4.1 ﬂow cell. The second half of the most of them corresponding to rDNA sequences. Because of their strong homology to scaffolds included in the chromosome sequences these scaffolds were discarded from the assembly. (ONT) and loaded on a PromethION R9.4.1 ﬂow cell. The second half of the library was loaded on the ﬂow cell after a Nuclease Flush using the Flow Cell Wash Kit EXP-WSH003 (ONT) according to the Oxford Nanopore protocol. Reads were basecalled using Guppy version 4.0.1. The nanopore long reads were not cleaned and raw reads were used for genome assembly. Gene prediction. Repeats in the genome assembly were masked using Tandem Repeat Finder52 for tandem repeats and RepeatMasker53 for simple repeats, as well as known repeats included in RepBase54. In addition, known Musa transposable elements (from D’Hont et al. 9), were detected using RepeatMasker. Gene prediction was done using proteomes from homologous species, Musa acuminata (UP000012960), Oryza. sativa (UP000059680), Phoenix dactylifera (UP000228380), Musa schizocarpa (www.genoscope.cns.fr/plants) and Musa balbisiana (banana-genome-hub.southgreen.fr). Optical mapping. The Direct Label and Stain (DLS) labeling (using the DLE-1 enzyme) and the Nick Label Repair and Stain (NLRS) labeling (using the BspQI enzyme) protocols were performed according to Bionano Genomics with 750 and 600 ng of DNA respectively. The Chip loadings were performed as recommended by Bionano Genomics. The proteomes were aligned against the genome assembly in two steps. ARTICLE Regions of both assemblies corresponding to these alignments were extracted and used as a second set that we used as input to merqury, to compare assemblies only in regions that are shared. The search for NLR loci was performed using NLR-annotator tools26 that scan speciﬁcally the 6 reading frames of the nucleotide sequence for the presence of 19 NLR-associated motifs and reconstruct a potential NLR locus which might correspond to a complete or partial gene and might also be a pseudogene. Transposable element detection. Transposable elements were detected using RepeatMasker53 associated with the TE Musa library9 and CR sequences18. The same procedure was used to detect TEs in the Musa acuminata V2, V4, Musa balbisiana and Musa schizocarpa assemblies. The gff output ﬁle was converted into a bed ﬁle and the TE coverage was calculated using bedtools61 coverage (version v2.29.2-17-ga9dc5335) on a 100 kbp window. Centromeric boundaries were deﬁned using the density of daterra-Maximus, ITS-5S, ITS-18S, ITS-26S, Nanica, maca-Angela, caturra-Reina elements. TEs were grouped following Wicker et al. classiﬁcation62. Transposable element detection. Transposable elements were detected using RepeatMasker53 associated with the TE Musa library9 and CR sequences18. The same procedure was used to detect TEs in the Musa acuminata V2, V4, Musa balbisiana and Musa schizocarpa assemblies. The gff output ﬁle was converted into a bed ﬁle and the TE coverage was calculated using bedtools61 coverage (version v2.29.2-17-ga9dc5335) on a 100 kbp window. Centromeric boundaries were deﬁned using the density of daterra-Maximus, ITS-5S, ITS-18S, ITS-26S, Nanica, maca-Angela, caturra-Reina elements. TEs were grouped following Wicker et al. classiﬁcation62. sequences. The ﬁrst one consists of the V1 and V4 assemblies, in order to compare them in their globality. As the V4 assembly is larger than V1, we aligned the V1 assembly to the V4 assembly using minimap2 and kept alignments that were larger than 50 kbp. Regions of both assemblies corresponding to these alignments were extracted and used as a second set that we used as input to merqury, to compare assemblies only in regions that are shared. Genome assemblies comparisons. The synteny relationships between Musa balbisiana, Musa schizocarpa and Musa acuminata V4 were determined using Assemblytics63. First, genome sequences were aligned against each other using nucmer64 version 3.23 (-maxmatch -l 100 -c 500) as recommended by the Assemblytics authors. Assemblytics was launched on the nucmer delta ﬁle (unique_length_required = 10000). Figures 1 and 2b were generated using the Circos software65. ARTICLE In the same way, each chromosome sequence of the Musa acuminata V4 was aligned against its relative chromosome sequence of Musa acuminata V2 using nucmer version 3.23 (-r −1 -l 10000) and dot plots were generated using the mummerplot command. Chromosome sequences reconstruction. DH-Pahang sequences were anchored on chromosomes using segregating markers obtained from the selﬁng of the ‘Pahang’ accession PT-BA-00267, described in Martin et al. 10 (Supplementary Table 12 and Supplementary Fig. 14). Data are available on the Banana Genome Hub47 in the download section under ‘AF-Pahang marker matrix ﬁle’ and ‘AF- Pahang marker sequence (FASTA)’ for coded segregating markers and marker sequence respectively. Sequences anchoring was performed following methodology described in Martin et al. 10. The complete process was performed using scaff- hunter tools48 available at the South Green platform. Detection of speciﬁc regions of the V4 assembly. New regions of the Musa acuminata V4 were determined using blast66 (ncbi-tools/6.1.20120620) alignment between the chromosomes of each assembly version. Regions of the V4 assembly larger than 100 kbp without any alignment to the V2 assembly were considered new. In addition, the Musa acuminata V2 gene predictions were aligned (see Gene prediction section) on the V4 assembly, and the positions of the genes were compared with the V4 gene catalog using bedtools61 (version bedtools-2.29.2) (-v option). Genes from the V4 assembly without any correspondence in the V2 were considered new. In addition, based on scaffold BLAST against Musa acuminata chloroplast sequence49, Musa acuminata putative 12 mitochondrial scaffolds10 and Phoenix dactylifera protein sequences50, 1 putative chloroplastic (corresponding to one initial contig), and 45 putative mitochondrial scaffolds (corresponding to 45 initial contigs) were identiﬁed in the assembly. The 45 putative mitochondrial scaffolds were grouped into a sequence named putative_mito_scaff. The chloroplastic scaffold was discarded from the assembly as the chloroplast genome of DH-Pahang was already fully assembled and published49. The 37 remaining scaffolds (cumulative size of 5.2 Mbp) (corresponding each to one initial contig) showed a strong BLAST homology to larger scaffolds included in the chromosome sequences (36 with more than 95% of their length and 1 with more than 88% of its length). Investigation (dot-plot analysis using gepard v1.3051 and BLAST against nr/nt of ncbi) of these scaffolds revealed a repetitive nature, Detection of tandemly duplicated genes. An all-against-all comparison of the Musa acuminata V4 proteins was performed using Diamond67 (version 0.9.24). ARTICLE This tool can ﬁnd CDSs based on genome located evidence without any calibration step. Brieﬂy, putative exons and introns, extracted from the alignments, were used to build a simpliﬁed graph by removing redundancies. Then, Gmove extracted all paths from the graph and searched for open reading frames (ORFs) consistent with the protein evidence. A selection step was applied to all candidate genes, essentially based on gene structure. Also, all gene predictions included in a genomic region tagged as transposable elements were removed from the gene set (Table 2). The completeness of the predicted genes was assessed with BUSCO60 version 5 (embryophyta dataset odb10). Assembly validation. The DLE-1 map was generated using the Direct Label and Stain (DLS) technology and the BspQI map using the Nick Label Repair and Stain (NLRS) technology. Genome map assemblies were performed using Bionano Solve Pipeline version 3.3 and Bionano Access version 1.3.0. We used the parameter “Add Pre-Assembly” which produced a rough assembly. This ﬁrst result was used as a reference for a second assembly, using the parameters “non-haplotype without extend and split”. We ﬁltered out molecules smaller than 150 kbp and molecules with less than nine labeling sites (Supplementary Tables 9 and 10). The nanopore contigs were then validated using the two Bionano maps and organized with the scaffolding procedure provided by Bionano Genomics (Supplementary Fig. 13). Negative gap sizes were checked and corrected using the BiscoT software15 to avoid artifactual genomic duplications (Supplementary Table 1). As recommended by the BiscoT authors, we performed a last iteration of Hapo-G14 to polish merged regions (Supplementary Table 11). In order to obtain a quality score used to compare the different versions of the assembly, we downloaded and used merqury16 version 1.3 (git commit 6b5405e). We ﬁrst used the included best_k.sh script with a tolerable collision rate of 0.0001 and a genome size of 500 Mbp, which gave us an estimated best k-mer size of 21. Then, we used meryl46 version 1.3 (git commit 3400615) to compute the reads k-mer counts via the meryl count com- mand with default parameters. Merqury was then launched on two sets of sequences. The ﬁrst one consists of the V1 and V4 assemblies, in order to compare them in their globality. As the V4 assembly is larger than V1, we aligned the V1 assembly to the V4 assembly using minimap2 and kept alignments that were larger than 50 kbp. ARTICLE Firstly, BLAT55 (default parameters) was used to quickly localize corresponding putative genes of the proteins on the genome. The best match and matches with a score ≥ 90% of the best match score were retained. Secondly, the alignments were reﬁned using Genewise56 (default parameters), which is more precise for intron/exon boundary detection. Alignments were kept if more than 80% of the length of the protein was aligned to the genome. Long reads-based genome assembly. We generated three samples of reads: all reads, 30X of the longest reads, and 30X of the ﬁltlong42 highest-score reads. We then applied four different assemblers, Smartdenovo43, Redbean44, Flye45, and NECAT11 on these three subsets of reads (Supplementary Table 7), with the exception of NECAT being only launched with all reads, as it applies a down- sampling algorithm in its pipeline. Smartdenovo was launched with -k 17, as advised by the developers in case of larger genomes and -c 1 to generate a con- sensus sequence. Redbean was launched with ‘-xont -X5000 -g450m’ and Flye with ‘-g 450m’. NECAT was launched with a genome size of 450 Mbp and other parameters were left as default. After the assembly phase, we selected the best assembly (NECAT with all reads) based on the cumulative size and contiguity. The assembler output was polished one time using Racon12 with Nanopore reads, then one time with Medaka13 and Nanopore reads, and two times with Hapo-G14 and Illumina PCR-free reads (Supplementary Table 8). To allow the detection of UTRs in the gene prediction step, we aligned not only the protein of M. acuminata, but also the virtual mRNAs of the M. acuminata predicted genes57 on a masked version of the genome assembly. Then, transcript sequences of M. acuminata predicted genes were aligned by BLAT (default parameters) on the masked genome assembly. Only the alignments with an identity percent greater or equal to 90% were kept. For each transcript, the best match was selected based on the alignment score. Finally, alignments were recomputed in the previously identiﬁed genomic regions by Est2Genome58 in order to deﬁne precisely intron boundaries. Alignments were kept if more than 80% of the length of the transcript was aligned to the genome with a minimal identity percent of 95%. To proceed to the gene prediction, we integrated the protein homologies and transcript mapping using a combiner called Gmove59. Methods Pl After the Illumina sequencing, an in-house quality control process was applied to the reads that passed the Illumina quality ﬁlters. The ﬁrst step discards low- quality nucleotides (Q < 20) from both ends of the reads. Next, Illumina sequencing adapters and primer sequences were removed from the reads. Then, reads shorter than 30 nucleotides after trimming were discarded. These trimming and removal steps were achieved using in-house-designed software based on the FastX package39. The last step identiﬁes and discards read pairs that are mapped to the phage phiX genome, using SOAP aligner40 and the Enterobacteria phage PhiX174 reference sequence (GenBank: NC_001422.1). This processing, described in Alberti et al. 41, resulted in high-quality data. Plant material. Double haploid Musa acuminata spp malaccensis (DH-Pahang) plant material was obtained from the CRB Plantes Tropicales Antilles CIRAD- INRA Guadeloupe under the collection number PT-BA-00461. DNA extraction. For Illumina sequencing libraries, DNA was extracted using a modiﬁed mixed alkyl trimethyl ammonium bromide (MATAB) procedure36. A total of 2 g freshly harvested leaves was ground in liquid nitrogen with a mortar and pestle and immediately transferred to 12 ml of 74 °C prewarmed extraction buffer containing 100 mM Tris-HCl, pH 8, 20 mM EDTA, 1.4 M NaCl, 2% w/v MATAB, 1% w/v PEG6000 (polyethylene glycol), 0.5% w/v sodium sulﬁte and 20 mgl−1 RNAse A. Crude extracts were maintained for 20 min at 74 °C, extracted with an equal volume of chloroform-isoamyl alcohol (24:1) and transferred to clean tubes. DNA was recovered by centrifugation after adding 10 ml isopropanol. DNA precipitates were brieﬂy dried, washed with 2 ml of 70% ethanol and resuspended in 1 ml sterile water. Extract quality was evaluated using pulse-ﬁeld gel electro- phoresis for size estimation and spectrophotometry (A260/A280 and A260/A230 PromethION library preparation and sequencing. The library was prepared according to the following protocol, using the Oxford Nanopore SQK-LSK109 kit. Genomic DNA fragments (4 µg) were repaired and 3′-adenylated with the NEB- Next FFPE DNA Repair Mix and the NEBNext® Ultra™II End Repair/dA-Tailing Module (New England Biolabs, Ipswich, MA, USA). Sequencing adapters provided by Oxford Nanopore Technologies (Oxford Nanopore Technologies Ltd, Oxford, MMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio 9 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 lower than 10e−20 and a coverage of the smallest protein greater than 80%. Genes were considered as tandemly duplicated if they were co-localized on the same chromosome and not distant from more than 10 genes to each other. Figure 4a was realized using the MCscan tool68 with the two following commands: jcvi.- compara.synteny (--iter=1) and jcvi.graphics.synteny (--glyphcolor = orientation). lower than 10e−20 and a coverage of the smallest protein greater than 80%. Genes were considered as tandemly duplicated if they were co-localized on the same 19. Neumann, P. et al. Plant centromeric retrotransposons: a structural and cytogenetic perspective. Mob. DNA 2, 4 (2011). were considered as tandemly duplicated if they were co-localized on the same chromosome and not distant from more than 10 genes to each other. Figure 4a was realized using the MCscan tool68 with the two following commands: jcvi - y p y chromosome and not distant from more than 10 genes to each other. Figure 4a was realized using the MCscan tool68 with the two following commands: jcvi.- y p y chromosome and not distant from more than 10 genes to each other. Figure 4a was realized using the MCscan tool68 with the two following commands: jcvi.- 20. Panchy, N., Lehti-Shiu, M. & Shiu, S.-H. Evolution of gene duplication in plants. Plant Physiol. 171, 2294–2316 (2016). g g j compara.synteny (--iter=1) and jcvi.graphics.synteny (--glyphcolor = orientation). g g j compara.synteny (--iter=1) and jcvi.graphics.synteny (--glyphcolor = orientation). g g j compara.synteny (--iter=1) and jcvi.graphics.synteny (--glyphcolor = orientation). 21. Del Terra, L. et al. Functional characterization of three Coffea arabica L. monoterpene synthases: Insights into the enzymatic machinery of coffee aroma. Phytochemistry 89, 6–14 (2013). Statistics and reproducibility. No statistical tests were used in this study, and to allow the reproducibility of our analysis and results, all the sequencing data are available in public databases and the scripts developed to generate the ﬁgures are available on Zenodo and on a Github repository, as described in the data and code availability sections. 22. Jiang, S.-Y., Jin, J., Sarojam, R. & Ramachandran, S. A comprehensive survey on the terpene synthase gene family provides new insight into its evolutionary patterns. Genome Biol. Evol. 11, 2078–2098 (2019). p 23. Falara, V. et al. The tomato terpene synthase gene family. Plant Physiol. 157, 770–789 (2011). 24. Martin, D. M. et al. References 35. Ahmad, F. et al. Genetic mapping of Fusarium wilt resistance in a wild banana Musa acuminata ssp. malaccensis accession. Theor. Appl. Genet. 133, 3409–3418 (2020). 1. Michael, T. P. & VanBuren, R. Building near-complete plant genomes. Curr. Opin. Plant Biol. 54, 26–33 (2020). 1. Michael, T. P. & VanBuren, R. 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COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 Functional annotation, genome organization and phylogeny of the grapevine (Vitis vinifera) terpene synthase gene family based on genome assembly, FLcDNA cloning, and enzyme assays. BMC Plant Biol. 10, 226 (2010). Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Data availability 25. Wersch, Svan & Li, X. Stronger when together: clustering of plant NLR disease resistance genes. Trends Plant Sci. 24, 688–699 (2019). y All the supporting data are included in three additional ﬁles which contain (a) Supplementary Tables 1−12 and Supplementary Figs. 1−14, (b) Supplementary Data 1 (position of NLR genes in the V4 assembly) and (c) Supplementary Data 2 (position of NLR genes in the V2 assembly). The genome assembly is freely available at http:// www.genoscope.cns.fr/plants and http://banana-genome-hub.southgreen.fr. The ONT, Illumina, and Bionano Genomics data are available in the European Nucleotide Archive under the following projects PRJEB35002. All the supporting data are included in three additional ﬁles which contain (a) Supplementary Tables 1−12 and Supplementary Figs. 1−14, (b) Supplementary Data 1 (position of NLR genes in the V4 assembly) and (c) Supplementary Data 2 (position of NLR genes in the V2 assembly). The genome assembly is freely available at http:// www.genoscope.cns.fr/plants and http://banana-genome-hub.southgreen.fr. The ONT, g ( ) 26. Steuernagel, B. et al. The NLR-annotator tool enables annotation of the g 26. Steuernagel, B. et al. The NLR-annotator tool enables annotation of the intracellular immune receptor repertoire. Plant Physiol. 183, 468–482 (2020 27. Belser, C. et al. Chromosome-scale assemblies of plant genomes using nanopore long reads and optical maps. Nat. Plants 4, 879–887 (2018). www.genoscope.cns.fr/plants and http://banana-genome-hub.southgreen.fr. The ONT, 28. Wang, Z. et al. Musa balbisiana genome reveals subgenome evolution and functional divergence. Nat. Plants 5, 810–821 (2019). Illumina, and Bionano Genomics data are available in the European Nucleotide Archive under the following projects PRJEB35002. Illumina, and Bionano Genomics data are available in the European Nucleotide Archive under the following projects PRJEB35002. 29. Lang, D. et al. Comparison of the two up-to-date sequencing technologies for genome assembly: HiFi reads of Paciﬁc Biosciences Sequel II system and ultralong reads of Oxford Nanopore. GigaScience 9, giaa123 (2020). Code availability y All the code and data used to generate the ﬁgures are available on Zenodo69 and on a Github repository https://github.com/institut-de-genomique/Pahang-associated-data All the code and data used to generate the ﬁgures are available on Zenodo69 and on a Github repository https://github.com/institut-de-genomique/Pahang-associated-data 30. Yang, X. et al. Ampliﬁcation and adaptation of centromeric repeats in polyploid switchgrass species. N. Phytol. 218, 1645–1657 (2018). polyploid switchgrass species. N. Phytol. 218, 1645–1657 (2018). 31. Miga, K. H. Centromere studies in the era of ‘telomere-to-telomere’ genomics. Exp. Cell Res. 394, 112127 (2020). Received: 11 June 2021; Accepted: 13 August 2021; Received: 11 June 2021; Accepted: 13 August 2021; 32. Comai, L., Maheshwari, S. & Marimuthu, M. P. A. Plant centromeres. Curr. Opin. Plant Biol. 36, 158–167 (2017). p 33. Bellaire, L., de, L., de, Fouré, E., Abadie, C. & Carlier, J. Black leaf streak disease is challenging the banana industry. Fruits 65, 327–342 (2010). 34. Kema, G. H. J. et al. Editorial: Fusarium wilt of banana, a recurring threat to global banana production. Front. Plant Sci. 11, 628888 (2021). ARTICLE ARTICLE COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio ARTICLE Mapping output was ﬁltered according to the following parameters: an e-value 10 COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio /doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio Author contributions 55. Kent, W. J. BLAT—the BLAST-like alignment tool. Genome Res. 12, 656–664 (2002). K.L. extracted the sequenced DNA. E.H., and J.D. prepared HMW DNA for optical mapping. K.L. extracted the plugs. C.C. realized the bionano experiments. K.L., C.C., and A.L. optimized and performed the sequencing. C.B., B.I., B.N., N.Y., F.C.B., G.M., and J.M.A. performed the bioinformatic analyses. A.D. and J.M.A. conceived the project. C.B., B.I., B.N., K.L., C.C., E.H., G.M., A.D., and J.M.A. wrote the article. A.D., P.W., and J.M.A. supervised the study. 56. Birney, E., Clamp, M. & Durbin, R. GeneWise and Genomewise. Genome Res. 14, 988 (2004). 57. Martin, G. et al. Improvement of the banana “Musa acuminata” reference sequence using NGS data and semi-automated bioinformatics methods. BMC Genomics 17, 243 (2016). 58. Mott, R. EST_GENOME: a program to align spliced DNA sequences to unspliced genomic DNA. Comput. Appl. Biosci. CABIOS 13, 477–478 (1997). p g g p q unspliced genomic DNA. Comput. Appl. Biosci. CABIOS 13, 477–478 (1997). 59. Dubarry, M. et al. Gmove a tool for eukaryotic gene predictions using various evidences. F1000Research 5 (2016). 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/ licenses/by/4.0/. Competing interests p g p pp 59. Dubarry, M. et al. Gmove a tool for eukaryotic gene predictions using various evidences. F1000Research 5 (2016). The authors declare the following competing interests, J.M.A. received travel and accommodation expenses to speak at Oxford Nanopore Technologies conferences. J.M.A. and C.B. received accommodation expenses to speak during Bionano Genomics user meetings. The authors declare no other competing interests. 60. Waterhouse, R. M. et al. BUSCO applications from quality assessments to gene prediction and phylogenomics. Mol. Biol. Evol. 35, 543–548 (2018). 61. Quinlan, A. R. & Hall, I. M. BEDTools: a ﬂexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010). 61. Quinlan, A. R. & Hall, I. M. BEDTools: a ﬂexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010). 62. Wicker, T. et al. A uniﬁed classiﬁcation system for eukaryotic transposable COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02559-3 Additional information 62. Wicker, T. et al. A uniﬁed classiﬁcation system for eukaryotic transposable elements. Nat. Rev. Genet. 8, 973–982 (2007). Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42003-021-02559-3. 63. Nattestad, M. & Schatz, M. C. Assemblytics: a web analytics tool for the detection of variants from an assembly. Bioinformatics 32, 3021–3023 (2016). 64. Kurtz, S. et al. Versatile and open software for comparing large genomes. Genome Biol. 5, R12 (2004). Correspondence and requests for materials should be addressed to J.-M. Peer review information Communications Biology thanks David Studholme and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editors: Caitlin Karniski and Brooke LaFlamme. Peer reviewer reports are available. 65. Krzywinski, M. I. et al. Circos: an information aesthetic for comparative genomics. Genome Res. https://doi.org/10.1101/gr.092759.109 (2009). 66. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990). g 67. Buchﬁnk, B., Xie, C. & Huson, D. H. Fast and sensitive protein alignment using DIAMOND. Nat. Methods 12, 59–60 (2015). Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints References Molecular analysis and genomic organization of major DNA satellites in banana (Musa spp.). PLoS One 8, e54808 (2013). 52. Benson, G. Tandem repeats ﬁnder: a program to Nucleic Acids Res. 27, 573–580 (1999). 52. Benson, G. Tandem repeats ﬁnder: a program to analyze DNA sequences. Nucleic Acids Res. 27, 573–580 (1999). 18. Tran, T. D. et al. Centromere and telomere sequence alterations reﬂect the rapid genome evolution within the carnivorous plant genus Genlisea.Plant J. Cell Mol. Biol. 84, 1087–1099 (2015). 53. Smit, A. F. A., Hubley, R. & Green, P. RepeatMasker http://repeatmasker.org/. 53. Smit, A. F. A., Hubley, R. & Green, P. RepeatMasker ht 54. Bao, W., Kojima, K. K. & Kohany, O. Repbase update, a database of repetitive elements in eukaryotic genomes. Mob. DNA 6, 11 (2015). 11 COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio Reprints and permission information is available at http://www.nature.com/reprints 68. Tang, H. et al. Synteny and collinearity in plant genomes. Science 320, 486–488 (2008). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 69. Belser, C. et al. Musa acuminata DH-Pahang genome assembly: associated data. Zenodo https://doi.org/10.5281/zenodo.5120019 (2021). Acknowledgements This work was supported by the Genoscope, the Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA), France Génomique (ANR-10-INBS-09-08), the Center de coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) and Agropolis Fondation (ID 1504-006) ‘GenomeHarvest’ project through the French Investissements d’avenir program (Labex Agro: ANR- 10-LABX-0001-01). EH and JD were supported by ERDF project ‘Plants as a tool for sustainable global devel- opment’ (No. CZ.02.1.01/0.0/0.0/16_019/0000827). The authors thank the staff of Oxford Nanopore Technology Ltd for technical help, Jitka Weiserová, Eva Jahnová and Dr. Jan Vrána for their help with the material preparation, the CRB Plantes Tropicales Antilles CIRAD-INRA Guadeloupe France for providing the plant materials and the CIRAD – UMR AGAP HPC Data Center of the South Green Bioinformatics platform (http:// www.southgreen.fr) for providing computational resources. © The Author(s) 2021 © The Author(s) 2021 12 COMMUNICATIONS BIOLOGY \| (2021) 4:1047 \| https://doi.org/10.1038/s42003-021-02559-3 \| www.nature.com/commsbio
https://openalex.org/W3137649123	https://www.researchsquare.com/article/rs-139177/latest.pdf	English	null	Difference of binding modes among three ligands to a receptor mSin3B corresponding to their inhibitory activities	Scientific reports	2,021	cc-by	12,239	Difference of Binding Modes Among Three Ligands to a Receptor mSin3B Corresponding to Their Inhibitory Activities Tomonori Hayami Osaka University Narutoshi Kamiya University of Hyogo Kota Kasahara Ritsumeikan University Takeshi Kawabata Osaka University Jun-ichi Kurita Yokohama City University Yoshifumi Fukunishi National Institute of Advanced Industrial Science a Yoshifumi Nishimura Yokohama City University Haruki Nakamura Osaka University Junichi Higo (  higo@protein.osaka-u.ac.jp ) University of Hyogo Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Scienti¦c Reports on March 17th, 2021. See the published version at https://doi.org/10.1038/s41598-021-85612-9. Tomonori Hayami1, Narutoshi Kamiya2, Kota Kasahara3, Takeshi Kawabata1, Jun-ichi Kurita4, Yoshifumi Fukunishi5, Yoshifumi Nishimura4,6, Haruki Nakamura1, Junichi Higo1,2,* Tomonori Hayami1, Narutoshi Kamiya2, Kota Kasahara3, Takeshi Kawabata1, Jun-ichi Kurita4, Yoshifumi Fukunishi5, Yoshifumi Nishimura4,6, Haruki Nakamura1, Junichi Higo1,2,* 1 Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita, Osaka 565- 0871, Japan 2 Graduate School of Simulation Studies, University of Hyogo, 7-1-28 Minatojima Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan 3 College of Life Sciences, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan 3 College of Life Sciences, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga 525-8577, Japan 4 Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro- cho, Tsurumi-ku, Yokohama, 230-0045, Japan 4 Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro- cho, Tsurumi-ku, Yokohama, 230-0045, Japan 5 Cellular and Molecular Biotechnology Research Institute, National Institute of 5 Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-3-26, Aomi, Koto-ku, Tokyo, 135-0064, Japan Advanced Industrial Science and Technology (AIST), 2-3-26, Aomi, Koto-ku, Tokyo, 135-0064, Japan 6 Graduate School of Integrated Sciences for Life, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8258, Japan Correspondence and requests for materials should be addressed to J.H. (e-mail: higo@protein.osaka-u.ac.jp) and K.K. (e-mail: ktkshr@fc.ritsumei.ac.jp) Difference of binding modes among three ligands to a receptor mSin3B corresponding to their inhibitory activities Tomonori Hayami1, Narutoshi Kamiya2, Kota Kasahara3, Takeshi Kawabata1, Jun-ichi Kurita4, Yoshifumi Fukunishi5, Yoshifumi Nishimura4,6, Haruki Nakamura1, Junichi Higo1,2, ABSTRACT A preceding experiment suggested that a compound, which inhibits binding of the REST/NRSF segment to the cleft of a receptor protein mSin3B, can be a potential drug candidate to ameliorate many neuropathies. We have recently developed an enhanced conformational sampling method, genetic-algorithm-guided multi-dimensional virtual- system-coupled canonical molecular dynamics, and in the present study, applied it to three systems consisting of mSin3B and one of three compounds, sertraline, YN3, and acitretin. Other preceding experiments showed that only sertraline inhibits the binding of REST/NRSF to mSin3B. The current simulation study produced the spatial distribution of the compounds around mSin3B, and showed that sertraline and YN3 bound to the cleft of mSin3B with a high propensity, although acitretin did not. Further analyses of the simulation data indicated that only the sertraline–mSin3B complex produced a hydrophobic core similar to that observed in the molecular interface of the REST/NRSF–mSin3B complex: An aromatic ring of sertraline sunk deeply in the mSin3B’s cleft forming a hydrophobic core contacting to hydrophobic amino-acid residues located at the bottom of the cleft. The present study proposes a step to design a compound that inhibits competitively the binding of a ligand to its receptor. Introduction Neural restrictive silencer factor (NRSF), which is also known as Repressor-element 1 silencing transcription factor (REST)1,2, is a fundamental repressor, which binds to repressor-element 1 (re1) or neural restrictive silencer element (nrse) of many neuronal genes3,4. Importantly, overexpression of REST/NRSF or dysregulation of its cellular expression pattern is related to many neuropathies: Medulloblastoma5,6, malignant pediatric brain tumor7, glioblastoma8,9, Huntington’s disease10–13, neuropathic pain14,15, Parkinson’s disease16, autism17, and fibromyalgia18. REST/NRSF mediates transcriptional repression recruiting two corepressor complexes: REST/NRSF binds to mSin3 at its N-terminus and to CoREST plus the histone H3K9 methyltransferase G9a at its C-terminus19. The mSin3 complex, which contains two histone deacetylases HDAC1 and HDAC220, was implicated as an important epigenetic regulator in cancer21. The corepressor mSin3B, an isoform of mSin3, consists of four paired amphipathic helix domains (PAH1–PAH4) connected by linkers among the domains, and an intrinsically disordered region of REST/NRSF binds to the cleft of PAH1 of mSin3B22. Interestingly, an NMR experiment has shown that the disordered regions of REST/NRSF folds into a helix when binding to the hydrophobic cleft of the PAH1 domain22 (coupled folding and binding23–25). See Figure S1 of Supplementary Information for the PAH1 structure and the cleft position in the domain. A microscopic mechanism for the coupled folding and binding of this system was elucidated by our earlier computational study26. The NMR work provided a useful strategy for drug discovery: A compound that inhibits the binding of REST/NRSF to the PAH1 cleft of mSin3B can be a potential drug candidate to ameliorate the neuropathies18,27–30, and many compounds have been examined18,22,31. In fact, Ueda et al. analyzed the NMR complex structure of REST/NRST and the PAH1 domain of mSin3B, and proposed a compound mS-11 to inhibit the binding of REST/NRSF to the PAH1 domain18. This compound mimics the helical structure of a four-residue segment (Leu46-Ile47-Met48-Leu49) of REST/NRSF in the bound form, and importantly, this compound inhibited actually the binding of REST/NRST to the PAH1 domain. We call this four-residue segment a LIML sequence in this study. Recently, we developed a generalized ensemble method, multi-dimensional virtual-system coupled molecular dynamics (mD-VcMD) simulation32,33. This method enhances conformational sampling of biomolecules in an explicit solvent: By introducing multiple reaction coordinates (RCs) in the molecular system, the conformational motions of the molecules are enhanced with controlling the values of the multiple RCs. The search region in the RC space is expanded through iterative simulation. Introduction Importantly, a thermodynamic weight is assigned to each of the sampled conformations, and various thermodynamic quantities of the system are computed from the weighted snapshots. Then, we extended mD-VcMD by using a genetic algorithm and named the method as a genetic- algorithm-guided mD-VcMD (GA-guided mD-VcMD) simulation34,35 where the genetic algorithm supports expansion of the search range effectively. We have shown that the sampling efficiency of the GA-guided mD-VcMD is significantly higher than that of the original mD-VcMD34. In the present study, we investigate the spatial distribution of three compounds sertraline, YN3, and acitretin, respectively, around the PAH1 domain of mSin3B, which are obtained from the GA-guided mD-VcMD simulation of the system. The chemical structures of the three compounds are shown in Supplementary Figures S2–S4. We note that preceding experiments on sertraline and YN3 have shown that only sertraline exhibited medulloblastoma cell growth inhibitory activity, although both compounds bound to the PAH1 domain of mSin3B31. Another preceding experiment has shown that no inhibitory activities were detected for acitretin whereas it also binds to the PAH1 domain (personal communication with Nishimura). We show that the spatial distribution of the three compounds from the simulations rationally explains why only sertraline exhibited the inhibitory activity. Based on these computational results, we discuss a strategy to develop a drug candidate. Materials and Methods In this study, we denote the PAH1 domain of mSin3B simply as “mSin3B” for convenience. Besides, a system composed of sertraline and mSin3B is referred to as a “sertraline–mSin3B” system even though the two molecules are apart to each other during the simulation. Similarly, a system of YN3 and mSin3B is done to as a “YN3–mSin3B” system, and that of acitretin and mSin3B as a “acitretin–mSin3B” system. Three Reaction Coordinates. First, we introduced multiple RCs in the system, where an RC was defined by the distance between centers of mass of two atom groups. Supplementary Section 1 presents a detailed method to set an RC in the system, and Supplementary Figure S5 shows schematically the RC set in a system. For each the of ligand–receptor systems, we introduced three RCs, denoted as 𝜆(#) (ℎ= 𝛼, 𝛽, 𝛾). We briefly explain here the RCs as follows: The two atom groups 𝐺,- (red-colored segments in Supplementary Figures S2c, S3c, and S4c) and 𝐺,. (blue-colored segments) define the first RC 𝜆(,). We can imagine readily that the move of 𝜆(,) opens/closes the cleft. Atom groups 𝐺/ - and 𝐺0- are respectively green-colored and cyan-colored segments in mSin3B, and 𝐺/ . and 𝐺0. are purple-colored and orange- colored portions in the ligand (Supplementary Figures S2b, S3b, and S4b). The moves of 𝜆(/) (distance between 𝐺/ - and 𝐺/ .) and 𝜆(0) (distance between 𝐺0- and 𝐺0.) control the ligand approaching/departing to mSin3B. When 𝜆(/) increases with decreasing 𝜆(0) or when 𝜆(/) decreases with increasing 𝜆(0), the ligand rotates. Supplementary Figure S6 illustrate schematically that the system moves according to the variations of RCs in the RC space. We note that the selection of RCs can be arbitrary in theory if a very long simulation is possible. However, the selection is essentially important to raise the efficiency in an actual simulation. Detailed information for the atom groups is given in Supplementary Table S1. To study molecular binding extensively, the multi-dimensional RC region should involve both the unbound and bound conformations. For this purpose, we set the variable ranges for the three RCs wide enough (Supplementary Table S2). The term “multi-dimensional (mD)” means three-dimensional (3D) in this study, whereas the current method is applicable to any dimensional RC space. Initial Conformations of Simulation. After setting the RCs above, the initial conformation of simulation was generated. First, the tertiary structure of the receptor mSin3B (PAH1 domain) was taken from the PDBj site (https://pdbj.org/) (PDB ID: 2CZY), in which the receptor binds to the REST/NRSF fragment. After removing REST/NRSF from the complex, we introduced a ligand (sertraline, YN3, or acitretin) near mSin3B. As explained later, we randomized the position of the ligand to generate the initial conformations of the GA-guided mD-VcMD simulation, where the ligand was distant from the binding cleft of mSin3B. Next, we put the two molecules (ligand and mSin3B) generated above in a periodic boundary box (size is 70.0Å5) filled by water molecules, and removed water molecules that overlapped to mSin3B or the ligand. Then Na+ and Cl- ions were introduced with randomly replacing water molecules by ions. The number of ions was set to a physiological ionic concentration with neutralizing the net charge of the whole system to zero. The resultant sertraline–mSin3B system consists of 33,543 atoms (1,200 atoms for mSin3B, 38 atoms for sertraline, 10,749 water molecules, 29 Na+, 29 Cl-), the YN3–mSin3B system does of 33,533 atoms (1,200 atoms for mSin3B, 40 atoms for YN3, 10,745 water molecules, 29 Na+, 29 Cl-), and the acitretin–mSin3B system does of 33,525 atoms (1,200 atoms for mSin3B, 50 atoms for acitretin, 10,739 water molecules, 29 Na+, 29 Cl-). After a short energy minimization, a short constant-volume and constant- temperature (300 K) simulation (NVT simulation) was performed. Then, a constant- pressure (1 atm) and constant-temperature (300 K) simulation (NPT simulation) was performed to relaxed the box size. The resultant box size was 68.9203 Å3 , 68.9093, and 68.9093 for the sertraline–mSin3B, YN3–mSin3B, and acitretin–mSin3B systems, respectively. Whereas the PAH1 domain is linked to the PAH2 domain by a long flexible linker in a real cell, only the PAH1 domain was computed in our simulation, and the inter- domain linker was treated as the C-terminal tail. This tail might be inserted into the binding cleft of PAH1 domain incidentally during the simulation. It is likely that the incidental insertion of the inter-domain linker into the cleft does not happen if the PAH1 and PAH domains are connected by the linker. Thus, to prevent this incidental and artificial event, we applied weak restraints to the C-terminal tail (see Supplementary Section 2). Initial Conformations of Simulation. By the restraints, the C-terminal did not move to the binding cleft, and fluctuated around the initial conformation (NMR structure; PDB ID: 2CZY) during the simulation. To generate the initial conformations of simulation, where the ligand is distant from the binding cleft of mSin3B, we applied interactions between mSin3B and the ligand so that the RCs fall in the following ranges: 15Å< 𝜆(,) < 16Å, 24Å< 𝜆(/) < 25Å, and 24Å< 𝜆(0) < 25Å. Then, with applying these interactions we performed 256 runs starting from the last snapshot of the NPT simulation done above. Supplementary Figures S7–S9 display some of the last conformations picked randomly from those 256 runs for the three systems. We used those 256 conformations for the initial conformation of GA- guided mD-VcMD. Apparently, the ligand in these conformations was distant from the cleft of mSin3B (i.e., REST/NRSF position). These figures also display the REST/NRSF fragment binding to the cleft of mSin3B (PDB ID: 2CZY), whereas REST/NRSF did not exist in the current simulation. The GA-guided mD-VcMD. Methodological details for GA-guided mD-VcMD are explained elsewhere34. Here, we explain the outline of GA-guided mD-VcMD and resultant quantities. This method controls the system’s motions by modulating the three RCs 𝜆(#) ( ℎ= 𝛼, 𝛽, 𝛾). Supplementary Figure S6b presents schematically a distribution of the system’s conformation in the 3D RC space resulted from the moves of 𝜆(#). The outline of the method is as follows: First, the entire 3D RC space is divided into many small zones. Then, the GA-guided mD-VcMD simulation provides a conformational distribution function 𝑄=>?@A𝜆(,), 𝜆(/), 𝜆(0)B of the system, where 𝑄=>?@A𝜆(,), 𝜆(/), 𝜆(0)B is the probability of existence at position [𝜆(,), 𝜆(/), 𝜆(0)] in the 3D RC space constructed by 𝜆(,), 𝜆(/), and 𝜆(0). Because the conformational space of a biological molecular system is wide, the GA-guided mD-VcMD is executed via iterative simulations, during which the sampled RC region is expanded. The simulation is terminated when 𝑄=>?@A𝜆(,), 𝜆(/), 𝜆(0)B has converged. We discuss the convergence quantitatively later. After the convergence, a thermodynamic weight is assigned to each of stored snapshots using 𝑄=>?@A𝜆(,), 𝜆(/), 𝜆(0)B, and the ensemble of the snapshots can be regarded as a thermally equilibrated conformational ensemble (canonical ensemble)34. If the GA-guided mD-VcMD simulation is done at temperature 𝑇, then the canonical ensemble at 𝑇 is obtained. We performed 256 runs for an iteration in the present study to raise the sampling efficiency further. A simple integration of the 256 trajectories can be regarded as a long single trajectory36,37. When the M-th iteration is finished, we have snapshots stored from the 1st to M-th iterations. The 256 initial conformations for the (M+1)-th iteration were selected from those stored snapshots so that the conformations distributed as even as possible in the 3D-RC space. On the other hand, when an RC region was sampled poorly, we prepared the initial conformations around the poorly sampled region34. Because we obtained the canonical ensemble at 300 K as a result of the GA-guided mD-VcMD, we calculated the distribution function of various quantities in equilibrium at 300 K. Simulations. The actual parameter values for the GA-guised mD-VcMD are presented in Supplementary Section 3. The simulation was performed using a computer program omegagene/myPresto38 with the following condition: SHAKE39 to fix the covalent-bond lengths related to hydrogen atoms, the Berendsen thermostat to control temperature40, the zero-dipole summation method41–43 to compute accurately and quickly the long-range electrostatic interactions, a time-step of 2 fs (∆𝑡= 2 fs), and simulation temperature of 300 K. The Berendsen thermostat produces an ensemble that can approximate a canonical distribution for a many-atom system, whereas it generates a non-physical distribution for a small system44. To compute the original potential energy of the system, the Amber hybrid force fields (mixture parameter 𝑤= 0.75)45 was used for mSin3B, the TIP3P potential model for water molecules46, and the Joung–Cheatham model for chloride and sodium ions47. The force fields for the sertraline, YN3, and acitretin were set as follows: First, the atomic partial charges were derived by quantum chemical calculations using Gaussian0348 at the HF/6-31G* level, followed by RESP fitting49. Then, those partial charges were incorporated into a GAFF (general amber force field) force-field file50. GAFF was designed to be compatible with conventional AMBER force-fields. The Amber hybrid force fields currently used for mSin3B was generated by ourselves with mixing Amber parm9451 and parm9652 force fields to treat both helical and stranded polypeptides45, and the difference between parm94 and parm96 exists only in the dihedral energy parameters. Therefore, the inter-molecular interaction energy between mSin3B and the ligands is invariant mechanically among the parm94, parm96, and hybrid force fields. Spatial density of a compound around mSin3B. As mentioned above, the GA-guided VcMD simulation produces a conformational ensemble, where a thermodynamic weight at 300 K is assigned to each constituent conformation. Thus, we can calculate a spatial distribution function of any structural quantity from the ensemble. In this study, we compute the spatial density 𝜌JK (L)(𝒓), which is the probability of detecting the geometrical center (GC) of the ligand in the vicinity of a three-dimensional position 𝒓= [𝒙, 𝒚, 𝒛] in the real space, where the superscript 𝑠 is the system specifier: 𝑠= sertraline–mSin3B, YN3–mSin3B, or acitretin–mSin3B. The detailed computational procedure to calculate 𝜌JK (L)(𝒓) is explained in Supplementary Section 4. Other spatial density functions were calculated with the same manner: See Supplementary Eq. S4. Distribution of the system’s conformation in 3D RC space. In GA-guided mD-VcMD, the distribution is defined originally by 𝑄=>?@(𝐿(,), 𝐿(/), 𝐿(0)), where 𝐿(,), 𝐿(/), and 𝐿(0) are respectively indices to specify the positions 𝜆(,), 𝜆(/) , and 𝜆(0) in the 3D-RC space. Then, we convert [𝐿Y (,), 𝐿Z (/), 𝐿[ (0)] to: 𝜆\ (,) = 0.5{^𝜆Y (,)_`Y? + ^𝜆Y (,)_`>L}, where 𝑖= 1, … , 𝑛fL(𝛼), 𝜆Z (/) = 0.5{g𝜆Z (/)h `Y? + g𝜆Z (/)h `>L}, where 𝑗= 1, … , 𝑛fL(𝛽), and 𝜆Z (/) = 0.5{^𝜆Z (0)_ `Y? + ^𝜆Z (0)_ `>L}, 𝑘= 1, … , 𝑛fL(𝛾). See Supplementary Table S3 for values of ^𝜆Y (#)_`Y?, ^𝜆Y (#)_`>k , and 𝑛fL(ℎ) (ℎ= 𝛼, 𝛽, 𝛾). Then, 𝑄=>?@(𝜆(,), 𝜆(/), 𝜆(0)) is normalized so that the highest density is set to 1. Contour levels are presented by colors in inset. 𝜆Z (/) = 0.5{^𝜆Z (0)_ `Y? + ^𝜆Z (0)_ `>L}, 𝑘= 1, … , 𝑛fL(𝛾). See Supplementary Table S3 for values of ^𝜆Y (#)_`Y?, ^𝜆Y (#)_`>k , and 𝑛fL(ℎ) (ℎ= 𝛼, 𝛽, 𝛾). Then, 𝑄=>?@(𝜆(,), 𝜆(/), 𝜆(0)) is normalized so that the highest density is set to 1. Contour levels are presented by colors in inset. 𝜆Z (/) = 0.5{^𝜆Z (0)_ `Y? + ^𝜆Z (0)_ `>L}, 𝑘= 1, … , 𝑛fL(𝛾). See Supplementary Table S3 for values of ^𝜆Y (#)_`Y?, ^𝜆Y (#)_`>k , and 𝑛fL(ℎ) (ℎ= 𝛼, 𝛽, 𝛾). Then, 𝑄=>?@(𝜆(,), 𝜆(/), 𝜆(0)) is normalized so that the highest density is set to 1. Contour levels are presented by colors in inset. Distribution of the system’s conformation in 3D RC space. We repeated 14, 13, and 27 iterations for the sertraline–mSin3B, YN3–mSin3B, and acitretin–mSin3B systems, respectively. For all of the three systems, an iteration was composed of 256 runs. Each run was performed for 3 × 10T steps (6 ns; time step ∆𝑡= 2 fs). Thus, the total simulation length was 21.504 𝜇𝑠 (= 14 × 256 × 6 ns), 19.968 𝜇𝑠 (= 13 × 256 × 6 ns), and 41.472 𝜇𝑠 (= 27 × 256 × 6 ns) for the sertraline–mSin3B, YN3– mSin3B, and acitretin–mSin3B systems, respectively. A snapshot was stored every 1 × 10V steps (200 ps), yielding 107,520, 99,840, and 207,360 snapshots, for the sertraline–mSin3B, YN3–mSin3B, and acitretin–mSin3B systems, respectively. Figure 1 demonstrates the conformational distributions of the three systems in the 3D RC space, where the density is normalized so that the largest density is set to 1.0. We checked the convergence of distribution during the iterations. The method to check the convergence is given in Supplementary Section 5. Supplementary Figures S10–S12 indicate that the regions with 𝑄=>?@A𝜆(,), 𝜆(/), 𝜆(0)B > 0.001 are converged well. Although Figure 1 is basically important to show that the sampling covered a wide conformational space, this figure is not useful for understanding the ligand’s distribution around mSin3B. In the next section, we analyze the ligand’s distribution using the canonical conformational ensemble. ----------------- Figure 1. Density 𝑄=>?@(𝜆(,), 𝜆(/), 𝜆(0)) of (a) the sertraline-mSin3B, (b) YN3-mSin3B, and (c) acitretin- mSin3B systems in the 3D-RC space. In GA-guided mD-VcMD, the distribution is defined originally by 𝑄=>?@(𝐿(,), 𝐿(/), 𝐿(0)), where 𝐿(,), 𝐿(/), and 𝐿(0) are respectively indices to specify the positions 𝜆(,), 𝜆(/) , and 𝜆(0) in the 3D-RC space. Then, we convert [𝐿Y (,), 𝐿Z (/), 𝐿[ (0)] to: 𝜆\ (,) = 0.5{^𝜆Y (,)_`Y? + ^𝜆Y (,)_`>L}, where 𝑖= 1, … , 𝑛fL(𝛼), 𝜆Z (/) = 0.5{g𝜆Z (/)h `Y? + g𝜆Z (/)h `>L}, where 𝑗= 1, … , 𝑛fL(𝛽), and 𝜆Z (/) = 0.5{^𝜆Z (0)_ `Y? + ^𝜆Z (0)_ `>L}, 𝑘= 1, … , 𝑛fL(𝛾). See Supplementary Table S3 for values of ^𝜆Y (#)_`Y?, ^𝜆Y (#)_`>k , and 𝑛fL(ℎ) (ℎ= 𝛼, 𝛽, 𝛾). Then, 𝑄=>?@(𝜆(,), 𝜆(/), 𝜆(0)) is normalized so that the highest density is set to 1. Contour levels are presented by colors in inset. Figure 1. Density 𝑄=>?@(𝜆(,), 𝜆(/), 𝜆(0)) of (a) the sertraline-mSin3B, (b) YN3-mSin3B, and (c) acitretin- mSin3B systems in the 3D-RC space. Fig. 2 Distribution of ligands around mSin3B. Displayed structure of mSin3B is that after NPT simulation for each system, where labels H2 and H3 represent helix 2 and helix 3, respectively. The high-density cluster (𝜌JK (L)(𝒓) > 0.5𝜌l) in the cleft of mSin3B (PAH1) is named as Cluster A, and one near the N-terminal of mSin3B as Cluster B. (d) NMR structure of REST/NRSF– mSin3B complex (PDB ID: 2CZY), where cyan-colored model is REST/NRSF, and the magenta-colored segment is the LIML sequence of REST/NRSF. Label C indicates the position of the C-terminal tail of mSin3B. Two magenta-colored side-chains are Leu 46 and Leu 49 of the LIML sequence. Black broken- line circle indicates the position of the two sidechains. Those circles in panels (a), (b), and (c) are presented to indicate the sidechain position of Leu 46 and Leu 49. Green-colored sidechains are Val 75, Phe 93, and Phe 96 of mSin3B (see also green-colored sidechains of Supplementary Figure S12a), which form a hydrophobic core with Leu 46 and Leu 49 of the LIML sequence. Radial distribution function of ligand-cleft distance Figure 2. Spatial density 𝜌JK (L)(𝒓) of the geometric center (GC) at position 𝒓 for (a) the sertraline-mSin3B, (b) YN3-mSin3B, and (c) acitretin-mSin3B systems in the 3D real space. See Supplementary Section 4 for procedure to calculate 𝜌JK (L)(𝒓). Contour levels are shown in inset where 𝜌l = 0.001. Displayed structure of mSin3B is that after NPT simulation for each system, where labels H2 and H3 represent helix 2 and helix 3, respectively. The high-density cluster (𝜌JK (L)(𝒓) > 0.5𝜌l) in the cleft of mSin3B (PAH1) is named as Cluster A, and one near the N-terminal of mSin3B as Cluster B. (d) NMR structure of REST/NRSF– mSin3B complex (PDB ID: 2CZY), where cyan-colored model is REST/NRSF, and the magenta-colored segment is the LIML sequence of REST/NRSF. Label C indicates the position of the C-terminal tail of mSin3B. Two magenta-colored side-chains are Leu 46 and Leu 49 of the LIML sequence. Black broken- line circle indicates the position of the two sidechains. Those circles in panels (a), (b), and (c) are presented to indicate the sidechain position of Leu 46 and Leu 49. Green-colored sidechains are Val 75, Phe 93, and Phe 96 of mSin3B (see also green-colored sidechains of Supplementary Figure S12a), which form a hydrophobic core with Leu 46 and Leu 49 of the LIML sequence. Figure 2. Distribution of ligands around mSin3B. Spatial density 𝜌JK (L)(𝒓) of the geometric center (GC) at position 𝒓 for (a) the sertraline-mSin3B, (b) YN3-mSin3B, and (c) acitretin-mSin3B systems in the 3D real space. See Supplementary Section 4 for procedure to calculate 𝜌JK (L)(𝒓). Contour levels are shown in inset where 𝜌l = 0.001. Displayed structure of mSin3B is that after NPT simulation for each system, where labels H2 and H3 represent helix 2 and helix 3, respectively. The high-density cluster (𝜌JK (L)(𝒓) > 0.5𝜌l) in the cleft of mSin3B (PAH1) is named as Cluster A, and one near the N-terminal of mSin3B as Cluster B. (d) NMR structure of REST/NRSF– mSin3B complex (PDB ID: 2CZY), where cyan-colored model is REST/NRSF, and the magenta-colored segment is the LIML sequence of REST/NRSF. Label C indicates the position of the C-terminal tail of mSin3B. Two magenta-colored side-chains are Leu 46 and Leu 49 of the LIML sequence. Black broken- line circle indicates the position of the two sidechains. Those circles in panels (a), (b), and (c) are presented to indicate the sidechain position of Leu 46 and Leu 49. Green-colored sidechains are Val 75, Phe 93, and Phe 96 of mSin3B (see also green-colored sidechains of Supplementary Figure S12a), which form a hydrophobic core with Leu 46 and Leu 49 of the LIML sequence. Distribution of ligands around mSin3B. We computed the spatial density 𝜌JK (L)(𝒓) for the geometric center of the ligands (Figure 2). At the low-contour level (𝜌JK (L)(𝒓) = 0.005𝜌l); green-colored contours), the ligands distributed almost everywhere around mSin3B for all systems. This result is natural indicating that the sampling was done widely. With increasing the density level, the ligands tended to be localized at some surface regions of mSin3B (𝜌JK (L)(𝒓) = 0.05𝜌l; blue-colored contours). At the high-density level (𝜌JK (L)(𝒓) = 0.5𝜌l; red-colored contours), a high-density conformational cluster (labeled Cluster A in the figure) can be identified in the cleft of mSin3B for sertraline and YN3, whereas acitretin did not show a remarkable cluster in the cleft. This indicates that the ligand–mSin3B binding for sertraline and YN3 is stronger than that for acitretin. Figure 2d displays the REST/NRSF–mSin3B complex structure solved by the NMR experiment22 from the same orientation of Figure 1a–c. A view from a different orientation is presented in Supplementary Figure S13a. The binding cleft of mSin3B is hydrophobic (Supplementary Figure S13), and two residues Leu 46 and Leu 49 of the LIML sequence bind to the cleft forming a hydrophobic core with Val 75, Phe 93, and Phe 96 of mSin3B. See Introduction for the LIML sequence. Interestingly, only the geometrical center of sertraline overlaps the position of Leu 46 and Leu 49 of the LIML sequence (black broken-line circle) in the REST/NRSF–mSin3B complex, when the spatial density is viewed at the contour level of 𝜌JK (L)(𝒓) = 0.5𝜌l. Therefore, sertraline forms a tighter hydrophobic core with the binding cleft of mSin3B than the other ligands do. We also observed a high-density cluster (Cluster B in Figure 2) near the C- terminal of mSin3B. Because the C-terminal tail was restrained (see the Materials–and– Methods and Supplementary Section 2), the C-terminal tail did not fluctuate largely in the simulation in spite that the C-terminal tail is exposed to solvent. Therefore, it is likely that Cluster B was induced by this less-fluctuating C-terminal tail: Cluster B is an artificial cluster. Figure 2. Spatial density 𝜌JK (L)(𝒓) of the geometric center (GC) at position 𝒓 for (a) the sertraline-mSin3B, (b) YN3-mSin3B, and (c) acitretin-mSin3B systems in the 3D real space. See Supplementary Section 4 for procedure to calculate 𝜌JK (L)(𝒓). Contour levels are shown in inset where 𝜌l = 0.001. Radial distribution function of ligand-cleft distance. Figure 2 was insightful to guess the ligand–mSin3B interaction. Nevertheless, 𝜌JK (L)(𝒓) was calculated using the geometrical centers of the ligands. There is a possibility that some parts of the ligands contacted to the hydrophobic cleft tightly even when the geometrical center was distant from the cleft. For instance, acitretin is a long molecule, and then the head or tail of acitretin (Supplementary Figure S4) may be inserted to the cleft keeping the geometrical center out of the cleft. To make clear this point, we calculated a radial distribution function (RDF) between the ligand and three residues Val 75, Phe 93, and Phe 96 located in the mSin3B cleft. The computation procedure for RDF is given in Supplementary Section 6. First, we define the minimum heavy-atomic distance between the ligand and one of the three residues: 𝑟o (L) where the subscript 𝑅 is the residue specifier (𝑅= Val 75, Phe 93, or Phe 96) and the superscript 𝑠 is the system specifier defined before. Then, the RDF 𝑝(𝑟o (L)) was calculated for each of the three distances. Figure 3 demonstrates the resultant RDFs. For all of the systems, the function 𝑝(𝑟r#stT (L) ) exhibited a peak at 3.5 Å regardless of the peak height. This result is plausible because the residue Phe 96 is located at the entrance of the cleft (Supplementary Figure S13): The ligand can contact to Phe 96 without sinking into the cleft. More important RDFs are 𝑝(𝑟u>\vV (L) ) and 𝑝(𝑟r#st5 (L) ) because Val 75 and Phe 93 are located at the bottom of the cleft. Apparently, the peaks of the RDFs at 𝑟o (L)~4Å for the acitretin–mSin3B system were considerably smaller than those for sertraline–mSin3B and YN3–mSin3B systems. This means that acitretin did not interact frequently or tightly with the bottom of the cleft. The highest peaks were from the sertraline–mSin3B system (Figure 3a), and the peaks from the YN3–mSin3B were intermediate between sertraline and acitretin (Figure 3b). These results suggest that sertraline may resemble the ligand–receptor interaction mode found in the REST/NRSF–mSin3B complex. Figure 3. Radial distribution functions (RDFs) 𝑝(𝑟o (L)) for (a) the sertraline–mSin3B, (b) YN3–mSin3B, and (c) acitretin–mSin3B systems. Three RDFs 𝑝(𝑟u>\vV (L) ), 𝑝(𝑟r#st5 (L) ), and 𝑝(𝑟r#stT (L) ) are shown by different colors as indicated in inset of panel (a). See Supplementary Section 6 for procedure to calculate RDF. Figure 3. Radial distribution function of ligand-cleft distance. Radial distribution functions (RDFs) 𝑝(𝑟o (L)) for (a) the sertraline–mSin3B, (b) YN3–mSin3B, and (c) acitretin–mSin3B systems. Three RDFs 𝑝(𝑟u>\vV (L) ), 𝑝(𝑟r#st5 (L) ), and 𝑝(𝑟r#stT (L) ) are shown by different colors as indicated in inset of panel (a). See Supplementary Section 6 for procedure to calculate RDF. To investigate more the ligand–receptor interactions for the sertraline–mSin3B system, we calculated the spatial density 𝜌KJ- (L) (𝒓) of the geometrical center of Ring A of sertraline: See Supplementary Figure S1b for the position of Ring A and Supplementary Eq. S4 for the procedure to calculate 𝜌KJ- (L) (𝒓). Figure 4 illustrates 𝜌KJ- (L) (𝒓) and 𝜌KJ (L)(𝒓) with the contour level of 0.5𝜌l. Apparently, Ring A occupied the deeper position of the cleft than the geometrical center of the entire sertraline did. Thus, Ring A was closer to Val 75, Phe 93, and Phe 96 of mSin3B. Figure 4. Spatial density 𝜌JK- (L) (𝒓) (blue-colored contours) for the geometric center of Ring A of sertraline in the sertraline-mSin3B system, where the contour level is 𝜌JK- (L) (𝒓) = 0.5𝜌l (𝜌l = 0.001). Chemical structure of sertraline is also shown. Red-colored contours are 𝜌JK (L)(𝒓) for the geometric center of the entire sertraline. Labels H2 and H3 represent helices 2 and 3, respectively. Green-colored residues are Val 75, Phe 93, and Phe 96 of mSin3B. See Supplementary Section 4 for procedure to calculate 𝜌JK- (L) (𝒓). Figure 4. Spatial density 𝜌JK- (L) (𝒓) (blue-colored contours) for the geometric center of Ring A of sertraline in the sertraline-mSin3B system, where the contour level is 𝜌JK- (L) (𝒓) = 0.5𝜌l (𝜌l = 0.001). Chemical structure of sertraline is also shown. Red-colored contours are 𝜌JK (L)(𝒓) for the geometric center of the entire sertraline. Labels H2 and H3 represent helices 2 and 3, respectively. Green-colored residues are Val 75, Phe 93, and Phe 96 of mSin3B. See Supplementary Section 4 for procedure to calculate 𝜌JK- (L) (𝒓). Orientation of ligands bound to mSin3B. We checked the sertraline–mSin3B complex structures detected in the cleft of mSin3B with the high-density region, 𝜌JK- (L) (𝒓) > 0.5𝜌l, of Ring A. Figure 5 demonstrates some conformations picked randomly from the high-density region. This figure also displays Val 75, Phe 93, and Phe 96 of mSin3B. Here we judged that Ring A was contacting to the residues when 𝑟o (L) < 4.0 Å was satisfied. In Figure 5a, Ring A contacted to Phe 93 and Phe 96, whereas that in Figure 5b did not to any of the three residues. In Figure 5c Ring A contacted to all of the three residues in Figure 5c and to Val 75 in Figure 5d. Examining a number of snapshots, we presumed that ligand–receptor contacts shown in Figure 3a were contributed mainly by Ring A. Figure 5. (a)–(d) Sertraline-mSin3B complexes picked randomly from regions of 𝜌JK- (L) (𝒓) > 0.5𝜌l (𝜌l = 0.001), which are shown by blue-colored contours. Magenta-colored portion is Ring A of Sertraline. Red- colored vectors are those pointing from Ring-BC geometrical center to that of Ring A (see Supplementary Figure S1b for positions of Ring A and BC). Green-colored residues are Val 75, Phe 93, and Phe 96 of mSin3B. Labels H1,…, H4 are helices 1–4 of mSin3B (PAH1 domain). Figure 5. (a)–(d) Sertraline-mSin3B complexes picked randomly from regions of 𝜌JK- (L) (𝒓) > 0.5𝜌l (𝜌l = 0.001), which are shown by blue-colored contours. Magenta-colored portion is Ring A of Sertraline. Red- colored vectors are those pointing from Ring-BC geometrical center to that of Ring A (see Supplementary Figure S1b for positions of Ring A and BC). Green-colored residues are Val 75, Phe 93, and Phe 96 of mSin3B. Labels H1,…, H4 are helices 1–4 of mSin3B (PAH1 domain). Fig. 5 Red-colored vectors in Figure 5 indicate the molecular orientations of the shown snapshots. See Supplementary Figure S2b for the definition of the sertraline’s molecular orientation. Figure 5 indicates that Ring A was inserted into the binding cleft of mSin3B and that Ring BC was left behind. Here, to investigate statistically the sertraline’s orientation, we introduced a unit vector 𝒆Y (L) parallel to the vector from ring BC to Ring A for snapshot 𝑖, and calculated average, < 𝒆(L)(𝒓) >, of the unit vectors in each cube centered at 𝒓. The detailed procedure for averaging is presented in Supplementary Section 7. Orientation of ligands bound to mSin3B. Figure 6 demonstrates the spatial pattern of < 𝒆(L)(𝒓) > for the sertraline– mSin3B system viewed from two different directions. This figure also indicates that Ring A tends to be inserted in the cleft of mSin3B, which is consistent to Figure 5. Fig. 6 ----------------- ----------------- Figure 6. Spatial pattern of < 𝒆(L)(𝒓) > for sertraline in the cleft of mSin3B viewed from two different directions. Red and black vectors are those with \| < 𝒆(L)(𝒓) > \| ≥0.5 and \| < 𝒆(L)(𝒓) > \| < 0.5 , respectively. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.5𝜌l (𝜌l = 0.001). The vectors < 𝒆(L)(𝒓) > are shown in regions with 𝜌JK (L)(𝒓) > 0.5𝜌l. This figure also displays REST/NRSF bound to mSin3B, and the magenta-colored segment is the LIML sequence of REST/NRSF. Note that REST/NRSF is not involved in the current simulation. The shown structure is the REST/NRSF–mSin3B complex structure (PDB ID: 2CZY). Labels H1,…, H4 are helices 1,…, 4 of mSin3B (PAH1 domain). Figure 6. Spatial pattern of < 𝒆(L)(𝒓) > for sertraline in the cleft of mSin3B viewed from two different directions. Red and black vectors are those with \| < 𝒆(L)(𝒓) > \| ≥0.5 and \| < 𝒆(L)(𝒓) > \| < 0.5 , respectively. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.5𝜌l (𝜌l = 0.001). The vectors < 𝒆(L)(𝒓) > are shown in regions with 𝜌JK (L)(𝒓) > 0.5𝜌l. This figure also displays REST/NRSF bound to mSin3B, and the magenta-colored segment is the LIML sequence of REST/NRSF. Note that REST/NRSF is not involved in the current simulation. The shown structure is the REST/NRSF–mSin3B complex structure (PDB ID: 2CZY). Labels H1,…, H4 are helices 1,…, 4 of mSin3B (PAH1 domain). Figure 6. Spatial pattern of < 𝒆(L)(𝒓) > for sertraline in the cleft of mSin3B viewed from two different directions. Red and black vectors are those with \| < 𝒆(L)(𝒓) > \| ≥0.5 and \| < 𝒆(L)(𝒓) > \| < 0.5 , respectively. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.5𝜌l (𝜌l = 0.001). The vectors < 𝒆(L)(𝒓) > are shown in regions with 𝜌JK (L)(𝒓) > 0.5𝜌l. This figure also displays REST/NRSF bound to mSin3B, and the magenta-colored segment is the LIML sequence of REST/NRSF. Note that REST/NRSF is not involved in the current simulation. The shown structure is the REST/NRSF–mSin3B complex structure (PDB ID: 2CZY). Fig. 5 ----------------- Fig. 8 ----------------- Fig. 8 Orientation of ligands bound to mSin3B. Labels H1,…, H4 are helices 1,…, 4 of mSin3B (PAH1 domain). Figure 6. Spatial pattern of < 𝒆(L)(𝒓) > for sertraline in the cleft of mSin3B viewed from two different directions. Red and black vectors are those with \| < 𝒆(L)(𝒓) > \| ≥0.5 and \| < 𝒆(L)(𝒓) > \| < 0.5 , respectively. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.5𝜌l (𝜌l = 0.001). The vectors < 𝒆(L)(𝒓) > are shown in regions with 𝜌JK (L)(𝒓) > 0.5𝜌l. This figure also displays REST/NRSF bound to mSin3B, and the magenta-colored segment is the LIML sequence of REST/NRSF. Note that REST/NRSF is not involved in the current simulation. The shown structure is the REST/NRSF–mSin3B complex structure (PDB ID: 2CZY). Labels H1,…, H4 are helices 1,…, 4 of mSin3B (PAH1 domain). We also calculated < 𝒆(•‚5)(𝒓) > for the YN3–mSin3B (Figure 7) and acitretin–mSin3B (Figure 8) systems. See Supplementary Figures S3b and S4b for the definition of the molecular orientations for YN3 and acitretin. Figure 8 shows that the acitretin’s orientation tends to be parallel or anti-parallel to the helical cylinder of REST/NRSF in the REST/NRSF–mSin3B complex. We presume that these acitretin’s orientations have an advantage to fit the whole acitretin’s framework to the binding cleft of mSin3B. To stabilize one of the parallel or anti-parallel orientations of acitretin, an additional inter-molecular interaction is required, which works differently between the two orientations. We consider that there is no such interaction to stabilize effectively one of the two orientations for the acitretin–mSin3B system. Figure 7. Spatial pattern of < 𝒆(L)(𝒓) > for YN3 in the cleft of mSin3B viewed from two different directions. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.5𝜌l (𝜌l = 0.001). See caption of Figure 6 for more information. Figure 7. Spatial pattern of < 𝒆(L)(𝒓) > for YN3 in the cleft of mSin3B viewed from two different directions. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.5𝜌l (𝜌l = 0.001). See caption of Figure 6 for more information. Figure 7. Spatial pattern of < 𝒆(L)(𝒓) > for YN3 in the cleft of mSin3B viewed from two different directions. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.5𝜌l (𝜌l = 0.001). See caption of Figure 6 for more information. Figure 8. Spatial pattern of < 𝒆(L)(𝒓) > for acitretin in the cleft of mSin3B viewed from two different directions. Orientation of ligands bound to mSin3B. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.1𝜌l (𝜌l = 0.001). See caption of Figure 6 for more information. Figure 8. Spatial pattern of < 𝒆(L)(𝒓) > for acitretin in the cleft of mSin3B viewed from two different directions. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.1𝜌l (𝜌l = 0.001). See caption of Figure 6 for more information. On the other hand, the YN3’s orientations are not aligned to the helical cylinder of REST/NRSF. We presume that this tilting of YN3 to the helical cylinder is because YN3 is smaller than acitretin: YN3 may be responding to undulations of mSin3B’s molecular surface in the cleft. This point is discussed later again. Flexibility of ligands’ framework From these values, the framework of sertraline is the most flexible, acitretin has a considerably stiffer framework than sertraline does, and YN3 is stiffer than acitretin. The flexibility of the framework may be related to the ligand’s molecular orientation as discussed later. Fig. 9 Figure 9. (a) Structures of the three compounds sertraline, YN3, and acitretin. For each compound, inter- atomic distance 𝑟>ƒ@`„…>Y† (L) (𝑠= sertraline, YN3, or acitretin) is defined between cyan- to orange-colored atoms. A cyan-colored atom, which is involved in a ring for each ligand, is not set on the ring rotation axis to detect the ring-rotational motions. (b) Distance distribution functions (DDFs) 𝑃(𝑟>ƒ@`„…>Y† (L) ) for the three distances. See Supplementary Section 6 for procedure to calculate DDF. Figure 9. (a) Structures of the three compounds sertraline, YN3, and acitretin. For each compound, inter- atomic distance 𝑟>ƒ@`„…>Y† (L) (𝑠= sertraline, YN3, or acitretin) is defined between cyan- to orange-colored atoms. A cyan-colored atom, which is involved in a ring for each ligand, is not set on the ring rotation axis to detect the ring-rotational motions. (b) Distance distribution functions (DDFs) 𝑃(𝑟>ƒ@`„…>Y† (L) ) for the three distances. See Supplementary Section 6 for procedure to calculate DDF. Figure 9. (a) Structures of the three compounds sertraline, YN3, and acitretin. For each compound, inter- atomic distance 𝑟>ƒ@`„…>Y† (L) (𝑠= sertraline, YN3, or acitretin) is defined between cyan- to orange-colored atoms. A cyan-colored atom, which is involved in a ring for each ligand, is not set on the ring rotation axis to detect the ring-rotational motions. (b) Distance distribution functions (DDFs) 𝑃(𝑟>ƒ@`„…>Y† (L) ) for the three distances. See Supplementary Section 6 for procedure to calculate DDF. the three compounds sertraline, YN3, and acitretin. For each compound, inter- Flexibility of ligands’ framework Figure 5 demonstrates the structural variety of the sertraline’s framework. To quantify the flexibility of the ligand’s framework, we calculated the distance distribution function, DDF, for a distance between two atoms set in each ligand. The procedure to calculate DDF for a distance is given in Supplementary Section 6. Here, we picked the cyan- and orange-colored atoms in Figure 9a from the framework of each ligand, and calculated the distance between the two atoms: 𝑟>ƒ@`„…>Y† (L) . he DDFs 𝑃(𝑟>ƒ@`„…>Y† (L) ) for these distances are displayed in Figure 9b. The average of e distance < 𝑟>ƒ@`„…>Y† (L) > and its standard deviation (amount of fluctuations) the distance < 𝑟>ƒ@`„…>Y† > and its standard deviation (amount of fluctuations) 𝑆𝐷(𝑟>ƒ@`„…>Y† (L) ) were: 7.12 Å and 0.37 Å for sertraline, 6.01 Å and 0.14 Å for YN3, and 13.15 and 0.47 Å for acitretin. The largest SD was assigned to acitretin. However, this does not mean that acitretin is the most flexible, because a long molecule may have generally a large SD even if the ligand is stiff, and because acitretin is the longest ligand of the three. We consider that the amount of fluctuations per unit length, 𝑠𝑑(L) = 𝑆𝐷(𝑟>ƒ@`„…>Y† (L) ) were: 7.12 Å and 0.37 Å for sertraline, 6.01 Å and 0.14 Å for YN3, and 13.15 and 0.47 Å for acitretin. The largest SD was assigned to acitretin. However, this does not mean that acitretin is the most flexible, because a long molecule may have generally a large SD even if the ligand is stiff, and because acitretin is the longest ligand of the three. We consider that the amount of fluctuations per unit length, 𝑠𝑑(L) = 𝑆𝐷(𝑟>ƒ@`„…>Y† (L) )/< 𝑟>ƒ@`„…>Y† (L) >, is a better quantity to quantify the flexibility of the 𝑆𝐷(𝑟>ƒ@`„…>Y† (L) )/< 𝑟>ƒ@`„…>Y† (L) >, is a better quantity to quantify the flexibility of th molecular flexibility. The resultant 𝑠𝑑(L) was 5.20 × 10„Œ for sertraline, 2.33 × 10„Œ for YN3, and 3.57 × 10„Œ for acitretin. From these values, the framework of sertraline is the most flexible, acitretin has a considerably stiffer framework than sertraline does, and YN3 is stiffer than acitretin. The flexibility of the framework may be related to the ligand’s molecular orientation as discussed later. molecular flexibility. The resultant 𝑠𝑑(L) was 5.20 × 10„Œ for sertraline, 2.33 × 10„Œ for YN3, and 3.57 × 10„Œ for acitretin. Discussion Starting from the initial conformations in that the ligands were distant from the cleft of mSin3B (Supplementary Figure S7–S9), only sertraline reproduced a similar intermolecular hydrophobic core as observed in the REST/NRSF–mSin3B complex (PDB ID: 2CZY): Ring A of sertraline, which is hydrophobic, bound deeply to the cleft of mSin3B with contacting to the hydrophobic sidechains sited in the cleft (Figure 5). Thus, the sertraline–mSin3B complex formation competes with the REST/NRSF– mSin3B complex formation. Now, we discuss the interactions of acitretin and YN3 with mSin3B. Acitretin has a low spatial density in the mSin3B’s cleft (Figure 2c), which weakens the inhibitory activity partly. Furthermore, the framework of acitretin tends to be parallel or anti-parallel to the mSin3B’s cleft (Figures 8 and 10c). Remember that acitretin can be regarded as a long and stiff rod as shown in the above section. The parallel or anti-parallel molecular orientation is advantageous to be fit to the cleft. If acitretin has a flexible framework, acitretin may insert a portion into the hydrophobic cleft by bending the framework. Although YN3 had a high spatial density around the mSin3B’s cleft (Figure 2c), YN3 did not sink deeply in the cleft and the hydrophobic core was not formed. This result is natural because YN3 can be regarded as a stiff rod. As discussed for acitretin above, the stiff rod fits well to the cleft taking the parallel or anti-parallel orientation. On the other hand, we presume that the slight tilt of < 𝒆(•‚5)(𝒓) > to the helix cylinder of NRSF/REST in the NRSF/RESTmSin3B complex is resulted from the small size of YN3 (Figure 9b). I.e., YN3 is can adopt to the jaggedness of the inside of the cleft. If YN3 has more flexibility, this compound may exert the inhibitory activity by inserting a molecular portion into the cleft with varying the molecular conformation. On the other hand, the added flexibility may induce binding of YN3 to the other surfaces of mSin3B than the cleft. These competitive effects of the molecular flexibility cannot be assessed only from the chemical structure of the compound. We also note another chemical property of YN3 and acitretin, which is disadvantageous for interacting with hydrophobic cleft of mSin3B: Both rings in these compounds have an oxygen atom (Supplementary Figures S3 and S4), which can interact to hydrophilic residues in mSin3B electrostatically or by hydrogen bonding. Discussion Therefore, we note that the current simulation is useful to judge the probable complex structure. The current simulation provides the thermally equilibrated conformational ensemble of the molecular system, where the atomic interactions, the structural flexibility, and the solvent effect are explicitly considered. The conventional structure–activity relationship (SAR) study has been based on the chemical structures and target-binding activity data. Figure 10a shows one of the possible alignments of the three compounds based on their chemical structures. Each compound has an aromatic ring corresponding to Ring A and each aromatic ring has side chains in the para position. While sertraline has Cl atoms on Ring A, YN3 and acitretin have methoxy groups whose volumes are close to that of the Cl atom. Instead of Ring C of sertraline, YN3 and acitretin have triple and double bonds, respectively. These bonds are 𝜋-electron rich, and can form CH-𝜋 interaction as same as aromatic rings. However, such SAR analysis could not explain presence and absence of the medulloblastoma cell- growth inhibition activities of their compounds (Figure 10b). The present MD simulation study predicted the molecular orientations of the compounds (Figure 10c) and the presence/absence of the hydrophobic core in the cleft of mSin3B (Figure 2). Importantly, only sertraline could reproduce the binding mode in the complex of NRSF/REST and mSin3B. We emphasize that the current simulation method, the GA-guided mD-vcMD simulation, produces a thermodynamically acceptable ensemble consisting of various conformations (bound and unbound conformations), and importantly a thermodynamic weight is assigned to each snapshot in the ensemble. Fig. 10 ----------------- Figure 10. (a) Chemical structures of mSin3B binders, and their orientation aligned from head to tail sides. Rings A, B and C of sertraline are depicted in magenta, black, and orange, respectively. Blue circles represent aromatic rings that are expected to interact with the hydrophobic cleft of mSin3B from conventional structure-activity relationship (SAR). Green circles represent 𝜋-electron rich regions. (b) Presence/absence of Medulloblastoma cell-growth inhibition activities for the compounds. (c) The actual orientations of the compounds with respect to the cleft of mSin3B, predicted by the present MD simulation study. Figure 10. (a) Chemical structures of mSin3B binders, and their orientation aligned from head to tail side Figure 10. (a) Chemical structures of mSin3B binders, and their orientation aligned from head to tail sides. Rings A, B and C of sertraline are depicted in magenta, black, and orange, respectively. Discussion Blue circles represent aromatic rings that are expected to interact with the hydrophobic cleft of mSin3B from conventional structure-activity relationship (SAR). Green circles represent 𝜋-electron rich regions. (b) Presence/absence of Medulloblastoma cell-growth inhibition activities for the compounds. (c) The actual orientations of the compounds with respect to the cleft of mSin3B, predicted by the present MD simulation study. The preceding study31 classified 52 compounds into two pharmacophores, A and B, based on their chemical structures (see Fig. 2 of Ref. 31), where sertraline belongs to Pharmacophore A and the YN3 to Pharmacophore B. Because acitretin has a structural similarity with YN3 apparently, acitretin belongs to Pharmacophore B. Based on Figures 6–8 and Figure 10c, the compounds belonging to Pharmacophore A have the parallel or anti-parallel orientation, and the compound belonging to Pharmacophore B has a perpendicular orientation. Therefore, the current MD procedure is useful if it is used with the pharmacophore analysis. Fig. 11 Conclusions Binding of the compounds to mSin3B (PAH1 domain) was investigated by the GA- guided mD-VcMD simulation. This method produced useful quantities such as the spatial density of the ligand around the receptor (Figure 2), the intermolecular contact patterns (Figure 3), the propensity of molecular orientation (Figures 5–8), and the ligand flexibility (Figure 9). From these analyses, we showed that only sertraline produces a similar inter- molecular binding mode observed in the REST/NRSF–mSin3B complex. Figure 11 is a schematic drawing to design an inhibitor. Given a framework of the compound, by adding a hydrophobic sidechain to the framework, the hydrophobic core is formed between the sidechain and the hydrophobic cleft of mSin3B. The flexibility of the compound’s framework may increase the binding affinity, although a long and stiff framework may decrease the binding affinity. In general, it is difficult to specify the effect of the modification to the biological activity (i.e., inhibitory activity in the present study) only from the compound’s chemical structure. In contrast, the GA-guided mD-VcMD is useful to identify the effect with analyzing the thermally equilibrated conformational ensemble. Fig. 11 Figure 11. Scheme of a compound that binds to the cleft of mSin3B. Figure 11. Scheme of a compound that binds to the cleft of mSin3B. Because REST/NRSF is an intrinsically disordered segment that can bind to multiple proteins3,4 as mentioned in Introduction, the current study is an example to design a compound that can inhibit binding of an intrinsically disorder segment to a protein receptor. The 3D models have been submitted to the Biological Structure Model Archive (BSM-Arc) of the PDBj under BSM-ID BSM00020 (https://bsma.pdbj.org/entry/20), which are freely available 53. References 1. Schoenherr, C. J. & Anderson, D. J. The neuron-restrictive silencer factor (NRSF): A coordinate repressor of multiple neuron-specific genes. Science 267, 1360–1363 (1995). 1. Schoenherr, C. J. & Anderson, D. J. 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Many human medulloblastoma tumors overexpress repressor element-1 silencing transcription (REST)/neuronrestrictive silencer factor, which can be functionally countered by REST-VP16. Mol. Cancer Ther. 4, 343–349 (2005). 7. Dhall, G. Medulloblastoma. J. Child Neurol. 24, 1418–1430 (2009). 8. Conti, L., Crisafulli, L., Caldera, V., Tortoreto, M., Brilli, E., Conforti, P., Zunino, F., Magrassi, L., Schiffer, D. & Cattaneo, E. REST controls self-renewal and tumorigenic competence of human glioblastoma cells. Plos One 7, e38486 (2012). 9. Acknowledgements J. H. was supported by JSPS KAKENHI Grant No. 16K05517 and by the Development of Core Technologies for Innovative Drug Development Based Upon IT from Japan Agency for Medical Research and Development (AMED). N. K. was supported by a JSPS KAKENHI Grant (No. JP20H03229). K. K. was supported by JSPS KAKENHI Grant No. 16K18526. 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Contour levels are presented by colors in inset. 0.5{g𝜆Z (0)h `Y? + g𝜆Z (0)h `>L}, 𝑘= 1, … , 𝑛fL(𝛾). See Supplementary Table S3 for values of g𝜆Y (#)h `Y? , g𝜆Y (#)h `>k , and 𝑛fL(ℎ) (ℎ= 𝛼, 𝛽, 𝛾). Then, 𝑄=>?@(𝜆(,), 𝜆(/), 𝜆(0)) is normalized so that the highest density is set to 1. Contour levels are presented by colors in inset. normalized so that the highest density is set to 1. Contour levels are presented by colors in inset. Figure 2. Spatial density 𝜌JK (L)(𝒓) of the geometric center (GC) at position 𝒓 for (a) the sertraline-mSin3B, (b) YN3-mSin3B, and (c) acitretin-mSin3B systems in the 3D real space. See Supplementary Section 4 for procedure to calculate 𝜌JK (L)(𝒓). Contour levels are shown in inset where 𝜌l = 0.001. Displayed structure of mSin3B is that after NPT simulation for each system, where labels H2 and H3 represent helix 2 and helix 3, respectively. The high-density cluster (𝜌JK (L)(𝒓) > 0.5𝜌l) in the cleft of mSin3B (PAH1) is named as Cluster A, and one near the N-terminal of mSin3B as Cluster B. (d) NMR structure of REST/NRSF–mSin3B complex (PDB ID: 2CZY), where cyan-colored model is REST/NRSF, and the magenta-colored segment is the LIML sequence of REST/NRSF. Label C indicates the position of the C-terminal tail of mSin3B. Two magenta-colored side-chains are Leu 46 and Leu 49 of the LIML sequence. Black broken-line circle indicates the position of the two sidechains. and S. & Cheatham, III, T. E. Determination of alkali and halide monovalent ion parameters for use in explicitly solvated biomolecular simulations. J. Phys. Chem. B 112, 9020–9041 (2008). 48. Frisch, M. J. et al. Gaussian 09, Revision D.01. 48. Frisch, M. J. et al. Gaussian 09, Revision D.01. 49. Bayly, C. I., Cieplak, P., Cornell, W. & Kollman, P. A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. 97, 10269–10280 (1993). 50. Wang, J. M., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. J. Comput. Chem. 25, 1157– 1174 (2004). 51. Cornell, W. D., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, K. M., Ferguson, D. M., Spellmeyer, D. C., Fox, T., Caldwell, J. W. & Kollman, P. A. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 117, 5179–5197 (1995). 52. Kollman, P. A., Dixon, R. W., Cornell, W. D., Chipot, C. & Pohorille, A. The development/application of a 'minimalist' organic/biochemical molecular mechanic force field using a combination of ab initio calculations and experimental data. In Computer Simulations of Biological Systems; van Gunsteren, W. F., Weiner, P. K. & Wilkinson, A. J., Eds., Springer, Dordrecht, The Netherlands, 1997; Volume 3, pp. 83–96. 53. Bekker, G., Kawabata, T & Kurisu G. The Biological Structure Model Archive (BSM-Arc): An Archive for in Silico Models and Simulations. Biophys Rev. 12, 371– 375 (2020). Figure legends Those circles in panels (a), (b), and (c) are presented to indicate the sidechain position of Leu 46 and Leu 49. Green-colored sidechains are Val 75, Phe 93, and Phe 96 of mSin3B (see also green-colored sidechains of Supplementary Figure S12a), which form a hydrophobic core with Leu 46 and Leu 49 of the LIML sequence. Figure 3. Radial distribution functions (RDFs) 𝑝(𝑟o (L)) for (a) the sertraline–mSin3B, (b) YN3–mSin3B, and (c) acitretin–mSin3B systems. Three RDFs 𝑝(𝑟u>\vV (L) ), 𝑝(𝑟r#st5 (L) ), and 𝑝(𝑟r#stT (L) ) are shown by different colors as indicated in inset of panel (a). See Supplementary Section 6 for procedure to calculate RDF. Figure 3. Radial distribution functions (RDFs) 𝑝(𝑟o (L)) for (a) the sertraline–mSin3B, (b) YN3–mSin3B, and (c) acitretin–mSin3B systems. Three RDFs 𝑝(𝑟u>\vV (L) ), 𝑝(𝑟r#st5 (L) ), and 𝑝(𝑟r#stT (L) ) are shown by different colors as indicated in inset of panel (a). See Supplementary Section 6 for procedure to calculate RDF. Figure 4. Spatial density 𝜌JK- (L) (𝒓) (blue-colored contours) for the geometric center of Ring A of sertraline in the sertraline-mSin3B system, where the contour level is 𝜌JK- (L) (𝒓) = 0.5𝜌l (𝜌l = 0.001). Chemical structure of sertraline is also shown. Red- colored contours are 𝜌JK (L)(𝒓) for the geometric center of the entire sertraline. Labels H2 and H3 represent helices 2 and 3, respectively. Green-colored residues are Val 75, Phe 93, and Phe 96 of mSin3B. See Supplementary Section 4 for procedure to calculate 𝜌JK- (L) (𝒓). Figure 5. (a)–(d) Sertraline-mSin3B complexes picked randomly from regions of 𝜌JK- (L) (𝒓) > 0.5𝜌l (𝜌l = 0.001), which are shown by blue-colored contours. Magenta- colored portion is Ring A of Sertraline. Red-colored vectors are those pointing from Ring- BC geometrical center to that of Ring A (see Supplementary Figure S1b for positions of Ring A and BC). Green-colored residues are Val 75, Phe 93, and Phe 96 of mSin3B. Labels H1,…, H4 are helices 1–4 of mSin3B (PAH1 domain). Figure 6. Spatial pattern of < 𝒆(L)(𝒓) > for sertraline in the cleft of mSin3B viewed from two different directions. Red and black vectors are those with \| < 𝒆(L)(𝒓) > \| ≥ 0.5 and \| < 𝒆(L)(𝒓) > \| < 0.5, respectively. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.5𝜌l (𝜌l = 0.001). The vectors < 𝒆(L)(𝒓) > are shown in regions with 𝜌JK (L)(𝒓) > 0.5𝜌l. Figure 11. Scheme of a compound that binds to the cleft of mSin3B. Figure legends This figure also displays REST/NRSF bound to mSin3B, and the magenta-colored segment is the LIML sequence of REST/NRSF. Note that REST/NRSF is not involved in the current simulation. The shown structure is the REST/NRSF–mSin3B complex structure (PDB ID: 2CZY). Labels H1,…, H4 are helices 1,…, 4 of mSin3B (PAH1 domain). Figure 7. Spatial pattern of < 𝒆(L)(𝒓) > for YN3 in the cleft of mSin3B viewed from two different directions. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.5𝜌l (𝜌l = 0.001). See caption of Figure 6 for more information. Figure 8. Spatial pattern of < 𝒆(L)(𝒓) > for acitretin in the cleft of mSin3B viewed from two different directions. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.1𝜌l (𝜌l = 0.001). See caption of Figure 6 for more information. Figure 8. Spatial pattern of < 𝒆(L)(𝒓) > for acitretin in the cleft of mSin3B viewed from two different directions. Gray contours represent the high-density regions of 𝜌JK (L)(𝒓) > 0.1𝜌l (𝜌l = 0.001). See caption of Figure 6 for more information. Figure 9. (a) Structures of the three compounds sertraline, YN3, and acitretin. For each compound, inter-atomic distance 𝑟>ƒ@`„…>Y† (L) ( 𝑠= sertraline, YN3, or acitretin) is defined between cyan- to orange-colored atoms. A cyan-colored atom, which is involved in a ring for each ligand, is not set on the ring rotation axis to detect the ring-rotational motions. (b) Distance distribution functions (DDFs) 𝑃(𝑟>ƒ@`„…>Y† (L) ) for the three distances. See Supplementary Section 6 for procedure to calculate DDF. Figure 10. (a) Chemical structures of mSin3B binders, and their orientation aligned from head to tail sides. Rings A, B and C of sertraline are depicted in magenta, black, and orange, respectively. Blue circles represent aromatic rings that are expected to interact with the hydrophobic cleft of mSin3B from conventional structure-activity relationship (SAR). Green circles represent 𝜋-electron rich regions. (b) Presence/absence of Medulloblastoma cell-growth inhibition activities for the compounds. (c) The actual orientations of the compounds with respect to the cleft of mSin3B, predicted by the present MD simulation study. Figure 11. Scheme of a compound that binds to the cleft of mSin3B. Figures Figure 1 “See the Supplemental Files section for the complete ¦gure caption”. Figures Figures Figure 1 Figure 1 e the Supplemental Files section for the complete ¦gure caption”. “See the Supplemental Files section for the complete ¦gure caption”. “See the Supplemental Files section for the complete ¦gure caption”. 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Figure 9 “See the Supplemental Files section for the complete ¦gure caption”. Figure 9 Figure 9 Figure 9 “See the Supplemental Files section for the complete ¦gure caption”. Figure 10 “See the Supplemental Files section for the complete ¦gure caption”. “See the Supplemental Files section for the complete ¦gure caption”. “See the Supplemental Files section for the complete ¦gure caption”. Figure 10 “See the Supplemental Files section for the complete ¦gure caption”. Figure 10 Figure 10 “See the Supplemental Files section for the complete ¦gure caption”. Figure 11 Scheme of a compound that binds to the cleft of mSin3B. Figure 11 Scheme of a compound that binds to the cleft of mSin3B. Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. mSin3B3compSISciRep.pdf FigureLegend.pdf
https://openalex.org/W4327616659	https://hal.science/hal-04241910/document	English	null	Excessive self-grooming, gene dysregulation and imbalance between the striosome and matrix compartments in the striatum of Shank3 mutant mice	Frontiers in molecular neuroscience	2,023	cc-by	16,991	To cite this version: Allain-Thibeault Ferhat, Elisabeth Verpy, Anne Biton, Benoît Forget, Fabrice de Chaumont, et al.. Excessive self-grooming, gene dysregulation and imbalance between the striosome and matrix com- partments in the striatum of Shank3 mutant mice. Frontiers in Molecular Neuroscience, 2023, 16, pp.1139118. 10.3389/fnmol.2023.1139118. hal-04241910 Excessive self-grooming, gene dysregulation and imbalance between the striosome and matrix compartments in the striatum of Shank3 mutant mice Allain-Thibeault Ferhat, Elisabeth Verpy, Anne Biton, Benoît Forget, Fabrice de Chaumont, Florian Mueller, Anne-Marie Le Sourd, Sabrina Coqueran, Julien Schmitt, Christelle Rochefort, et al. Distributed under a Creative Commons Attribution 4.0 International License OPEN ACCESS Allain-Thibeault Ferhat 1,2,3†, Elisabeth Verpy 1†, Anne Biton 4†, Benoît Forget 1, Fabrice De Chaumont 1, Florian Mueller 5, Anne-Marie Le Sourd 1, Sabrina Coqueran 1, Julien Schmitt 6, Christelle Rochefort 6, Laure Rondi-Reig 6, Aziliz Leboucher 1, Anne Boland 7, Bertrand Fin 7, Jean-François Deleuze 7,8, Tobias M. Boeckers 9,10, Elodie Ey 1,11‡ and Thomas Bourgeron 1‡ 1 Génétique Humaine et Fonctions Cognitives, Institut Pasteur, CNRS UMR 3571, IUF, Université Paris Cité, Paris, France, 2 Department of Neuroscience, Columbia University Irving Medical Center, New York, NY, United States, 3 Zuckerman Mind Brain Behavior Institute, Columbia University, New York, NY, United States, 4 Bioinformatics and Biostatistics Hub, Institut Pasteur, Université Paris Cité, Paris, France, 5 Imagerie et Modélisation, Institut Pasteur, CNRS UMR 3691, Paris, France, 6 Cerebellum Navigation and Memory Team, Institut de Biologie Paris Seine, Neurosciences Paris Seine, CNRS UMR 8246, Inserm UMR-S 1130, Sorbonne Université, Paris, France, 7 Centre National de Recherche en Génomique Humaine, CEA, Université Paris-Saclay, Evry, France, 8 Centre d’Étude du Polymorphisme Humain, Paris, France, 9 Institute of Anatomy and Cell Biology, Ulm University, Ulm, Germany, 10 Deutsches Zentrum für Neurodegenerative Erkrankungen, Ulm, Germany, 11 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, Inserm UMR-S 1258, Université de Strasbourg, Illkirch-Graffenstaden, France CITATION Ferhat A-T, Verpy E, Biton A, Forget B, De Chaumont F, Mueller F, Le Sourd A-M, Coqueran S, Schmitt J, Rochefort C, Rondi-Reig L, Leboucher A, Boland A, Fin B, Deleuze J-F, Boeckers TM, Ey E and Bourgeron T (2023) Excessive self-grooming, gene dysregulation and imbalance between the striosome and matrix compartments in the striatum of Shank3 mutant mice. Front. Mol. Neurosci. 16:1139118. doi: 10.3389/fnmol.2023.1139118 Autism is characterized by atypical social communication and stereotyped behaviors. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are detected in 1–2% of patients with autism and intellectual disability, but the mechanisms underpinning the symptoms remain largely unknown. Here, we characterized the behavior of Shank3Δ11/Δ11 mice from 3 to 12 months of age. We observed decreased locomotor activity, increased stereotyped self- grooming and modification of socio-sexual interaction compared to wild-type littermates. We then used RNAseq on four brain regions of the same animals to identify differentially expressed genes (DEGs). DEGs were identified mainly in the striatum and were associated with synaptic transmission (e.g., Grm2, Dlgap1), G-protein-signaling pathways (e.g., Gnal, Prkcg1, and Camk2g), as well as excitation/inhibition balance (e.g., Gad2). HAL Id: hal-04241910 https://hal.science/hal-04241910v1 Submitted on 18 Oct 2023 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 TYPE Original Research PUBLISHED 16 March 2023 DOI 10.3389/fnmol.2023.1139118 OPEN ACCESS Downregulated and upregulated genes were enriched in the gene clusters of medium-sized spiny neurons expressing the dopamine 1 (D1-MSN) and the dopamine 2 receptor (D2-MSN), respectively. Several DEGs (Cnr1, Gnal, Gad2, and Drd4) were reported as striosome markers. By studying the distribution of the glutamate decarboxylase GAD65, encoded by Gad2, we showed that the striosome compartment of Shank3Δ11/Δ11 mice was enlarged and displayed much higher expression of GAD65 compared to wild-type mice. Altogether, these results indicate altered gene expression in the striatum of Shank3-deficient mice and strongly suggest, for the first time, that the excessive self-grooming of these mice is related to an imbalance in the striatal striosome and matrix compartments. Abbreviations: dd-PCR, droplet digital Polymerase Chain Reaction; DEGs, differentially expressed genes; DRD1/2, D1/D2 dopamine receptor; D1/2-MSN, medium-sized spiny neurons expressing the D1/D2 receptor; GABA, γ-aminobutyric acid; ORA, over-representation analysis; PPI, protein–protein interaction; PSD, postsynaptic density; q-PCR, real time quantitative PCR; RNAseq, RNA sequencing; SNP, single nucleotide polymorphism; SNpc, substantia nigra pars compacta; USV, ultrasonic vocalisations. COPYRIGHT © 202 COPYRIGHT © 2023 Ferhat, Verpy, Biton, Forget, De Chaumont, Mueller, Le Sourd, Coqueran, Schmitt, Rochefort, Rondi-Reig, Leboucher, Boland, Fin, Deleuze, Boekers, Ey and Bourgeron. 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. 01 Frontiers in Molecular Neuroscience frontiersin.org Ferhat et al. 10.3389/fnmol.2023.1139118 10.3389/fnmol.2023.1139118 KEYWORDS autism, Shank3, mouse model, stereotyped behavior, striatum compartmentation, GAD65, striosomes, matrix Introduction basal ganglia) and cellular (e.g., decreased striatal expression of GluN2B, a NMDA receptor subunit) levels (Peixoto et al., 2016; Reim et al., 2017; Wang et al., 2017; Bey et al., 2018; Schoen et al., 2019; Jin et al., 2021). The biological pathways underpinning the observed abnormalities remain however largely unknown. Autism is a neurodevelopmental condition characterized by atypical social communication and interactions associated with stereotyped behaviors and restricted interests. More than 200 genes have been robustly associated with autism pointing at biological pathways such as chromatin remodeling, protein translation and synaptic function (Leblond et al., 2021). Among these genes, SHANK3 (SH3 and multiple ankyrin repeat domain 3) codes for a scaffolding protein located at the postsynaptic density (PSD) of glutamatergic synapses where it interacts with other scaffolding proteins, cytoskeletal proteins, glutamate receptors and signaling molecules (Monteiro and Feng, 2017). In the present work, we first conducted a thorough longitudinal behavioral characterization of Shank3∆11/Δ11 mice, carrying a deletion of exon 11 of Shank3 (supplementary information of Schmeisser et al., 2012), at 3, 8, and 12 months of age, focusing on social interactions, locomotion and stereotyped behaviors. We then compared gene expression in four brain structures - cortex, hippocampus, cerebellum and striatum - in Shank3+/+ and Shank3Δ11/Δ11 male littermates characterized in the behavioral study. Compared to the other brain regions, the striatum appeared to be highly sensitive to the loss of SHANK3. We also identified a correlation between the level of self- grooming and an increased striatal expression of several genes, including Gad2 encoding GAD65, which catalyzes the transformation of glutamate into γ-aminobutyric acid (GABA) at the synapse. Finally, using immunofluorescence on striatum sections at 12 months, we found that, compared to wild-type littermates, the Shank3Δ11/Δ11 mice displayed an enlargement of the compartment formed by striosomes/patches which also markedly over-expressed GAD65. These alterations of the striosomal compartment were also found in the Shank3Δ4–22/Δ4-22 mouse (deletion from exon 4 to 22) with a complete lack of all SHANK3 isoforms. Altogether our results point toward a correlation between excessive self-grooming and signaling imbalance between the striosome and matrix compartments of the striatum in SHANK3-deficient mice. Heterozygous de novo mutations deleting SHANK3 on chromosome 22q13.3 or affecting its coding region are associated with Phelan-McDermid syndrome (Phelan and McDermid, 2012) and observed in 1–2% of patients with both autism and intellectual disability, making this gene a major cause of neurodevelopmental disorder (Durand et al., 2007). Introduction Remarkably, a relatively high proportion of patients (>60%) carrying SHANK3 mutations display regression (i.e., substantial loss of language and social skills) during adolescence and adulthood (Rubeis et al., 2018; Kohlenberg et al., 2020). Through the use of multiple intragenic promoters and alternative splicing, several SHANK3 protein isoforms are differentially expressed according to developmental stage or brain region (Wang et al., 2014). Numerous Shank3 mutant mice have been generated (for reviews see Ferhat et al., 2017; Monteiro and Feng, 2017). Most of them bear deletion of specific exons still allowing the expression of some isoforms (Wang et al., 2014; Drapeau et al., 2018). The behavioral deficits reported for Shank3 mutant mice depend on the strains and experimental conditions. However, the presence of stereotyped-like behaviors, such as excessive self-grooming, was reported in all Shank3 mutant mice and could be reminiscent of the stereotyped behaviors observed in patients with autism (Peça et al., 2011; Wang et al., 2011; Monteiro and Feng, 2017). Stereotyped behaviors are frequently associated with anomalies of the striatum (Kalueff et al., 2016), a region that coordinates multiple aspects of cognition, including action planning, decision-making, motivation, reinforcement, and reward perception as well as motor control. Several studies identified anomalies in the striatum of Shank3 mutant mice at the anatomical (e.g., larger volume), circuit (e.g., disruption of indirect pathway of Frontiers in Molecular Neuroscience KEYWORDS autism, Shank3, mouse model, stereotyped behavior, striatum compartmentation, GAD65, striosomes, matrix Animals Shank3∆11/∆11 mice were generated by Genoway (Lyon, FRANCE) using 129S1/SVImJ ES cells. The Shank3 mutation, deletion of exon 11, was then transferred onto a C57BL/6 J background with more than 15 backcrosses (supplementary information of Schmeisser et al., 2012). In this model, some protein isoforms are still expressed (supplementary information of Schmeisser et al., 2012). Mice were weaned at 22 ± 1 days of age and housed in same-sex mixed-genotype groups of three to five animals unless otherwise specified. They were housed under constant temperature (22 ± 1°C) with a 12:12 light/dark cycle (light on from 07:00 AM to 07:00 PM) and food and water were provided ad libitum. The experimenters were blind to the genotype of the tested animals for data collection and analyzes. We bred Shank3∆11/+ males and females to obtain Shank3+/+, Shank3+/∆11 and Shank3∆11/∆11 littermates. For behavioral tests, we used three cohorts (including both males and females) that we analyzed separately. Cohort 1 involved 44 males (13 Shank3+/+, 19 Shank3+/∆11, and 12 Shank3∆11/∆11) and 38 females (10 Shank3+/+, 16 Shank3+/∆11, and 12 02 Frontiers in Molecular Neuroscience Frontiers in Molecular Neuroscience frontiersin.org Ferhat et al. 10.3389/fnmol.2023.1139118 Observation of stereotyped behavior Mice were individually placed in a new test cage (Plexiglas, 50 × 25 × 30 cm; 100 lux; clean sawdust bedding) in a soundproof chamber. After 10 min habituation, we recorded their behavior for 10 min (camera Logitech C920). We manually scored the time spent self-grooming and digging (The Observer, Noldus Information Technology). When scoring self-grooming, we did not take into account the scratching behavior. The total duration and the average event duration of each behavioral category were calculated. i Shank3∆4–22/∆4–22 mice (Drapeau et al., 2018), with a C57BL/6 N background, were obtained from The Jackson Laboratory Repository. Frontiers in Molecular Neuroscience Occupant/New-comer social test with ultrasonic vocalization recordingh At weaning, we measured weight and observed hind limb clasping as well as the physical aspect (fur, injury, or malformation) of the mice. At 3 and 12 months of age in Cohort 1, we again measured weight and noted the physical aspect of all mice. The tested mouse was isolated socially for 3 days (females) or 3 weeks (males) to increase motivation for affiliative social contacts (Ferhat et al., 2016). From this test on at 3 months of age, males remained singly housed. After 20 min of habituation for the tested mouse (the occupant) to the test cage (Plexiglas, 50 × 25 × 30 cm; 100 lux; clean sawdust bedding) in a soundproof chamber, an unfamiliar age- and sex-matched C57BL/6 J mouse (new-comer, NC; Charles River Laboratories, France) was introduced. The two mice were allowed to freely interact for 4 min. Social interactions (video camera Logitech C920, 30 fps) were semi-automatically analyzed using the 2D tracking module Mice Profiler from the ICY platform (de Chaumont et al., 2012a,b) (Institut Pasteur, Paris, France). We quantified, for both the occupant and the new-comer, the time spent in contact, the types of contact (oral-oral contact, oro-genital contact), the “approach then escape” sequences, the follow behavior, and the time spent in the vision field of the other one (Ferhat et al., 2015). At the same time, ultrasonic vocalizations (USV) were recorded (Condenser ultrasound microphone Polaroid/CMPA, UltraSoundGate 416–200, Avisoft Bioacoustics, Glienicke, Germany; sampling frequency: 300 kHz; FFT-length: 1024 points; 16-bit format). Vocalization files were analyzed automatically using the vocalization analysis plugin from LMT USV Toolbox (de Chaumont et al., 2021). Elevated plus-maze anxiety-like testh The animal (Cohort 2) was allowed to freely explore the setup for 10 min. The elevated plus-maze (four arms of 7 cm by 30 cm, 50 cm above the floor) consisted of two open arms (no walls) and two closed arms (with walls), all connected by a neutral zone in the center (100 lux). We measured the time spent in the open and closed arms, as well as the number of transitions using Ethovision (Noldus Information Technologies, Wageningen, Netherlands). We interpret that the time spent in the closed arms is positively correlated with the anxiety level of the mice. Locomotion and exploratory test in the open field Shank3∆11/∆11). Mice from Cohort 1 were evaluated at Institut Pasteur at 3 months of age -the standard age for behavioral testing- for dark– light, Y-maze, open field tests, self-grooming observation, 3-chambered test, free social interactions, as well as at 8 and 12 months of age for open field test, self-grooming and free social interaction. After the tests at 3 months of age, males were housed individually, while females remained group-housed. This difference of housing conditions between males and females was related to the increased aggressivity of males (C57BL/6 J background) after sexual experience with females in socio-sexual interactions at 3 months of age. Cohort 2 involved 42 males (15 Shank3+/+, 14 Shank3+/∆11, and 13 Shank3∆11/∆11) and 22 females (4 Shank3+/+, 11 Shank3+/∆11, and 7 Shank3∆11/∆11). Male and female mice from Cohort 2 were evaluated at Institut Pasteur at 3 months of age for self-grooming observation, open field, elevated plus maze, and dark–light anxiety tests. Cohort 3 involved 14 Shank3+/+ and 7 Shank3∆11/∆11 males that were bred at Institut Pasteur and sent at 2–2.5 months to Institut Biologie Paris Seine (IBPS). They were subjected to the open field, water maze and starmaze tests. The mouse (Cohorts 1 and 2) was allowed to freely explore for 30 min a round open field arena of 1 m in diameter (100 lux in the center of the arena). We recorded the total distance traveled (Ethovision, Noldus Information Technology, Wageningen, Netherlands). Spontaneous locomotor activity of Cohort 3 was quantified in an arena made of gray perspex (45 × 45 cm) surrounded by red Plexiglas walls (30 cm height). Mice were first positioned in the center of the arena and were allowed to freely explore for 10 min. Data acquisition was performed at a frequency of 25 Hz using the SMART® video recording system and tracking software and traveled distance was computed using NAT (Navigation Analysis Tool), a custom Matlab-based software (Jarlier et al., 2013). Y-mazeh The animal (Cohort 1) was allowed to freely explore a y-maze during 5 min. The number of entrances as well as the sequence of entrances into each arm were manually noted by an experimented scientist. The sequence of correct alternations, i.e., when the subject does not go back to the previous arm visited, are used to determine the working memory. Dark/light anxiety-like testht The mouse was left to freely explore a test cage separated into two compartments connected by a small door (5 × 5 cm; dark side: 3 lux; light side: 1300 lux obtained through a desk lamp directly above the compartment) for 5 min. The latency to enter the dark compartment and the time spent in each compartment were measured (Ethovision, Noldus Information Technologies, Wageningen, Netherlands). More anxious mice are expected to spend shorter time in the light compartment and do less transitions between the compartments. Male behavior in presence of an estrous femaleh The tested male was placed in the presence of a female for 48 h. Then, the male was isolated again for 1 day. During the test, the male was placed in the test cage (Plexiglas, 50 × 25 × 30 cm; 100 lux; with clean sawdust bedding) during 10 min for habituation (Ferhat et al., 2015). After this period, an unknown C57BL/6 J female in estrus (tested through vaginal smears in the morning) was introduced into 03 frontiersin.org Ferhat et al. 10.3389/fnmol.2023.1139118 pathway.” Mice were tracked by using the Smart Software (Bioseb, Vitrolles, France). pathway.” Mice were tracked by using the Smart Software (Bioseb, Vitrolles, France). the test cage for 4 min. Both mice were allowed to freely interact. Social interactions as well as USV were recorded, as described above. Behavioral statistics A Plexiglas cage was divided in three connected compartments (side compartments: 150 lux; central compartment: 140 lux) as previously described (Nadler et al., 2004). Both side compartments contained an empty wire cup. First, the tested mouse was allowed to freely explore the setting, with doors open for 10 min (phase 1) for habituation. Then, the mouse was restricted in the central compartment, while an unfamiliar C57BL/6 J mouse of the same sex (stranger 1) was placed under one of the cups (sides alternated between each mouse). The tested mouse was then allowed to explore the apparatus for 10 min (phase 2). In all phases, the time spent in each compartment and the number of transitions between compartments were automatically recorded. The time spent in contact with the cup containing the mouse (stranger 1) and the time spent in contact with the empty cup were manually measured in phase 2 to evaluate social interest. All group data are represented as mean ± standard error of the mean (s.e.m.), as well as the individual points. All statistics were performed with R software (R Core Team (2020), R Foundation for Statistical Computing). In all behavioral tests, Shank3+/Δ11 mice were not significantly different from Shank3+/+ unless otherwise specified (see Supplementary Table S1). Given the limited sample size and the non-normality of the data, comparisons between genotypes were performed using non-parametric Mann–Whitney U-tests. We used the non-parametric Friedman test to examine the effect of age within each genotype. Between age points, post-hoc tests were performed using paired Wilcoxon signed-rank tests. If required, a Bonferroni correction for multiple testing was performed. Differences between groups were considered significant when p < 0.05. Tissue collection Mice were trained in a circular water tank (150 cm in diameter, 40 cm high) to swim toward a visible platform (12 cm in diameter, 1 cm under the water surface) marked with an object (10 cm above the water surface), as previously described (Burguiere et al., 2005; Lefort et al., 2019). The platform was randomly placed at different locations across trials and the pool was surrounded by blue curtains to occlude extramaze cues. Training consisted in one training session per day, four trials per session, during 2 days. The starting position (North, East, West, or South) was randomly selected with each quadrant sampled once a day. At the beginning of each trial, the mouse was released at the starting point and made facing the inner wall. Then, it was given a maximum of 90 s to locate and climb onto the escape platform. If the mouse was unable to find the platform within the 90 s period, it was guided to the platform by the experimenter. In either case, the mouse was allowed to remain on the platform for 30 s. Data acquisition was performed at a frequency of 25 Hz using the SMART® video recording system and tracking software. Mice were killed during the light phase, specifically between 9:00 and 11:00 AM, by CO2 intoxication. The brain was removed and macro-dissected on ice (4°C) into HBSS solution by an experienced practitioner. After separation of the hemispheres, six brain structures were extracted: whole cortex, hippocampus, whole striatum, cerebellum, diencephalon, and brainstem. Samples were snap frozen in liquid nitrogen and stored at −80°C. Total RNA extraction and sequencing Total RNA was extracted from four over six brain structures (whole cortex, hippocampus, whole striatum, cerebellum) using the miRNeasyPlus Micro Kit (Qiagen), following the manufacturer’s instructions, including DNAse digestion. After first quality assessment using the Nanodrop spectrophotometer ND-1000 (Thermoscientific), the samples were analyzed by the CNRGH (Centre National de Recherche en Génomique Humaine, CEA, Evry, France). RNA integrity was assessed using the Bioanalyzer RNA 6000 Nano assay and 2,100 Bioanalyzer (Agilent Technologies). Then, an oriented mRNA sequencing was performed on samples with a RNA Integrity Number (RIN) larger than eight. Eight Shank3∆11/∆11 and 7 Shank3+/+ 12-month old mice were used for the differential expression analysis. For each of these mice, we studied four brain regions (cerebellum, cortex, hippocampus, and striatum), except for one in each group for which only three of the four brain regions were available. The description of the samples is available in Supplementary Table S5. The data were obtained from two batches of sequencing performed at 8 months of interval. For the first batch, RNA extractions were performed on the same day while for the second sequencing batch, RNAs were extracted on two different days. We refer to this variable containing three levels as the batch variable. Each sequencing run included two flow cells. The RNA-seq datasets presented in the study are deposited in the Sequence Read Archive (SRA) repository, accession number PRJNA934367. Starmazeh The starmaze consisted of five alleys radiating from the vertices of a central pentagonal ring. All the alleys were filled with water, and the water was made opaque with an inert nontoxic product (AccuScan OP 301, Brenntag). The maze was surrounded by a square black curtain with 2D and 3D patterns affixed to provide configurations of spatial cues. To avoid the possible use of a guidance strategy (i.e., animals could rely on the use of a single distal cue), cue was given in duplicate. White noise was used to cover all other sounds that the mice could have used to orientate themselves. To solve the task, animals had to swim to a trapezoidal platform (10 and 24 cm for top and bottom parallel sides, 11 cm of lateral sides) hidden 1 cm below the water surface and located 10 cm from the end of one alley. Departure and arrival points were always the same. All animals ran one session of five trials per day over 2 days using a 40-min inter-trial interval. If an animal did not locate the escape platform within 90 s, the experimenter placed the animal onto the platform for 30 s. During the protocol, one central alley and two peripheral alleys were blocked, forcing the mice to use the “left Frontiers in Molecular Neuroscience frontiersin.org Calling variants from the RNAseq dataf To see whether differences between the C57BL/6 J and the 129S1/ SvImj genomes in the region around Shank3 could explain the larger number of DEGs detected in the vicinity of Shank3, we used the RNAseq data to identify single nucleotide polymorphisms (SNPs) in each mouse. We followed the Gatk Best Practices (Van der Auwera et al., 2013) workflow for SNP and indel calling on RNAseq data2 which includes the following steps: (i) map to the reference genome with STAR in multi-sample 2-pass mode to get the most sensitive novel junction discovery; (ii) add read groups, sorting, marking duplicates, and create index, using Picard’s tools3; (iii) split reads into exon segments (removing Ns but maintaining grouping information) and hard clipping sequences overhanging into the intronic regions, using the SplitNCigarReads Gatk tool; (iv) realign indels and recalibrate Base quality; (v) call variant with HaplotypeCaller, and finally filter the variants with VariantFiltration. The last step was adapted to our project where several samples coming from the same mouse were available. HaplotypeCaller (with parameter -ERC BP_ RESOLUTION) was called for each mouse individually using as input the processed BAM files coming from different brain tissues of the same mouse. Mice with the same Shank3 status were then genotyped together by inputting the GVCF files to the Gatk tool GenotypeGVCFs. Shank3∆11/∆11- and Shank3+/+-specific VCF files were finally combined with the Gatk tool CombineVariants, which provides allele frequency and specificity of the variants to Shank3∆11/∆11 or Shank3+/+ mice populations. The resulting VCF file was filtered using the Gatk tool VariantFiltration using the parameters recommended in the Gatk workflow. Gene-set over-representation analyses were performed using Fisher’s hypergeometric tests. Plots were made using the R packages ggplot2 (Wickham, 2009), upsetR (Conway et al., 2017), and ggbio (Yin et al., 2012). Differential expression analysish The 18,194 genes with at least one count-per-million (CPM) in two samples were selected. The samples flagged by the QC Analyzer were filtered out. Multidimensional scaling plots showed separation of the samples according to brain regions, batches, and flow cells (Supplementary Figure S13). Differential gene expression analysis was performed with limma-voom v3.34.8 (Law et al., 2014), and the version of voom using sample-quality weights (Liu et al., 2015) (function voomWithQualityWeights) in order to take into account Protein-protein interaction network analysis (PPI) Using the BioGRID database for Mus musculus4, we analyzed, for the 4 brain structures independently, the protein–protein interaction (PPI) network of the DEGs. The majority of DEGs for each structure was not annotated, i.e., not associated with a known pathway in BioGRID database (fraction of annotated DEGs: striatum: 71/186; hippocampus: 7/33, cerebellum: 4/24, cortex: 3/22). Cystoscape v3.6.1 was used to visualize the PPI network to find out key genes. A randomized protein interaction network was created using all DEGs to determine the probability of finding a network. 4 https://thebiogrid.org/ Mapping and reference genomeh The RNAseq reads were mapped onto the genome with the STAR aligner v2.5.3a (Dobin et al., 2013) in 2-pass mode to a masked version 04 frontiersin.org Ferhat et al. 10.3389/fnmol.2023.1139118 10.3389/fnmol.2023.1139118 the sample heterogeneity observed within and across brain regions (Supplementary Figure S14). The Trimmed Mean of M-values (TMM) method was used to calculate normalization factors between samples. Three factors were included in the design matrix: the batch, the flow cell, and the brain tissue and Shank3 status combined into one factor of eight levels. Since we were making comparisons both within and between mice, we treated the mouse as a random effect to adjust for baseline differences between subjects. To do so, the mouse was used as a blocking factor and the correlation between measurements made on the same mouse was computed using the function duplicateCorrelation and was input into the linear model fit, as suggested in the section “Multi-level Experiments” of the limma user guide. For each contrast of interest, the linear model was fitted for each gene using the function lmFit, and empirical Bayes smoothing was applied to the standard errors using the function eBayes with robust mode set to TRUE. of the Mus Musculus GRCm38 genome. During a first round of the differential gene expression analysis, we observed enrichment of the DEGs in genes located on the same arm of chromosome 15 as Shank3. Differences in genetic backgrounds around the Shank3+/+ (C57BL/6 J) and the Shank3∆11/∆11 alleles (129S1/SvImJ, from the ES cells used to generate the Shank3∆11 mutation followed by 15 backcrosses on C57BL/6 J) could affect the mapping by impacting the mapped reads on the remaining region of 129S1/SvImJ around Shank3. To avoid this bias, we masked the GRCm38 genome for variants of the 129S1/SvImJ mouse strain before mapping the sequencing reads. Variants were extracted from the VCF file provided by The Mouse Genome Project (Yalcin et al., 2012)1 and masked in the reference genome using the SNPsplit software (Krueger and Andrews, 2016). Gene set analysis Gene set over-representation analyses (ORA) were performed with the egsea.ora function available in the Bioconductor R package EGSEA (Ensemble of Gene Set Enrichment Analyses) v1.6.1 (Alhamdoosh et al., 2017) without considering the genes around the Shank3 gene. All collections of the databases MSigDB (Subramanian et al., 2005), GeneSetDB (Araki et al., 2012), and KEGG (Kanehisa et al., 2012) were used. Frontiers in Molecular Neuroscience 1 https://ftp.ebi.ac.uk/pub/databases/mousegenomes/REL-1505-SNPs_ Indels/mgp.v5.merged.snps_all.dbSNP142.vcf.gz 2 https://software.broadinstitute.org/gatk/guid#e/article?id=3891 3 http://broadinstitute.github.io/picard Quantitative RT-PCR Acquisition parameters were adjusted for each brain hemi- section in order to have no saturating signal for GAD65 in the striatal region of interest. Images of the dorsal striatum were reconstructed by stitching multiple maximum-intensity projected z-stacks. by immersion in 2-methyl butane (Sigma-Aldrich) chilled in liquid nitrogen. Samples were kept at −80°C until use. Forty μm coronal sections, obtained using the CM3050S cryostat (Leica Biosystems), were rapidly transferred into PBS in 12-well-culture plates. The samples for GAD65 immunostaining were collected every five sections and grouped together in a separate single well and stored in PBS at 4°C until use. The free-floating sections were rinsed three times in PBS (pH 7.4) and then incubated in NH4Cl (Sigma-Aldrich) 50 mM in PBS for 15 min. After three PBS washes (5 min each), sections were incubated for 1 h at room temperature in PBS containing 1% bovine serum albumin (Applichem) and 0.3% Triton X-100 (Sigma-Aldrich) (PBS/BSA/TX) before incubation for ≈ 20 h at room temperature with the following primary antibodies diluted in PBS/BSA/TX: rabbit polyclonal anti-GAD65 (Invitrogen PA5-77983, 1/200), mouse monoclonal anti-GAD65 (Millipore MAB351, 1/500), guinea pig polyclonal anti MOR (Millipore AB5509, 1/100), rabbit polyclonal anti-VGLUT1 (Synaptic Systems 135303, 1/1000), and mouse monoclonal anti-SHANK3 (Santa Cruz Biotechnology sc-377088, 1/1000). This latter antibody is directed against the C-terminal region of most SHANK3 isoforms. After three PBS washes (10 min each), sections were incubated in secondary antibodies (Alexa fluor 555™-conjugated goat anti-rabbit IgG, Alexa fluor488™-conjugated goat anti-mouse IgG and Alexa fluor488™-conjugated goat anti- guinea-pig IgG (all from Invitrogen)) diluted 1/500 in PBS/BSA/TX for 1 h at room temperature, washed in PBS (3 washes of 10 min) and mounted in FluorSave™ Reagent (Calbiochem). To stain nuclei, a 10 min incubation step in DAPI (1 μg/ml in H20, Thermoscientific) followed by a PBS wash was added before mounting. Images were acquired using a LSM 700 confocal microscope (Carl Zeiss) equipped with a Plan-Apochromat 10X/0.45 M27 objective (Carl Zeiss). For each animal, 5 to 7 coronal sections spanning the anterior–posterior axis from Bregma 1.10 mm to Bregma 0.14 mm were imaged bilaterally. Acquisition parameters were adjusted for each brain hemi- section in order to have no saturating signal for GAD65 in the striatal region of interest. Images of the dorsal striatum were reconstructed by stitching multiple maximum-intensity projected z-stacks. Quantitative analysis of GAD65 immunoreactivity To deal with sample to sample variations in labeling efficiency inherent to immunofluorescent labeling methods we determined, for each image, the ratio of the labeling intensities of the striosome and matrix compartments to the adjacent cortex, in which Gad2 is not differentially expressed in Shank3∆11/∆11 and Shank3+/+ mice, according to the transcriptome analysis. For each image, the regions of interest (dorsal striatum and adjacent cortex) were delimited using the Icy software (Institut Pasteur, Paris). In case of edge effect along the lateral ventricle, the concerned edge was excluded from the region of interest. We developed a method to automatically detect three areas in the striatum: the unlabeled myelinated fibers, the matrix (lower expression of GAD65), and the patches/striosomes (higher expression of GAD65). To deal with possible acquisition artifacts, we first applied a mean filter on the image by using a box blur (size 7 × 7). We then pre-computed two maps corresponding to the local mean and standard deviation of the image. For each point, we computed those values considering a window of 1000 × 1000 pixels centered on the Quantitative RT-PCR For dd-PCR, total RNA was extracted as described above and the cDNA library was generated using the iScript advanced cDNA kit (Bio-Rad). The dd-PCR was performed with the dd-PCR supermix for probes (no dUTP, Bio-Rad) and probes labeled with the FAM (for the genes of interest) and HEX (for the Gapdh housekeeping gene) fluorophores using the QX100 droplet digital PCR system (Bio-Rad). Results were analyzed using the QuantaSoft Software. tt For q-PCR, total RNA (200 ng) was reverse-transcribed with oligo-dT primers using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher). Quantitative PCR was performed in triplicate with a 7,500 real time PCR system (Applied Biosystems) using LightCycler SYBR Green I Master Mix (Roche) and specific pairs of primers. Individual data were normalized using a combination of two housekeeping genes (Ppia and Rpl13a). The results are reported as fold changes. All statistical analyses were performed using R software (R Core Team (2020), R Foundation for Statistical Computing). The comparison between genotypes was performed using Mann–Whitney U-tests. Single-molecule fluorescent in situ hybridization (smFISH) and image analysis Three month-old mice, deeply anesthetized by an intraperitoneal injection of a mixture of Ketamine (Imalgen®, 200 mg/kg, Merial) and Xylazine (Rompun®, 8 mg/kg, Bayer), were transcardially perfused with 10 ml of PBS, followed by 50 ml 4% paraformaldehyde (PFA) in PBS (Santa Cruz Biotechnology) at 4°C. Each brain was snap frozen in liquid nitrogen and then stored at −80°C. Cryosections (16 μm thick), obtained using the CM300 cryostat (Leica Biosystem) were collected on Superfrost Plus microscope slides (Thermo Fisher Scientific) and stored at −20°C before hybridization. The brain sections (from bregma 1.7 to −0.58) were treated as described by Tsanov et al. (2016) using 48 probes along Drd1 RNA and 40 probes along Drd2 RNA (see Supplementary Table S6). Images were acquired in the dorso-medial striatum and in the nucleus accumbens core regions using an Axio observer Z1 inverted microscope (Carl Zeiss) equipped with a Plan- Apochromat 20X/0.8 M27 objective (Carl Zeiss). Images were manually analyzed using imageJ software (NIH). The statistical analyses were performed using R software (R Core Team (2020), R Foundation for Statistical Computing). The comparison between genotypes was performed using Mann–Whitney U-tests. Frontiers in Molecular Neuroscience Quantitative RT-PCR We selected several genes of interest by cross checking the DEGs, ORA (gene ontology – GO –, Kyoto encyclopaedia of genes and genomes – KEGG– and Pathway), and data from the literature. To validate these genes, we used q-RT-PCR, either with the droplet digital Polymerase Chain Reaction (dd-PCR) or with the real time quantitative PCR (q-PCR) technology. Frontiers in Molecular Neuroscience 1 https://ftp.ebi.ac.uk/pub/databases/mousegenomes/REL-1505-SNPs_ Indels/mgp.v5.merged.snps_all.dbSNP142.vcf.gz 2 https://software.broadinstitute.org/gatk/guid#e/article?id=3891 3 http://broadinstitute.github.io/picard 05 frontiersin.org Ferhat et al. Ferhat et al. 10.3389/fnmol.2023.1139118 by immersion in 2-methyl butane (Sigma-Aldrich) chilled in liquid nitrogen. Samples were kept at −80°C until use. Forty μm coronal sections, obtained using the CM3050S cryostat (Leica Biosystems), were rapidly transferred into PBS in 12-well-culture plates. The samples for GAD65 immunostaining were collected every five sections and grouped together in a separate single well and stored in PBS at 4°C until use. The free-floating sections were rinsed three times in PBS (pH 7.4) and then incubated in NH4Cl (Sigma-Aldrich) 50 mM in PBS for 15 min. After three PBS washes (5 min each), sections were incubated for 1 h at room temperature in PBS containing 1% bovine serum albumin (Applichem) and 0.3% Triton X-100 (Sigma-Aldrich) (PBS/BSA/TX) before incubation for ≈ 20 h at room temperature with the following primary antibodies diluted in PBS/BSA/TX: rabbit polyclonal anti-GAD65 (Invitrogen PA5-77983, 1/200), mouse monoclonal anti-GAD65 (Millipore MAB351, 1/500), guinea pig polyclonal anti MOR (Millipore AB5509, 1/100), rabbit polyclonal anti-VGLUT1 (Synaptic Systems 135303, 1/1000), and mouse monoclonal anti-SHANK3 (Santa Cruz Biotechnology sc-377088, 1/1000). This latter antibody is directed against the C-terminal region of most SHANK3 isoforms. After three PBS washes (10 min each), sections were incubated in secondary antibodies (Alexa fluor 555™-conjugated goat anti-rabbit IgG, Alexa fluor488™-conjugated goat anti-mouse IgG and Alexa fluor488™-conjugated goat anti- guinea-pig IgG (all from Invitrogen)) diluted 1/500 in PBS/BSA/TX for 1 h at room temperature, washed in PBS (3 washes of 10 min) and mounted in FluorSave™ Reagent (Calbiochem). To stain nuclei, a 10 min incubation step in DAPI (1 μg/ml in H20, Thermoscientific) followed by a PBS wash was added before mounting. Images were acquired using a LSM 700 confocal microscope (Carl Zeiss) equipped with a Plan-Apochromat 10X/0.45 M27 objective (Carl Zeiss). For each animal, 5 to 7 coronal sections spanning the anterior–posterior axis from Bregma 1.10 mm to Bregma 0.14 mm were imaged bilaterally. frontiersin.org Hypoactivity and atypical motor abilities in Shank3Δ11/Δ11 mice Impairment in locomotor activity is often observed in Shank3 mutant mice (Ferhat et al., 2017). In the open field at 3 months of age, Shank3Δ11/Δ11 males traveled significantly shorter distances in comparison with Shank3+/+ littermates (Cohort 1: W = 130, p = 0.007; Figure 1A, left panel). This observation was not significant for Shank3Δ11/Δ11 females of Cohort 1 but was confirmed for Shank3Δ11/Δ11 males of Cohort 2 (Supplementary Figure S2A) and Cohort 3 (Supplementary Figure S2B) at 3 months of age. This reduction of distance traveled in Shank3Δ11/Δ11 males was not present at older age; however, Shank3+/+ and Shank3+/Δ11 mice reduced their activity with increasing age (Figure 1A). A similar trend was present but did not reach significance levels in females (Cohort 1: W = 92, p = 0.07; Figure 1A). Remarkably, in stressful swimming conditions, Results pp y g In contrast, we observed that Shank3Δ11/Δ11 males spent significantly more time in contact with an estrous female compared to Shank3+/+ males at 3 months of age (W = 32, p = 0.034; Figure 1C), especially in oro-oral contact (W = 22, p = 0.005; Supplementary Figures S3, S4G). We also observed a reduction of time keeping the female in the visual field for Shank3Δ11/Δ11 males compared to Shank3+/+ males (W = 133, p = 0.006; Supplementary Figure S4G). These altered behaviors were not accompanied with atypical vocal behavior (Supplementary Figure S4B). At later age, the increase of overall social interaction disappeared during interaction between Shank3Δ11/Δ11 males and estrous females. However, at 8 months of age, the duration of oro-oral contact in Shank3Δ11/Δ11 males with an estrous female was still significantly increased (W = 29, p = 0.025), but these differences were no longer significant at 12 months of age (Supplementary Figures S4H,I). Nevertheless, at this age, we detected an increase of the “approach then escape” behavioral sequence displayed by Shank3Δ11/Δ11 males toward a C57BL/6 J female in comparison with Shank3+/+ males (W = 15.5, p = 0.005, Supplementary Figure S4I). At 3 months of age, Shank3Δ11/Δ11 mice, from Cohort 1 and 2, displayed typical body weight, anxiety-like behavior levels and working memory, compared to Shank3+/+ littermates (Supplementary Figure S1). Considering the frequent regression of patients carrying SHANK3 mutations, we tested whether a phenotypic deterioration occurred in Shank3Δ11/Δ11 mice. For that purpose, we tested the main impairments observed in the Shank3Δ11 mouse model over time, at 3, 8, and 12 months of age: exploratory activity, social communication and stereotyped behaviors in mice. Confocal fluorescence microscopy on brain sections Immunofluorescence analyses on striatum sections were performed on 1 year old (52–70 weeks) Shank3∆11 and 20–28 weeks old Shank3∆4–22 male mice. Animals, deeply anesthetized as described above, were transcardially perfused with 20 ml of PBS, followed by 50 ml 4% paraformaldehyde (PFA) in PBS (Santa Cruz Biotechnology). Brains were removed and fixed overnight at 4°C in 4% PFA, rapidly washed in PBS, and then immersed in 15% sucrose in PBS for overnight incubation at 4°C. The 15% sucrose solution was then replaced by a 30% sucrose solution for a second overnight incubation at 4°C. Brains were then transferred in Shandon Cryomatrix™ Frozen Embedding Medium (Thermo Scientific™) in cryomolds and frozen 06 frontiersin.org Ferhat et al. 10.3389/fnmol.2023.1139118 10.3389/fnmol.2023.1139118 point, with a computation step of 10px. Then, for each pixel of the smoothed image, we labeled as fibers all points of intensity value below localMean-localStd. The final fiber mask was eroded morphologically (2px) to remove spurious segmentations and connectivities. To compute the patches, we performed the same computation, but the pixel intensity had to be over localMean+localStd and below localMean + localStd5 to remove super-bright spots corresponding to fluorescent dust. We also eroded the result by a factor of 2 pixels. Fiber and Patch masks were then processed with a 2D 8-way connected component process to get all individual fibers and patches. We then cleaned up the detection by removing patches of a surface inferior to 100 pixels, and fibers of a surface inferior to 50 pixels. To determine the intensity ratio of matrix versus cortex, we created a matrix segmentation corresponding to the striatal region of interest subtracted by the patches and the fibers detected. The meanMatrix was computed over this surface while the meanPatch was the mean intensity of all pixels contained in the patch mask. For each image, we computed the ratio meanPatch over cortex and meanMatrix over cortex. We also computed the surface of the patch mask relative to the surface of the (patch mask + matrix mask). To determine the effect of genotype on the data extracted by the image analysis, we applied a linear mixed model (LMM) using the method “restricted maximum likelihood (REML)” from the Python library “statsmodels. formula.api.mixedlm.” In all presented results, the LMM has converged and a value of p indicating if the datasets from the Shank3- mutated and Shank3+/+ mice are significantly different is provided. Confocal fluorescence microscopy on brain sections Shank3Δ11/Δ11 mice, from Cohort 3, swam at a significantly higher speed compared to wild-type mice in the water maze with visible platform (Cohort 3: p < 0.01 over all training) and in the star maze (p < 0.01), without any significant difference to find the direct path in the star maze (Supplementary Figures S2E–G). Shank3Δ11/Δ11 males also spent less time digging in the bedding in comparison with Shank3+/+ mice at 3 months (Cohort 1: W = 123, p = 0.027; Figure 1B; Cohort 2: W = 173, p < 0.001; Supplementary Figure S2C). Genotype-related differences in digging behavior persisted with increasing age (8 months: W = 140, p < 0.01; 12 months; W = 107; p = 0.01; Figure 1B). Atypical socio-sexual behavior in Shank3Δ11/Δ11 male mice at early stage Impairments in social interactions and communication, some of the core symptoms of autism, were not always observed in the different Shank3 mouse models (Ferhat et al., 2017). We analyzed the preference for a conspecific during three-chambered test and the time of interaction and types of contact during free dyadic interaction (Supplementary Figure S3). During the three-chambered test, mice of all genotypes and both sexes displayed a typical preference for a same-sex conspecific (Supplementary Figure S4). Furthermore, no significant difference was observed during free dyadic social interaction, at different ages for Cohort 1, in the latency for the first contact (not shown), in the emission of ultrasonic vocalizations (USVs) (Supplementary Figure S4B), and in the total time spent in contact and in the different social behaviors with same-sex conspecific for both sexes (Supplementary Figures S3, S4C–F). Frontiers in Molecular Neuroscience Differential expression of genes related to synaptic function, signaling and cytoskeleton dynamics in the striatum of Shank3Δ11/Δ11 mice (Kalueff et al., 2016). Here, we investigated whether this behavior was exacerbated in mutant mice compared to WT littermates in an unfamiliar test environment. At 3 months of age, Shank3Δ11/Δ11 male mice already displayed a significant increase in the time spent self- grooming in comparison with Shank3+/+ males for Cohort 1 (W = 21, p = 0.002) and 2 (W = 43, p = 0.022). Similar trends did not reach significance in female mice (Figures 1D–F and Supplementary Figure S2D). Furthermore, a subsample of Shank3Δ11/Δ11 males (5 individuals over 12) and females (6 individuals over 12) presented hair removal that evolved into self-injuries. (Kalueff et al., 2016). Here, we investigated whether this behavior was exacerbated in mutant mice compared to WT littermates in an unfamiliar test environment. At 3 months of age, Shank3Δ11/Δ11 male mice already displayed a significant increase in the time spent self- grooming in comparison with Shank3+/+ males for Cohort 1 (W = 21, p = 0.002) and 2 (W = 43, p = 0.022). Similar trends did not reach significance in female mice (Figures 1D–F and Supplementary Figure S2D). Furthermore, a subsample of Shank3Δ11/Δ11 males (5 individuals over 12) and females (6 individuals over 12) presented hair removal that evolved into self-injuries. To investigate the impact of the loss of the major SHANK3 isoforms on the transcriptome, we performed RNAseq on four brain structures (whole cortex, striatum, hippocampus, and cerebellum) of seven Shank3+/+ and eight Shank3Δ11/Δ11 male mice after their behavioral characterization at 12 months of age. We observed a reduction of the transcript levels of the major isoforms of Shank3 in Shank3Δ11/Δ11 mice (Supplementary Figure S5). We also observed an enrichment of DEGs in the vicinity of Shank3 locus on chromosome 15 (before: 10.4 Mb; after: 7.5 Mb) (Supplementary Table S2). This enrichment is most likely due to the residual genomic region from the 129S1/SvImJ ES cells used to generate the Shank3Δ11/Δ11 mice (Supplementary Figure S6; supplementary information of Schmeisser et al., 2012). We therefore excluded these genes from the analyzes. As expected, Shank3 expression levels in all four brain structures were significantly reduced in Shank3Δ11/Δ11 mice in comparison with Shank3+/+ mice (FDR < 0.001; Supplementary Figure S7). Notably, the Shank3 sequence reads still present in the Shank3Δ11/Δ11 mice were aligned to exons downstream of exon 11 (Supplementary Table S2). Shank3 mutant mice display excessive self-grooming behavior worsening with age Self-grooming is a spontaneous and natural behavior displayed by mice for hygienic purposes or in reaction to stressful conditions 07 frontiersin.org 10.3389/fnmol.2023.1139118 Ferhat et al. C A B C D E F FIGURE 1 Hypoactivity, reduced exploration and increased stereotyped behaviors in Shank3Δ11/Δ11 mice at 3, 8, and 12 months of age. (A) Total distance traveled during 30-min free exploration of an open field in wild-type (green), heterozygous (blue) and Shank3Δ11/Δ11 (orange) for males (left panel) and females (right panel). (B) Total time spent digging in fresh bedding during 10 min observation in a new test cage, after 10 min habituation. (C) Total time spent in contact during male/female and female/female social interaction. (D) Total time spent self-grooming during 10 min observation in a new test cage, after 10 min habituation. (E) Z-score for the time spent self-grooming. (F) Proportion and number of individuals displaying different levels of severity in self-grooming. “Dead” corresponds to animals that had to be euthanized due to severe self-inflicted injuries. (A–D) Black points represent mean with standard error of the mean (s.e.m). Mann–Whitney U test with Bonferroni correction for multiple testing (two tests): Corrected value of p < 0.05; ** corrected value of p < 0.01. A B C B A C D E F D E F E F F FIGURE 1 Hypoactivity, reduced exploration and increased stereotyped behaviors in Shank3Δ11/Δ11 mice at 3, 8, and 12 months of age. (A) Total distance traveled during 30-min free exploration of an open field in wild-type (green), heterozygous (blue) and Shank3Δ11/Δ11 (orange) for males (left panel) and females (right panel). (B) Total time spent digging in fresh bedding during 10 min observation in a new test cage, after 10 min habituation. (C) Total time spent in contact during male/female and female/female social interaction. (D) Total time spent self-grooming during 10 min observation in a new test cage, after 10 min habituation. (E) Z-score for the time spent self-grooming. (F) Proportion and number of individuals displaying different levels of severity in self-grooming. “Dead” corresponds to animals that had to be euthanized due to severe self-inflicted injuries. (A–D) Black points represent mean with standard error of the mean (s.e.m). Mann–Whitney U test with Bonferroni correction for multiple testing (two tests): * Corrected value of p < 0.05; ** corrected value of p < 0.01. Frontiers in Molecular Neuroscience Abnormal expression of striosome markers Using RT-PCR, we investigated the correlation between the mRNA levels in the striatum of DEGs identified by RNAseq analysis and other genes of interest (Shank1, Shank2, and Drds) and the level of self-grooming observed in seven 12 months-old Shank3Δ11/Δ11 mice (Supplementary Figure S11). We found that excessive grooming correlated with higher levels of transcripts for four of the DEGs identified by RNAseq or RT-PCR: Cnr1, Gnal, Gad2, and Drd4 (Figure 3B). Cnr1 encodes the cannabinoid receptor 1; Gnal encodes a stimulatory G-alpha subunit of a heterotrimeric G-protein (Gαolf); Gad2 codes for GAD65, one of the two enzymes that convert glutamate into GABA, and Drd4 encodes the dopamine receptor 4. Interestingly, these proteins have been reported to be heterogeneously distributed in the dorsal striatum, being more abundant in the spatial microzones known as striosomes or patches than in the much larger matrix surrounding them, especially Gad2/GAD65 (Rivera et al., 2002; Lévesque et al., 2004; Sako et al., 2010; Davis et al., 2018). Therefore, given the function of GAD65 (i.e., converting glutamate into GABA, an inhibitory transmitter), we studied its distribution in the dorsal striatum, using immunofluorescence on brain sections (Figures 3C–I). Using two different anti-GAD65 antibodies, we found that GAD65 is enriched in striosomes, identified by co-labeling of μ-opioid receptor (MOR), the most widely used marker for striosomes of the rostral striatum (Miyamoto et al., 2018; Figures 3C,D). GAD65 can thus be considered as a marker of the striosomal compartment in mice. Quantitative analysis revealed that the relative surface occupied by the GAD65-enriched striosomes in the dorsal striatum is increased in the Shank3Δ11/Δ11 mice compared to the Shank3+/+ mice (p = 0.0018, Figures 3E–G). Moreover, in agreement with the overexpression of Gad2 in the striatum but not in the cortex (Supplementary Table S2) of Shank3Δ11/Δ11 mice, GAD65 labeling intensity relative to cortex was increased in the whole Shank3Δ11/Δ11 striatum, but at much higher levels in the striosomes (p = 6.5 10−5, Figure 3H) than in the matrix (p = 2.9 10−5, Figure 3I and Supplementary Figures S12A,B). In the striatum of Shank3Δ11/Δ11 mice, SHANK3 staining is still detected in a small fraction of glutamatergic synapses but the number of puncta and their intensity are highly reduced compared to Shank3+/+ mice (Supplementary Figures S12A,C). In both Shank3+/+ and Shank3Δ11/ Δ11 mice, the intensity of the SHANK3 staining is not different Differential expression of genes related to synaptic function, signaling and cytoskeleton dynamics in the striatum of Shank3Δ11/Δ11 mice Moreover, using Single Molecule RNA Fluorescence In Situ Hybridization with Drd1 and Drd2 RNA probes, we observed no significant difference in the number of D1 and D2 striatal neurons between the Shank3Δ11/Δ11 and Shank3+/+ mice (Supplementary Figure S10). Altogether, these results indicate that the deletion of exon 11 of Shank3 leads to differential transcriptional alterations in the D1- and D2-MSN, without major difference in the number of these types of neurons throughout the whole striatum. striatum with 140 DEGs. The hippocampus displayed 26 DEGs, the cortex 13 and the cerebellum 16 (Figure 2A). After removing the genes and pseudogenes located around the Shank3 locus, Shank3 was the only gene differentially expressed in the four brain structures (Figure 2A). The DEGs in the striatum displayed an increased dispersion compared to the other structures, indicating an increased variability of response between animals (Supplementary Figure S8). Altogether, these results suggest that, among the four structures studied, the transcriptome of the striatum is by far the most impacted by the loss of the major isoforms of SHANK3. We performed quantitative RT-PCR (q-RT- PCR) to confirm up- or downregulation of 30 DEGs (Spearman correlation R = 0.809, p < 0.0001; Supplementary Figure S9). We investigated the functions of the proteins encoded by the DEGs in the striatum using in the striatum using ORA and PPI. The ORA (Supplementary Table S3) revealed enrichment in DEGs associated with, among others, two pathways of interest: synaptic transmission (negative fold-change in Shank3Δ11/Δ11 mice in comparison with Shank3+/+ mice), and G-protein activity (positive and negative fold-change in Shank3Δ11/Δ11 mice in comparison with Shank3+/+ mice according to the type of G-protein pathway) (Figure 2B and Supplementary Table S3). PPI analysis of the striatal DEGs highlighted one network of 15 proteins (value of p from randomized network: <0.01; Figure 2C), some of them directly interacting with SHANK3 in the PSD, such as the metabotropic glutamate receptor 2 (encoded by Grm2, highly decreased) and Disks large-associated protein 1, a scaffolding protein (encoded by Dlgap1, also decreased) interacting with AMPA and NMDA receptors. In addition to these synaptic proteins, the network contains proteins involved in signal transduction (e.g., Prkcg1, Camk2g) and cytoskeleton dynamics (e.g., Slk). Other small networks identified by the PPI analysis contain proteins involved in glutamate transportation (Slc17a7) and decarboxylation (Gad2), and cytoskeleton dynamics (Actbl2). Frontiers in Molecular Neuroscience Differential expression of genes related to synaptic function, signaling and cytoskeleton dynamics in the striatum of Shank3Δ11/Δ11 mice No significant changes in the expression of Shank1 and Shank2 were found (Supplementary Figure S7 and Supplementary Table S2). At 8 and 12 months of age, Cohort 1 Shank3Δ11/Δ11 males still displayed increased self-grooming in comparison with Shank3+/+ mice (8 months: W = 27, p = 0.009; 12 months: W = 37, p = 0.041; Figure 1D). The same was true for Shank3Δ11/Δ11 females who also groomed themselves significantly more than Shank3+/+ females (8 months: W = 11, p = 0.002; 12 months: W = 20, p = 0.014; Figure 1D). With age, an increasing number of Shank3Δ11/Δ11 mice were outliers, performing self-grooming during a period between one and two standard deviation(s) (medium phenotype) or exceeding two standard deviations (severe phenotype) or had to be euthanized because of severe self-inflicted injuries. Hereafter, we denominate mice with a period of self-grooming over one standard deviation as excessive self-groomers. Nevertheless, for 4 out of 12 Shank3Δ11/Δ11 males and 3 out of 12 Shank3Δ11/Δ11 females, we did not observe excessive self-grooming at any of the three time points (Figures 1D,E). We examined the DEGs (FDR < 0.05) within each of the four brain structures. The largest number of DEGs was detected in the 08 frontiersin.org 10.3389/fnmol.2023.1139118 Ferhat et al. proportion of D1-MSN (decrease) and D2-MSN (increase) in the striatum of the Shank3Δ11/Δ11 mice. However, many genes commonly considered as specific to D1-MSN (Drd1, Isl1, Sfxn1, and Tac1) or D2-MSN (Drd2, Adora2a, Penk, Gpr6, Sp9) were not found to be differentially expressed in the Shank3Δ11/Δ11 animals (Supplementary Table S2). Moreover, using Single Molecule RNA Fluorescence In Situ Hybridization with Drd1 and Drd2 RNA probes, we observed no significant difference in the number of D1 and D2 striatal neurons between the Shank3Δ11/Δ11 and Shank3+/+ mice (Supplementary Figure S10). Altogether, these results indicate that the deletion of exon 11 of Shank3 leads to differential transcriptional alterations in the D1- and D2-MSN, without major difference in the number of these types of neurons throughout the whole striatum. proportion of D1-MSN (decrease) and D2-MSN (increase) in the striatum of the Shank3Δ11/Δ11 mice. However, many genes commonly considered as specific to D1-MSN (Drd1, Isl1, Sfxn1, and Tac1) or D2-MSN (Drd2, Adora2a, Penk, Gpr6, Sp9) were not found to be differentially expressed in the Shank3Δ11/Δ11 animals (Supplementary Table S2). Frontiers in Molecular Neuroscience Cellular expression pattern of the DEGs expressed in the striatum of Shank3Δ11/Δ11 mice The inhibitory medium-sized spiny neurons (MSN) constitute the major type of striatal neuronal population. They receive glutamatergic inputs and are the target of dopamine innervation from cortex, thalamus, amygdala and substantia nigra pars compacta (SNpc) (Park et al., 2020). Most of these MSN express either dopamine 1 receptor DRD1 (D1-MSN, belonging to the direct pathway), dopamine 2 receptor DRD2 (D2-MSN, belonging to the indirect pathway), or both receptors for a small fraction of MSN. In order to identify the striatal cell types which are the most impacted by SHANK3 deficiency, we compared the expression pattern of the DEGs with marker genes of striatal cell types reported in two single-cell RNA sequencing studies (Gokce et al., 2016; Montalban et al., 2020). We observed that DEGs under- expressed in Shank3Δ11/Δ11 compared to Shank3+/+ mice were enriched in the D1-MSN clusters while overexpressed genes were enriched in the D2-MSN clusters (Figure 3A and Supplementary Tables S2, S4). This could reflect a modified 09 frontiersin.org Ferhat et al. 10.3389/fnmol.2023.1139118 A A A B C FIGURE 2 The striatum displays the largest gene expression differences between Shank3+/+ and Shank3Δ11/Δ11 mice and the largest number of impacted pathways. (A) Upset plot displaying the intersection between brain structures for the genes differentially expressed between Shank3Δ11/Δ11 and Shank3+/+ mice (adjusted value of p < 0.05; determined by limma-voom using observation quality weights). Genes located around the Shank3 region on chromosome 15 are shown in blue. The horizontal bar plots on the left show the number of differentially expressed genes in the different brain structures according to the value of their log-fold changes (logFC). (B) GO (Gene Ontology), GeneSetDB Pathway (gsdbpath), and KEGG (Kyoto Encyclopaedia of Genes and Genomes) gene sets found to be over-represented (top 20 gene sets with adjusted value of p < 0.05) in the genes detected as differentially expressed in the striatum. (C) Protein–protein interaction (PPI) network for the differentially expressed genes (DEGs). Nodes, colored ovals, represent DEGs; edges, black lines, represent direct protein–protein interactions (from BioGrid) between DEGs products. The color of the node represents the Log2FC of DEGs: red means up-regulation and blue means down-regulation in Shank3Δ11/Δ11 mice compared to wild-type mice. B B B C FIGURE 2 The striatum displays the largest gene expression differences between Shank3+/+ and Shank3Δ11/Δ11 mice and the largest number of impacted pathways. Cellular expression pattern of the DEGs expressed in the striatum of Shank3Δ11/Δ11 mice (A) Upset plot displaying the intersection between brain structures for the genes differentially expressed between Shank3Δ11/Δ11 and Shank3+/+ mice (adjusted value of p < 0.05; determined by limma-voom using observation quality weights). Genes located around the Shank3 region on chromosome 15 are shown in blue. The horizontal bar plots on the left show the number of differentially expressed genes in the different brain structures according to the value of their log-fold changes (logFC). (B) GO (Gene Ontology), GeneSetDB Pathway (gsdbpath), and KEGG (Kyoto Encyclopaedia of Genes and Genomes) gene sets found to be over-represented (top 20 gene sets with adjusted value of p < 0.05) in the genes detected as differentially expressed in the striatum. (C) Protein–protein interaction (PPI) network for the differentially expressed genes (DEGs). Nodes, colored ovals, represent DEGs; edges, black lines, represent direct protein–protein interactions (from BioGrid) between DEGs products. The color of the node represents the Log2FC of DEGs: red means up-regulation and blue means down-regulation in Shank3Δ11/Δ11 mice compared to wild-type mice. C FIGURE 2f / Δ11/Δ11 C C FIGURE 2 The striatum displays the largest gene expression differences between Shank3+/+ and Shank3Δ11/Δ11 mice and the largest number of impacted pathways. (A) Upset plot displaying the intersection between brain structures for the genes differentially expressed between Shank3Δ11/Δ11 and Shank3+/+ mice (adjusted value of p < 0.05; determined by limma-voom using observation quality weights). Genes located around the Shank3 region on chromosome 15 are shown in blue. The horizontal bar plots on the left show the number of differentially expressed genes in the different brain structures according to the value of their log-fold changes (logFC). (B) GO (Gene Ontology), GeneSetDB Pathway (gsdbpath), and KEGG (Kyoto Encyclopaedia of Genes and Genomes) gene sets found to be over-represented (top 20 gene sets with adjusted value of p < 0.05) in the genes detected as differentially expressed in the striatum. (C) Protein–protein interaction (PPI) network for the differentially expressed genes (DEGs). Nodes, colored ovals, represent DEGs; edges, black lines, represent direct protein–protein interactions (from BioGrid) between DEGs products. The color of the node represents the Log2FC of DEGs: red means up-regulation and blue means down-regulation in Shank3Δ11/Δ11 mice compared to wild-type mice. Cellular expression pattern of the DEGs expressed in the striatum of Shank3Δ11/Δ11 mice Frontiers in Molecular Neuroscience 10 frontiersin.org Ferhat et al. 10.3389/fnmol.2023.1139118 A C G E D H F I B FIGURE 3 Altered transcriptome of D1- and D2-MSN, over-expression of GAD65, and modification of the striosome/matrix balance in the striatum of Shank3Δ11/Δ11 mice. (A) Enrichment of RNAseq DEGs in the striatum of Shank3Δ11/Δ11 mice compared to cell-specific gene clusters suggested by Gokce et al. (2016) and Montalban et al. (2020). Genes under-expressed in Shank3Δ11/Δ11 striatum are enriched in D1-MSN clusters while genes over-expressed in Shank3Δ11/Δ11 striatum are enriched in D2-MSN clusters. Ratio genes correspond to the proportion of DEGs enriched in the cluster. Over-representation analyses were performed with Fisher’s hypergeometric tests and p-values were adjusted for multiple testing with the Benjamini–Hochberg procedure within each category of DEGs. (B) Comparison of the log of the fold change of gene expression of selected genes in Shank3Δ11/Δ11 vs. Shank3+/+ mice (LogFC RNAseq, Y axis) and the β values (slope of the linear regression) for low self-grooming vs. high self-grooming (β value grooming, X axis). (C,D) Confocal images of striatum coronal sections of one-year old Shank3+/+ male mice. (C) GAD65 immunoreactivity (red) is enriched in striatal microzones identified as striosomes by immunostaining for μ-opioid receptor (MOR, in green), a canonical marker of striosomes. (D) Increased GAD65 immunoreactivity in striosomes (arrows) and subcallosal streak (two-headed arrows) compared to surrounding matrix is observed with two different antibodies: the rabbit polyclonal antibody (Invitrogen PA5-77983 in red) used in (C,E,F) and a mouse monoclonal antibody (Millipore MAB351, in green). (E,F) Distribution and quantification of GAD65 immunoreactivity in dorsal striatum sections of a Shank3+/+ (E) and a Shank3Δ11/Δ11 (F) mouse (Continued) B A A B A G C C G D D H E E H I I F FIGURE 3 Altered transcriptome of D1- and D2-MSN, over-expression of GAD65, and modification of the striosome/matrix balance in the striatum of Shank3Δ11/Δ11 mice. (A) Enrichment of RNAseq DEGs in the striatum of Shank3Δ11/Δ11 mice compared to cell-specific gene clusters suggested by Gokce et al. (2016) and Montalban et al. (2020). Genes under-expressed in Shank3Δ11/Δ11 striatum are enriched in D1-MSN clusters while genes over-expressed in Shank3Δ11/Δ11 striatum are enriched in D2-MSN clusters. Ratio genes correspond to the proportion of DEGs enriched in the cluster. Over-representation analyses were performed with Fisher’s hypergeometric tests and p-values were adjusted for multiple testing with the Benjamini–Hochberg procedure within each category of DEGs. Cellular expression pattern of the DEGs expressed in the striatum of Shank3Δ11/Δ11 mice (B) Comparison of the log of the fold change of gene expression of selected genes in Shank3Δ11/Δ11 vs. Shank3+/+ mice (LogFC RNAseq, Y axis) and the β values (slope of the linear regression) for low self-grooming vs. high self-grooming (β value grooming, X axis). (C,D) Confocal images of striatum coronal sections of one-year old Shank3+/+ male mice. (C) GAD65 immunoreactivity (red) is enriched in striatal microzones identified as striosomes by immunostaining for μ-opioid receptor (MOR, in green), a canonical marker of striosomes. (D) Increased GAD65 immunoreactivity in striosomes (arrows) and subcallosal streak (two-headed arrows) compared to surrounding matrix is observed with two different antibodies: the rabbit polyclonal antibody (Invitrogen PA5-77983 in red) used in (C,E,F) and a mouse monoclonal antibody (Millipore MAB351, in green). (E,F) Distribution and quantification of GAD65 immunoreactivity in dorsal striatum sections of a Shank3+/+ (E) and a Shank3Δ11/Δ11 (F) mouse (Continued) (Continued) 11 Frontiers in Molecular Neuroscience frontiersin.org Ferhat et al. 10.3389/fnmol.2023.1139118 FIGURE 3 (Continued) (images generated by stitching multiple maximum-intensity projected z-stacks). The arrows point to striosomes and the arrowheads to subcallosal streak. In the dorsal striatum ROI (surrounded in green), the unlabeled myelinated fibers are colored in red, the regions of higher expression of GAD65 (striosomes) are colored in green, and the regions of lower expression of GAD65 (matrix) are uncolored. The cortex ROI used as a reference is surrounded in red. Note that the acquisition parameters were adjusted for each brain hemi-section in order to have no saturating signal for GAD65 in the striatal ROI. Increased intensity in the striatum thus leads to a staining which appears weaker in the cortex. (G–I) Comparison of GAD65 immunoreactivity in the striosome and matrix compartments of the dorsal striatum in 5 Shank3+/+ (green) and 9 Shank3Δ11/Δ11 (orange) 1-year old male mice. (G) Relative surface of the GAD65-enriched striosome compartment (surface of striosomes/surface of (striosomes + matrix). (H) Relative GAD65 labeling intensity in the striosomal compartment of the striatum compared to cortex. (I) Relative GAD65 labeling intensity in the matrix compartment of the striatum compared to cortex. Data, generated from analysis of 9–14 images per animal, are presented as box-plots (median, first, and third quartiles). between the striosomes and the matrix. Finally, we investigated GAD65 distribution in the striatum of another Shank3 mouse model, lacking all SHANK3 isoforms (Drapeau et al., 2018). Discussion The present study highlighted robust behavioral deficits in Shank3Δ11/Δ11 mutant mice, more specifically a reduced activity in the open field, atypical social behavior in males interacting with estrous females, and increased self-grooming compared to wild-type littermates. The majority of Shank3Δ11/Δ11 mutant mice displayed a tendency to a worsening of the self-grooming phenotype with increasing age for mice showing an early emergence of this trait. At 12 months of age, the striatum had the most impacted transcriptomic profile compared to other brain regions. Further molecular characterizations pointed towards possible imbalances between the striosome and matrix compartments in the dorsal striatum. Excessive grooming Excessive self-grooming has been observed in the majority of Shank3 mutant mice over their lifetime and therefore represents the most robust behavioral phenotype in all models. Extreme self-grooming is most likely not due to increased skin sensitivity since rescuing normal tactile reactivity in Shank3b+/− mice does not improve over-grooming behavior (Orefice et al., 2019). In our study, we also observed an important inter-individual variability in this trait with 30% of the mutant mice that did not seem to display excessive grooming even after 1 year. Increased self-grooming might reflect a response to stressful conditions, which might vary from one individual to another (difference in susceptibility and/or exposure to stressful conditions). Such increased reactivity to novel or stressful conditions is reminiscent of what is observed in patients and deserves further systematic testing in the different Shank3 mutant models (Krüttner et al., 2022). Abnormal striatal transcriptomeh The massive impact of the Shank3Δ11 mutation on the transcriptome profile of the striatum suggests that this region could be the centerpiece of the behavioral deficits observed in Shank3Δ11/∆11 mutant mice. Several studies have already reported functional alterations of the striatum and more specifically of the MSN in the diverse Shank3 mutant strains (Peça et al., 2011; Bariselli et al., 2016; Peixoto et al., 2016; Bariselli and Bellone, 2017; Wang et al., 2017; Bey et al., 2018). Our report of an alteration of the striatal transcriptome could be due to a direct role of the SHANK3 protein in gene transcription. Indeed, a previous study, using transfected cells expressing EGFP-SHANK3 fusion proteins, has reported the presence of SHANK3 in the nucleus, especially the isoform SHANK3b (Wang et al., 2014), which is disrupted in our model. Using immunohistofluorescence, we did not observe SHANK3 in the nucleus of the striatal cells in Shank3+/+ adult mice, but the epitope recognized by the antibody we used is absent from the SHANK3b isoform. Alternatively, the abnormal striatal transcriptome profile could be the consequence of the alteration of the glutamatergic synapses that interfere with signal transduction, such as G protein (Qin et al., 2018) Cellular expression pattern of the DEGs expressed in the striatum of Shank3Δ11/Δ11 mice These 20–28 weeks-old Shank3Δ4–22/Δ4-22 male mice were also used to validate the specificity of the anti-SHANK3 antibody (Supplementary Figure S12). We confirmed enlargement and GAD65 over-expression in the striosomal compartment. However, in the absence of all SHANK3 isoforms, GAD65 is also markedly over-expressed in the matrix (Supplementary Figure S12). have shown that SHANK3 controls maturation of social circuits in the ventral tegmental area (VTA) (Bariselli et al., 2016; Bariselli and Bellone, 2017) and synaptic strength of D2-MSN in the striatum (Wang et al., 2017; Bey et al., 2018). Frontiers in Molecular Neuroscience Atypical social interaction Results from the literature have highlighted either reduced duration of social contact and reduced number of USVs emitted [e.g., Shank3∆ex4-22 (Wang et al., 2016)] or no significant difference in socio-sexual behaviors [e.g., Shank3Q321R (Yoo Y.-E. et al., 2019)]. Here, we observed limited difference in social interaction with an increase in the time spent in contact for Shank3Δ11/Δ11 males interacting with an oestrous female (observed at 3 and 8 months of age), and more specifically in nose-to-nose contacts, as already highlighted in previous studies [Shank3∆ex14 − 16cKO (Yoo T. et al., 2019); Shank3Δ11/Δ11 females (de Chaumont et al., 2021)]. Testing the specificity of this type of contact would help to understand its significance in mouse social communication. Further experiments should be designed to test whether this specific behavior is associated with a deficit in social reward processing [as suggested for Shank2Δ6–7/Δ6–7 mice (Ey et al., 2018)]. Indeed, several studies 12 frontiersin.org Ferhat et al. 10.3389/fnmol.2023.1139118 and mTOR (Moutin et al., 2021) signalling, and then with gene transcription or mRNA stability. We found that several transcripts encoding proteins involved in glutamatergic synaptic transmission were under-expressed in Shank3∆11/∆11 mice. For example, we observed a decrease of Grm2, encoding mGluR2, a G-protein-coupled glutamate receptor which interacts with the proline-rich domain of SHANK3 and which has been found decreased in a valproate-induced rat model of autism (Chen et al., 2014). We also observed significant under- expression of Dlgap1, coding for DAP1/GKAP. This protein connects SHANK3 to the scaffolding protein PSD-95 (Boeckers et al., 1999) which recruits AMPAR and NMDAR. In contrast, we did not observe diminution of transcript levels for several proteins (GluA, GluN, or mGluR5) previously shown as decreased in the synaptosomes or PSD fractions of Shank3 mutant mice compared to wild type. A possible explanation is that the absence of SHANK3 decreases the translation or the recruitment of these proteins at the synapse or increases their degradation without affecting gene transcription and mRNA degradation. Interestingly, we observed a sexual dimorphism in behavior in mice, especially for cohort 1, that is not reported in patients (Leblond et al., 2014). This aspect could be explained by the impact of estradiol on striatal function and behavior in mice (Meitzen et al., 2018), as well as by differential gene expression between males and females after an acute stress in mice as, for instance, in the CA3 subregion of hippocampus (Marrocco et al., 2017). A link between abnormal gene transcription, striatum organization and behavioral impairmen We first confirmed that GAD65, which had been reported to be a striosome marker in primates, but not in rats (Lévesque et al., 2004) was indeed a striosome marker in the mouse. We then found that the GAD65-enriched striosomal compartment is enlarged in Shank3Δ11/Δ11 mice compared to Shank3+/+ mice and that striatal GAD65 overexpression in the absence of the major SHANK3 isoforms is much more pronounced in the striosomes than in the matrix. Whereas the majority of GABA in brain is produced by the cytosolic isoform of glutamate decarboxylase (GAD67, encoded by Gad1), GAD65, which is anchored to the membrane of synaptic vesicles, can supply GABA in situations of high demand (Kaufman et al., 1991), such as stress or fear, and for fine tuning of inhibitory transmission. It is thus possible that the altered compartmentation and/or GAD65 over-expression in the striatum are responsible for some of the behavioral features of the Shank3Δ11/Δ11 mice, especially excessive self-grooming. Future studies, notably by modulating GAD65 expression in the dorsal striatum of wild-type and Shank3 mice, are needed to establish causality between the striatal alterations and the excessive self-grooming of Shank3 mice. Moreover, testing, at the molecular and cellular levels in the striatum and at the behavioral level, the impact of stress in Shank3 mice may help understand the inter-individual variability in the severity of excessive self-grooming. g p We observed that the expression of Gad2, Cnr1, Gnal, and Drd4 was positively correlated with excessive self-grooming. Remarkably, three of these genes have been previously associated with self-grooming, but not always in the same direction. In contrast to our study, Cnr1 deletions (as well as CB1R antagonists) have been reported to increase self-grooming behavior (Litvin et al., 2013; Gogolla et al., 2014). The overexpression of Cnr1 in Shank3∆11/∆11 mice might therefore result from a compensatory upregulation in response to the alterations of the endocannabinoid system which has been previously reported in SHANK3-deficient mice (Wang et al., 2017; Folkes et al., 2020), in valproic acid induced rat models of autism and also in some autistic individuals (Zou et al., 2021). Consistent with our findings, Gnal haploinsufficiency is associated with a reduction of self-grooming behavior in a mouse model of dystonia (Pelosi et al., 2017). A link between abnormal gene transcription, striatum organization and behavioral impairmen Links between Gad2 and Shank3 were previously reported with an increase of GAD65 immunoreactivity in the Shank3ß−/− mice (Gogolla et al., 2014) and a decrease of Gad2 transcript in the striatum of a Shank3-overexpressing model (Lee et al., 2017). GAD65 is associated with different disorders related to anxiety, such as obsessive–compulsive disorder, panic disorder or generalized anxiety disorder (Karunakaran et al., 2021). To our knowledge, there is no direct evidence in the literature for an association between Drd4 expression and grooming behavior. However, Drd4 is a candidate gene for obsessive–compulsive disorder and panic disorder (Taj et al., 2013). It would thus be interesting in future studies to investigate this potential link. Atypical social interaction Analyzing the brain transcriptome of Shank3+/+ and Shank3∆11/∆11 females should shed light on the differential effect of the lack of Shank3 between sexes. contain early born MSN and are embedded into the much larger surrounding matrix including late-born MSN (Hagimoto et al., 2017). The two compartments, initially distinguished by differential expression of numerous molecular markers [for a review see (Crittenden and Graybiel, 2011)], also differ in their input and output connectivity and electrophysiological characteristics (Prager and Plotkin, 2019), such as D1-MSN excitability (Prager et al., 2020), rates of dopamine release (Jaquins-Gerstl et al., 2021), and response to chronic stress (Friedman et al., 2017). Imbalances in striosome to matrix activity have been suggested in several movement disorders [Huntington’s disease, Parkinson’s disease, dyskinesia, dystonia, for a review see (Crittenden and Graybiel, 2011)], psychostimulant-induced motor stereotypies (Canales and Graybiel, 2000), and more recently in anxiety disorder (Karunakaran et al., 2021). Corticostriatal path targeting striosomes also control decision-making under cost–benefit conflict (Friedman et al., 2015, 2020) and habit formation (Nadel et al., 2021). Our finding that four striosome markers had increased expression associated with excessive self-grooming in Shank3Δ11/Δ11 mice prompted us to explore the compartmental architecture of the striatum in these mice. This investigation was also motivated by previous studies that have shown that enhanced striosomal activation is highly correlated with increased repetitive behaviors, including self-grooming in both non-human primates and rodents [for review see (Kalueff et al., 2016)]. Studies by Kuo and Liu (2017, 2020) have recently suggested that aberrant striatal compartmentation may be involved in autism. Furthermore, through the investigation of protein–protein interactions, a role for the striosomes in anxiety disorder such as social anxiety has been recently proposed (Karunakaran et al., 2021).ii Frontiers in Molecular Neuroscience A model for striosomes/matrix imbalance as a possible cause of excessive grooming in the Shank3∆11/∆11 mice It is well established that the striatum and the dopamine-containing nigrostriatal tract control self-grooming behavior (Kalueff et al., 2016), and that striosomal MSN are the predominant striatal population Interestingly, these four genes have also been reported to be enriched in striosomes (Rivera et al., 2002; Lévesque et al., 2004; Sako et al., 2010; Davis et al., 2018). These microzones of the dorsal striatum 13 frontiersin.org 10.3389/fnmol.2023.1139118 Ferhat et al. innervating dopamine neurons of the nigrostriatal tract (Watabe- Uchida et al., 2012). An imbalance between the striosome and the matrix compartments may then explain, via a modification of dopamine signalling, the excessive grooming of Shank3Δ11/Δ11 mice (Figure 4). Indeed, in contrast to MSN located in the matrix of the striatum, striosomal MSN projects directly to the dopamine-producing neurons in the SNpc (Crittenden and Graybiel, 2011; McGregor et al., 2019). Then, SNpc dopamine neurons project back to the entire dorsal striatum. An increased activity of GAD65 in striosomes, in a situation of stress for example, would lead to an inhibition of dopamine release by the SNpc and thus to a reduced dopamine modulation of both the direct and indirect pathways in the dorsal striatum. Because dopamine activation has opposite effects on D1- and D2-MSN by increasing and decreasing cell excitability, respectively, a decreased dopamine release by SNpc is expected to result in diminishing D1-MSN activity and boosting D2-MSN activity. The differential enrichment in up- and down-regulated DEGs in D2-MSN and D1-MSN, respectively, could be related to previous observation of an impairment of long-term depression in D2-MSN, but not in D1-MSN (Wang et al., 2017) in Shank3 mutant mice. Importantly, striosome cells are generated before matrix cells during development (Hagimoto et al., 2017) and SHANK3 seems to be involved in the early stages of neuronal development (Peixoto et al., 2016; Jaramillo et al., 2020). Therefore, the enlargement of the total relative surface of the GAD65-enriched striosomal compartment observed first in the Shank3Δ11/Δ11 and then in the Shank3Δ4–22/Δ4-22 mice may be due to a default in the compartmentation of the striatum during early development. A future longitudinal study, at the cellular and molecular levels, of striatal development during the late embryonic and early postnatal stages in Shank3 mice should help determine whether SHANK3 is involved in one of the sequential steps (generation of D1- and D2-MSN, cell migration, segregation of striosome and matrix cells) of striosome/matrix mosaic organisation in the developing striatum. Frontiers in Molecular Neuroscience Conclusion and perspectives Altogether, the present study describes the effects over time of the Shank3Δ11 mutation on mouse social and repetitive behaviors and suggests a critical role of the striatum in excessive self-grooming, probably through imbalances in two intertwined systems, the striosome/matrix compartments and the direct/indirect pathways. Our results suggest that SHANK3 deficiency/Phelan-McDermid syndrome and possibly to some extent autism could integrate the growing list of neurological conditions implicating an imbalance in the striosome/matrix compartmentation of the dorsal striatum. Future FIGURE 4 Proposed model linking increased activity in the striosomal pathway and imbalance between the direct and indirect pathways in the Shank3Δ11/Δ11 mice. The modifications of connection strength (line thickness) induced by striosomal compartment alterations in Shank3Δ11/Δ11 mice are only represented between the striatum and its output target areas (colored boxes). Increased activity of the striosomal pathway in Shank3Δ11/Δ11 mice, due to both larger size and increased GAD65 activity of the striosomal compartment (light/dark orange) enhances GABAergic inhibition of the dopamine-producing neurons of SNpc (green). This inhibition of the SNpc neurons (light green) results in an imbalance between the direct and indirect pathways by decreasing both the activation of D1-MSN (D1) and the inhibition of D2-MSN (D2). GPI, internal globus pallidus; GPE, external globus pallidus; SNpr, substantia nigra pars reticulata; SNpc, substantia nigra pars compacta; STN, subthalamic nucleus. FIGURE 4 Proposed model linking increased activity in the striosomal pathway and imbalance between the direct and indirect pathways in the Shank3Δ11/Δ11 mice. The modifications of connection strength (line thickness) induced by striosomal compartment alterations in Shank3Δ11/Δ11 mice are only represented between the striatum and its output target areas (colored boxes). Increased activity of the striosomal pathway in Shank3Δ11/Δ11 mice, due to both larger size and increased GAD65 activity of the striosomal compartment (light/dark orange) enhances GABAergic inhibition of the dopamine-producing neurons of SNpc (green). This inhibition of the SNpc neurons (light green) results in an imbalance between the direct and indirect pathways by decreasing both the activation of D1-MSN (D1) and the inhibition of D2-MSN (D2). GPI, internal globus pallidus; GPE, external globus pallidus; SNpr, substantia nigra pars reticulata; SNpc, substantia nigra pars compacta; STN, subthalamic nucleus. Acknowledgments The authors thank the members of the Génétique Humaine et Fonctions Cognitives lab for helpful discussions, especially Jean-Pierre Bourgeois for his advice and expertise, Thomas Rolland for his assistance with data analysis and Nathalie Lemière for her assistance with sample collection. The authors thank Jean-Antoine Girault, Véronique Brault, and Sonia Garel for their advice and expertise. The content of this manuscript has previously appeared online (Ferhat et al., 2021). Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Ethics statement The animal study was reviewed and approved by the ethical committees of Institut Pasteur (CEEA n°89) and Sorbonne Université (CEEA n°5). Conclusion and perspectives 14 Frontiers in Molecular Neuroscience frontiersin.org Ferhat et al. 10.3389/fnmol.2023.1139118 10.3389/fnmol.2023.1139118 Agency (ANR) part of the Investment for the Future program], the BioPsy Labex (ANR-10-LABX-BioPsy, ANR-11-IDEX-0004-02), AIMS-2-TRIALS which received support from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 777394 and the Inception program (Investissement d’Avenir grant ANR-16-CONV-0005). We gratefully acknowledge the UtechS Photonic Bioimaging (Imagopole), C2RT, Institut Pasteur, supported by the French National Research Agency (France BioImaging; ANR-10-INSB-04; Investments for the future). studies in mice, but also in other species such as rat (Harony-Nicolas et al., 2017) or non-human primates (Zhao et al., 2017; Zhou et al., 2019), are necessary to establish causality between the here-reported striatal defects and the stereotyped behaviors of Shank3 mice, to understand the mechanism at the origin of the striosome/matrix imbalance and finally consider the possibility of its reversibility. Supplementary material The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol.2023.1139118/ full#supplementary-material Author contributions ATF, ABi, EV, EE, and TB conceived the experiments. ATF and EE designed and acquired behavioral data from cohorts 1 and 2. ATF extracted and dissected brains, extracted RNA from cohorts 1 and 2, acquired the data from smFISH, performed the analysis and statistics of behavior, PPI and smFISH. ATF and FM designed the smFISH experiments. ABo, BFi, and JFD performed the RNA sequencing. ABi and ATF performed the analyses of ORA. ATF, BFo, and AL performed and analyzed the results of quantitative RT-PCR. EV performed the immunofluorescence experiments, with the help of SC for brain collection. FdC performed quantitative image analysis. SC and AMLS genotyped the mice. JS, CR, and LRR conducted the experiments on cohort 3. TMB generated the Shank3Δ11/Δ11 mouse model. ATF, ABi, EV, BFo, FdC, EE, and TB wrote the manuscript. All authors contributed to the article and approved the submitted version. 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. The reviewer MY declared a shared affiliation with the author ATF to the handling editor at the time of review. Funding This research was supported by Institut Pasteur, the Bettencourt- Schueller Foundation, CNRS, Université de Paris, the Conny-Maeva Charitable Foundation, the the Fondation Cognacq-Jay, the Fondation de France, the Fondation pour la Recherche Médicale (FRM), the Association Française du Syndrome Phelan-McDermid, the Association Téhani et les enfants Phelan-McDermid, the GIS “Autisme et Troubles du Neuro-Développement,” the Roger de Spoelberch Foundation, the Eranet-Neuron (ALTRUISM) project, the Laboratory of Excellence GENMED (Medical Genomics) [grant no. ANR-10-LABX-0013 managed by the National Research The views expressed here are the responsibility of the authors only. The EU Commission takes no responsibility for any use made of the information set out. Data availability statement The datasets presented in this study can be found in online repositories. 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https://openalex.org/W4206096235	https://www.researchprotocols.org/2022/2/e31928/PDF	English	null	Defining the Pre-exposure Prophylaxis Care Continuum Among Recently Incarcerated Men at High Risk for HIV Infection: Protocol for a Prospective Cohort Study	JMIR research protocols	2,022	cc-by	8,129	KEYWORDS IV; PrEP; criminal justice system; incarceration; criminal justice; pre-exposure prophylaxis; prison system most frequent mechanisms of HIV acquisition are sexual contact and injection drug use [1]. Men who have sex with men (MSM), including men who participate in transactional sex with other men, have a high burden of HIV and comprise approximately 70% of all HIV diagnoses [1,2]. There are also significant racial disparities within the United States among incident cases of Corresponding Author: Corresponding Author: Matthew Murphy, MPH, MD Brown University Open Door Health 7 Central Street Providence, RI, 02907 United States Phone: 1 401 648 4700 Corresponding Author: Matthew Murphy, MPH, MD Brown University Open Door Health 7 Central Street Providence, RI, 02907 United States Phone: 1 401 648 4700 Email: matthew murphy@brow Email: matthew_murphy@brown.edu Murphy et al Murphy et al JMIR RESEARCH PROTOCOLS JMIR Res Protoc 2022 \| vol. 11 \| iss. 2 \| e31928 \| p. 1 (page number not for citation purposes) https://www.researchprotocols.org/2022/2/e31928 Defining the Pre-exposure Prophylaxis Care Continuum Among Recently Incarcerated Men at High Risk for HIV Infection: Protocol for a Prospective Cohort Study Defining the Pre-exposure Prophylaxis Care Continuum Among Recently Incarcerated Men at High Risk for HIV Infection: Protocol for a Prospective Cohort Study Matthew Murphy1, MPH, MD; Collette Sosnowy1, PhD; Brooke Rogers1, MPH, PhD; Siena Napoleon1, MPH; Drew Galipeau2, BA; Ty Scott1; Jun Tao1, PhD; Justin Berk1, MPH, MBA, MD; Jennifer Clarke1, MPH, MD; Amy Nunn1, ScD; Philip A Chan1, MSc, MD 1Brown University, Providence, RI, United States 2The Miriam Hospital, Providence, RI, United States 1Brown University, Providence, RI, United States 2The Miriam Hospital, Providence, RI, United States Abstract Background: HIV disproportionately impacts criminal justice–involved individuals, including men who experience incarceration. Men make up the vast majority of those experiencing incarceration as well as those newly diagnosed with HIV infection. Pre-exposure prophylaxis (PrEP) is a highly effective biomedical intervention that significantly reduces the risk of HIV acquisition. However, implementation in criminal justice systems is limited. Little is known about effective PrEP implementation and use in this unique public health context. Objective: The aim of this study is to characterize the experience of implementing PrEP clinical care in a criminal justice setting for men vulnerable to HIV acquisition. Methods: This article describes a PrEP care continuum for men experiencing incarceration who are at increased risk of HIV acquisition, which can help conceptualize approaches to evaluating PrEP implementation. Results: The outlined study will enroll 100 men experiencing incarceration at high risk for HIV acquisition prior to release into the community. The goal is to initiate PrEP prior to release and link individuals to PrEP providers in the community, capturing barriers and facilitators to PrEP use during this uniquely vulnerable time period for HIV acquisition. Conclusions: Based on the proposed care continuum and what is known about HIV risk and prevention efforts in the criminal justice context, we outline key future research efforts to better understand effective approaches to preventing HIV infection among this vulnerable population. The described approach presents a powerful public health opportunity to help end the HIV epidemic. International Registered Report Identifier (IRRID): DERR1-10.2196/31928 International Registered Report Identifier (IRRID): DERR1-10.2196/31928 International Registered Report Identifier (IRRID): DERR1-10.2196/31928 (JMIR Res Protoc 2022;11(2):e31928) doi: 10.2196/31928 Introduction There are approximately 38,000 new HIV diagnoses in the United States annually [1]. Men make up the vast majority of new diagnoses, accounting for over 80% of these cases [1]. The https://www.researchprotocols.org/2022/2/e31928 XSL•FO RenderX JMIR RESEARCH PROTOCOLS Murphy et al through receptive vaginal intercourse [21] as well as injection drug use [22]. Importantly, research has shown different concentrations of TDF/FTC in vaginal compared to anorectal tissue, leading to different approaches to initiate PrEP and measure adherence among men and women [23]. HIV annually, with racial and ethnic minorities comprising a disproportionate number of new cases compared to White individuals. Black/African American men account for 39% of new HIV diagnoses, yet represent only 18% of the general male population, and Hispanic/Latino men accounted for 29% of new HIV diagnoses while only making up 13% of the general population [3]. Among MSM, Whites have a 1 in 11 lifetime risk of HIV acquisition, compared to Black/African American men, who have a 1 in 2 risk and Hispanic/Latino MSM, who have a 1 in 5 lifetime risk [4]. The most frequent mechanisms of HIV acquisition are sexual contact (88%) and injection drug use (7%-11%) [1]. Despite its effectiveness, PrEP uptake in real-world settings among multiply marginalized populations, including criminal justice–involved individuals, remains low [24]. Although some research has demonstrated PrEP interest among criminal justice–involved individuals [25,26], little is known about the barriers to PrEP implementation during incarceration or the period immediately postrelease from incarceration [27,28]. What is known is that there are expressed reasons to decline PrEP initiation unique to the correctional setting such as institutional mistrust and the high degree of uncertainty in the postrelease period [29,30]. A PrEP care continuum is a useful conceptual tool for identifying facilitators and barriers to PrEP use, adherence, retention, and persistence in PrEP care [31]. In this article, we propose a modified PrEP care continuum for criminal justice–involved populations and then describe an approach to implementing and assessing PrEP care among criminal justice–involved men at high risk of HIV infection. Criminal justice–involved individuals in the United States are among the most vulnerable to and heavily impacted by HIV [5]. Individuals with a history of incarceration have a rate of HIV infection that is 3-5 times that of their nonincarcerated counterparts [6]. One in 7 people living with HIV pass through the criminal justice system each year [7]. Introduction The rate of incarceration for men is 10 times greater than that for women [8]. Black/African American men are also 6 times as likely and Hispanic/Latino men are more than twice as likely as White men to be incarcerated [9]. Criminal justice involvement is highly prevalent among people who inject drugs, with an estimated 72.2% reporting a history of incarceration [5]. Following release, criminal justice–involved individuals are more likely to participate in sexual and substance use behaviors, including injection drug use, that place them at increased risk of HIV infection and overdose [10-17]. Complex, intersecting social and structural forces place racial and ethnic minority populations in particular at increased vulnerability to both criminal justice involvement and HIV acquisition. Defining the PrEP Care Continuum for Criminal Justice–Involved Populations The PrEP care continuum can help to conceptualize and structure PrEP programming in the criminal justice system, including PrEP awareness, uptake, and adherence, and retention in PrEP care [31]. The PrEP care continuum has been used as a tool to better understand the barriers and facilitators to PrEP care for other vulnerable populations [32-34], but it has yet to be applied to or extensively studied in the criminal justice–involved population [35]. Adapting a PrEP continuum unique to criminal justice environments may help clinical providers and policy makers to identify gaps and barriers to uptake and develop specific programming to address them. Ideally, these efforts will enable successful identification of individuals who meet the clinical criteria for PrEP use, as well as frame the development of effective interventions to increase PrEP uptake, adherence, and retention in care, particularly during the postrelease period (Figure 1). Although incarceration represents an opportunity to test criminal justice–involved individuals who are at risk for HIV and link them to HIV and/or pre-exposure prophylaxis (PrEP) care in the community as needed, surveys conducted in criminal justice settings show uneven uptake of HIV testing, preventive, and treatment services among criminal justice–involved individuals [18]. PrEP is currently available as an oral medication in two forms: tenofovir disoproxil fumarate and emtricitabine (TDF/FTC) and tenofovir alafenamide and emtricitabine (TAF/FTC). Both medications are highly effective at preventing HIV transmission among MSM [19,20]. TDF/FTC has also been shown to be effective at preventing HIV transmission Figure 1. PrEP care continuum for incarcerated populations. PrEP: pre-exposure prophylaxis. There are a number of elements to consider for the successful implementation of its use within the criminal justice setting. Although many individuals who pass through the criminal justice system are at increased risk for HIV, the Centers for Disease Control and Prevention (CDC) provides guidance for the clinical criteria for PrEP use that should be used when identifying individuals that are indicated for PrEP [36]. Additionally, for PrEP to be effective, adherence to the medication is crucial [22,37]. Finally, persistence in PrEP care is defined as maintaining all aspects of recommended PrEP JMIR Res Protoc 2022 \| vol. 11 \| iss. 2 \| e31928 \| p. 2 https://www.researchprotocols.org/2022/2/e31928 (page number not for citation purposes) FO There are a number of elements to consider for the successful implementation of its use within the criminal justice setting. https://www.researchprotocols.org/2022/2/e31928 JMIR Res Protoc 2022 \| vol. 11 \| iss. 2 \| e31928 \| p. 2 (page number not for citation purposes) Identify Incarcerated Individuals at Highest Risk for Contracting HIV Identify Incarcerated Individuals at Highest Risk for Contracting HIV The variability in the availability of HIV screening and preventive services in the correctional setting presents one of the biggest challenges to successfully implementing PrEP care in the criminal justice system. This challenge is compounded in part by the potentially high volume of criminal justice–involved individuals who meet CDC criteria for PrEP initiation, but who may not self-identify as being at risk for HIV. There may be a number of reasons why individuals do not self-identify as being at risk for HIV acquisition including stigma, fear of legal repercussions for disclosing criminal behavior, or a lack of awareness of the risk of acquiring HIV from certain behaviors [40-42]. Perhaps the greatest public health benefit of PrEP use would be among pretrial individuals who are detained in the country’s jail systems [43]. With a high volume of individuals who often spend a short period of time in confinement, quickly identifying individuals who are at increased risk of HIV acquisition and then linking them to HIV preventive care in the community poses significant challenges but could have a significant public health benefit. A different approach for identifying individuals who would benefit from HIV preventive care among the sentenced population, many of whom spend months or years incarcerated, warrants tailored clinical tools that acknowledge unique HIV acquisition risks Assess Interest in, Willingness to, and Barriers to Taking PrEP Assess Interest in, Willingness to, and Barriers to Taking PrEP The PrEP Care Continuum Although there is interest among incarcerated individuals in taking PrEP while in the criminal justice setting [45], it is important to characterize PrEP initiation patterns, hesitancy related to its use, and the influence of barriers both within the criminal justice system and upon reentry into the community. Components of this element of the PrEP care continuum are largely unknown and are likely to vary given the diverse makeup of those who experience incarceration, their socioeconomic context in the community, and the behaviors that put individuals at increased risk of HIV acquisition. https://www.researchprotocols.org/2022/2/e31928 Defining the PrEP Care Continuum for Criminal Justice–Involved Populations Although many individuals who pass through the criminal justice system are at increased risk for HIV, the Centers for Disease Control and Prevention (CDC) provides guidance for the clinical criteria for PrEP use that should be used when identifying individuals that are indicated for PrEP [36]. Additionally, for PrEP to be effective, adherence to the medication is crucial [22,37]. Finally, persistence in PrEP care is defined as maintaining all aspects of recommended PrEP XSL•FO RenderX XSL•FO RenderX JMIR RESEARCH PROTOCOLS Murphy et al clinical care during a defined period (ie, attending care appointments, attending lab visits, taking medication as prescribed) [38]. For individuals with ongoing HIV risk, persistence in PrEP care should extend for as long as it is indicated. not currently addressed by CDC guidelines, which focus on risk behaviors during the prior 6 months [43]. For individuals incarcerated for greater than 6 months, their risk for HIV acquisition is unlikely to be captured using this criterion, particularly as individuals prepare for reentry into the community postincarceration [18]. Within the three phases of the continuum (PrEP awareness, uptake, and adherence), the steps are the following: (1) identify incarcerated individuals at highest risk for contracting HIV, (2) refer to health care provider within the criminal justice system, (3) increase PrEP awareness among those individuals, (4) assess willingness to take PrEP, (5) prescribe, and (6) initiate PrEP while still incarcerated, (7) link to PrEP care in the community, (8) support adherence to PrEP, (9) retain individuals in care, and (10) support persistence in PrEP care. This multistep process highlights the potential complexity of implementing a clinical intervention within a criminal justice context and then linking individuals to clinical care in the community upon release. JMIR Res Protoc 2022 \| vol. 11 \| iss. 2 \| e31928 \| p. 3 (page number not for citation purposes) Increase PrEP Awareness Among Those Individuals The little that is known about PrEP awareness in the criminal justice system has demonstrated a lack of knowledge but significant interest [24-26]. Encounters with health care providers and public health support staff in the criminal justice setting can serve as important opportunities to discuss the benefits of PrEP use and improve an individual’s understanding of their own risk of HIV acquisition. Refer to Health Care Provider Within the Criminal Justice System Refer to Health Care Provider Within the Criminal Justice System For individuals at an increased risk of HIV acquisition, referral to a health care provider in the criminal justice setting who could order HIV, sexually transmitted infection, and other lab testing necessary to initiate PrEP is an important and often limiting step. Given the variability in access to medical providers in the criminal justice setting, particularly providers who are knowledgeable about PrEP, this may present a unique challenge to PrEP care implementation [44]. The identification and training of health care providers who could assess for PrEP eligibility and initiate the clinical care associated with its use will be important in this setting [43,44]. The challenges of successfully defining and implementing a care continuum have been more thoroughly studied among individuals who are living with HIV. Interventions to diagnose, initiate treatment for, and link people living with HIV to care in the community have become an important public health priority with a significant impact on the trajectory of the epidemic in the United States [5,39]. In order to broaden the public health impact of using the criminal justice system as a potential focal point for effective HIV prevention interventions, it is important to understand effective implementation strategies and interventions to promote PrEP uptake among the diverse populations that are both disproportionately impacted by HIV and criminal justice involvement. Increase PrEP Awareness Among Those Individuals Prescribe PrEP Many criminal justice–involved individuals experience barriers to accessing primary and preventive clinical care in the community [46]. Therefore, completing the clinical assessments and laboratory testing necessary to initiate PrEP during a period of detention provides significant advantages in promoting PrEP use. Ensuring individuals are HIV-negative, evaluating individuals for renal dysfunction, and assessing them for hepatitis B infection—disease processes that all disproportionately impact the criminal justice–involved population—facilitates identification of the few potential clinical limitations for PrEP use [47,48]. In addition to limited accessibility of PrEP providers and the clinical assessment needed to safely initiate PrEP, cost of the medication to criminal justice institutions may pose a barrier to its implementation. Onsite availability and prompt dispensation, particularly for XSL•FO RenderX XSL•FO RenderX JMIR RESEARCH PROTOCOLS Murphy et al approach will allow for the monitoring of changes in HIV acquisition risk behaviors as well as indications for continued PrEP use. pretrial individuals who may be spending only a brief period in detention, may pose a logistical challenge to criminal justice institutions and their clinical service providers [43]. Link to PrEP Care in the Community Linkage to care in the community poses another significant challenge to PrEP care among criminal justice–involved individuals. Currently, providing continuity of care for both pretrial individuals as well as those sentenced to a period of confinement poses a challenge, particularly for those who are HIV-positive, are impacted by substance use disorder, or have general medical needs [49,50]. The availability and accessibility of PrEP care providers is likely to vary significantly based on geographic region, an individual’s insurance status, and the existence of other health linkage programs unrelated to PrEP care [51,52]. Support Adherence to PrEP Although providing incarcerated individuals with a supply of medication upon release is likely to improve adherence by reducing a crucial barrier to PrEP access, adherence during the postrelease period is largely unknown and unstudied [43,53]. Given the importance of adherence to PrEP’s effectiveness, adherence patterns during the postrelease period represent an important knowledge gap and one that is likely to require tailored interventions to improve. This study uses both biological markers of PrEP adherence through dried blood spot measurements of TDF/FTC concentrations as well as self-reported measures of PrEP adherence. During the meeting with the PrEP clinical provider, the provider and patient discuss HIV risk, the provider assesses the patient’s baseline knowledge and interest in using PrEP, and the provider requisitions the necessary lab work and obtains the patient’s medical history to determine whether the patient is a candidate for PrEP use per CDC guidelines. For those interested in participating in the study, written informed consent is obtained and clinical documentation is shared with the research team at a community partner site. A PrEP navigator then subsequently meets with the patient and helps to coordinate postrelease PrEP care including measures of adherence and ongoing HIV acquisition risk. PrEP clinical care and enrollment in this study are centered largely on the pretrial population housed in RIDOC’s intake facility, which saw approximately 13,000 commitments during 2019 [55]. Results Given that men make up more than 90% of individuals who are incarcerated, and also comprise a majority of new HIV cases, we have proposed a study that focuses on PrEP uptake among men currently experiencing incarceration. This includes all men experiencing incarceration, including those detained in pretrial facilities and those sentenced to a period of confinement. The study is an open, prospective cohort study of 100 men who are currently incarcerated at the Rhode Island Department of Corrections (RIDOC) and who are scheduled to be released within one month of being enrolled in the study. RIDOC is a unique study environment, as it is a unified correctional system with all statewide pretrial and sentenced individuals detained on one campus under a single administrative structure. Every individual processed at RIDOC undergoes a nurse-led medical evaluation at intake (Figure 2). Individuals who report behaviors that would place them at increased HIV risk (condomless sex with multiple sexual partners; sex work and/or transactional sex; or injection drug use) are then referred to a general medical provider for potential study enrollment and/or initiation of PrEP clinical care through standard medical screening procedures. The existing nurse evaluation is brief and not intended to be a comprehensive evaluation of HIV acquisition risk. Other staff and medical providers can also refer individuals to the PrEP provider and program if they determine an individual to be at increased risk of HIV acquisition. Ethics Approval There is a well-documented risk of HIV infection among criminal justice–involved individuals during the period immediately following release [46]. Ideally, criminal justice–involved individuals at increased risk for HIV acquisition would initiate PrEP prior to release. This strategy would also allow for observation and evaluation of side effects, while providing an opportunity to initiate a protective biomedical intervention that can be continued in the community. Providing individuals with medication upon release may improve adherence in the crucial postrelease period, while reducing a potential barrier to HIV preventive care in this potentially chaotic period [29,37]. The study underwent review and approval by the Lifespan Institutional Review Board's special committee on research conducted among incarcerated populations. Additional review and approval was provided by the Rhode Island Department of Corrections' Ethical Review Advisory Group. JMIR Res Protoc 2022 \| vol. 11 \| iss. 2 \| e31928 \| p. 4 (page number not for citation purposes) Support Retention and Persistence in PrEP Care The goals of this study are to increase our knowledge of the characteristics and HIV risk of criminal justice–involved men, improve our understanding of the PrEP care continuum in a criminal justice setting to address these risks, and evaluate real-world barriers to PrEP care among this vulnerable population. This study is expected to conclude on November 30th, 2022. https://www.researchprotocols.org/2022/2/e31928 Support Retention and Persistence in PrEP Care Once an interested individual is clinically screened and determined to be eligible for PrEP, TDF/FTC is prescribed, and the medication is delivered to the detention facility so that the individual may begin taking the medication prior to being released into the community; they are provided with a short-term supply upon release. Individuals are referred to a PrEP clinic in the community where they will continue their PrEP care postrelease. Enrolled individuals are provided with an appointment date and time prior to their release, which is facilitated by a patient navigator who also helps to ensure the communication of important clinical information between the criminal justice clinical providers and the community site. The patient navigator also helps individuals address barriers that arise postrelease to facilitate retention in postrelease PrEP clinical care [56]. The primary clinical outcomes are the following: (1) PrEP uptake (yes/no prescription), (2) adherence (measured by self-report and dried blood spot), and (3) attendance at 1 follow up visit (yes/no attendance). The goals of this study are to increase our knowledge of the characteristics and HIV risk of criminal justice–involved men, improve our understanding of the PrEP care continuum in a criminal justice setting to address these risks, and evaluate real-world barriers to PrEP care among this vulnerable population. This study is expected to conclude on November 30th, 2022. Once an interested individual is clinically screened and determined to be eligible for PrEP, TDF/FTC is prescribed, and the medication is delivered to the detention facility so that the individual may begin taking the medication prior to being released into the community; they are provided with a short-term supply upon release. Individuals are referred to a PrEP clinic in the community where they will continue their PrEP care postrelease. Enrolled individuals are provided with an appointment date and time prior to their release, which is facilitated by a patient navigator who also helps to ensure the communication of important clinical information between the criminal justice clinical providers and the community site. The patient navigator also helps individuals address barriers that arise postrelease to facilitate retention in postrelease PrEP clinical care [56]. The primary clinical outcomes are the following: (1) PrEP uptake (yes/no prescription), (2) adherence (measured by self-report and dried blood spot), and (3) attendance at 1 follow up visit (yes/no attendance). Support Retention and Persistence in PrEP Care Retention and persistence in PrEP care pose a number of challenges in “real-world” community settings [31]. It is likely, although largely unknown, that retention and persistence in PrEP care will also pose a significant albeit unique challenge to those with criminal justice involvement given the barriers that have been well documented in retaining individuals with criminal justice involvement in HIV care [31], hepatitis C care, general primary care, and preventive care, to name a few [54]. By establishing a prospective cohort of individuals returning to the community postincarceration, the outlined methodological JMIR Res Protoc 2022 \| vol. 11 \| iss. 2 \| e31928 \| p. 4 (page number not for citation purposes) https://www.researchprotocols.org/2022/2/e31928 XSL•FO RenderX XSL•FO RenderX Murphy et al JMIR RESEARCH PROTOCOLS Figure 2. Implementing PrEP care continuum for criminal justice–involved populations. ACI: adult correctional institutions; PrEP: pre-exposure prophylaxis Figure 2. Implementing PrEP care continuum for criminal justice–involved populations. ACI: adult correctional institutions; PrEP: pre-exposure prophylaxis. igure 2. Implementing PrEP care continuum for criminal justice–involved populations. ACI: adult correctional institutions; PrEP: pre-exposu rophylaxis. incarcerated and recently incarcerated men. This study will help characterize several key elements related to PrEP implementation that will need consideration in order for PrEP use to be implemented to scale in the criminal justice system in the United States. One important consideration for public health policy makers that work within the criminal justice system is the identification of individuals who meet clinical criteria for PrEP usage. Many individuals who pass through the criminal justice system are held for a brief period of time prior to sentencing (eg, pretrial). The study protocol presented here is designed to address gaps in the existing literature by documenting the number and type of individuals who meet the clinical criteria for PrEP, the organizational resources in the criminal justice setting, and the processes required to successfully implement PrEP care in this setting. For individuals who are detained for a significant period of time, particularly those who are sentenced to prolonged periods of detention, understanding how to incorporate HIV prevention into the criminal justice setting would be particularly helpful. Research on health-promoting interventions for HIV-negative criminal justice–involved individuals has generally focused on chronic care management during the period of transition from detention to community reentry [57]. This approach may benefit from incorporating HIV preventive care into the design of clinical linkage interventions. Discussion Support from correctional leadership is key to the success of effective HIV prevention and PrEP programming. The commitment to effectively implement PrEP may vary The results from this study will help to identify predictors for initiation, adherence, and retention in PrEP care among JMIR Res Protoc 2022 \| vol. 11 \| iss. 2 \| e31928 \| p. 5 (page number not for citation purposes) JMIR Res Protoc 2022 \| vol. 11 \| iss. 2 \| e31928 \| p. 5 (page number not for citation purposes) https://www.researchprotocols.org/2022/2/e31928 XSL•FO RenderX JMIR RESEARCH PROTOCOLS Murphy et al significantly between different correctional departments, particularly given the cost of medications as well as the clinical and support personnel that might be required. In RIDOC, support from administrative and medical leadership has been key to the feasibility of conducting this study. Moving forward, a greater understanding of this variability and its impact on effective PrEP implementation will be critical. This will be particularly true if an injectable version of PrEP becomes available. A long-acting version of PrEP may provide greater protection from HIV acquisition, particularly in the chaotic postrelease period when adhering to a daily pill may be difficult for this population. represents an opportunity to reach and intervene with MSM who may not be traditionally reached otherwise (eg, LGBTQ+ engaged in health care). This is because criminal justice–involved men may be less likely to seek medical care in the community and more likely to have risk behaviors that are more highly stigmatized (eg, having sex with men for money, for drugs, or to meet other needs). There are likely to be some important differences among different populations experiencing incarceration who are eligible for PrEP, and characterizing these nuances in clinical care will be important to fully understanding what is required to encourage PrEP uptake and engagement in care among criminal justice–involved individuals. Finally, this population experiences many challenges in being linked to care while also experiencing extremely high risk for HIV acquisition [10,17,58,59]. Successful implementation of PrEP care within the criminal justice system and linking criminal justice–involved individuals to PrEP care in the community has the potential to significantly reduce the spread of HIV both within the criminal justice setting and the broader community, and is an important step to ending the HIV epidemic in the United States [60,61]. Discussion Beyond the consideration of adapting PrEP clinical processes to the criminal justice context, encouraging PrEP uptake for individuals from multiply marginalized backgrounds, linking individuals to care in the community postrelease, and addressing HIV risk and PrEP use in the context of the complex health needs of criminal justice–involved individuals are likely to require different approaches than may have been successful in other populations. Importantly, the criminal justice system Conflicts of Interest BR receives funding from Gilead Sciences for a research project (number IN-US-276-5463). BR receives funding from Gilead Sciences for a research project (number IN-US-276-5463 References 1. Diagnoses of HIV Infection in the United States and Dependent Areas, 2018. Centers for Disease Control and Prevention. 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URL: http://www. doc.ri.gov/docs/FY19%20Annual%20Population%20Report.pdf [accessed 2021-01-13] JMIR Res Protoc 2022 \| vol. 11 \| iss. 2 \| e31928 \| p. 8 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX JMIR RESEARCH PROTOCOLS JMIR Res Protoc 2022 \| vol. 11 \| iss. 2 \| e31928 \| p. 9 (page number not for citation purposes) Abbreviations CDC: Centers for Disease Control and Prevention MSM: men who have sex with men PrEP: pre-exposure prophylaxis RIDOC: Rhode Island Department of Corrections TAF/FTC: tenofovir alafenamide and emtricitabine TDF/FTC: tenofovir disoproxil fumarate and emtricitabine Edited by G Eysenbach; submitted 12.07.21; peer-reviewed by B Castonguay, C Khosropour; comments to author 25.10.21; revised version received 07.12.21; accepted 08.12.21; published 10.02.22 Please cite as: Murphy M, Sosnowy C, Rogers B, Napoleon S, Galipeau D, Scott T, Tao J, Berk J, Clarke J, Nunn A, Chan PA Defining the Pre-exposure Prophylaxis Care Continuum Among Recently Incarcerated Men at High Risk for HIV Infection: Protocol for a Prospective Cohort Study JMIR Res Protoc 2022;11(2):e31928 URL: https://www.researchprotocols.org/2022/2/e31928 doi: 10.2196/31928 PMID: ©Matthew Murphy, Collette Sosnowy, Brooke Rogers, Siena Napoleon, Drew Galipeau, Ty Scott, Jun Tao, Justin Berk, Jennifer Clarke, Amy Nunn, Philip A Chan. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 10.02.2022. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on https://www.researchprotocols.org, as well as this copyright and license information must be included. https://www.researchprotocols.org/2022/2/e31928 https://www.researchprotocols.org/2022/2/e31928 JMIR Res Protoc 2022 \| vol. 11 \| iss. 2 \| e31928 \| p. 9 (page number not for citation purposes)
https://openalex.org/W2160471436	https://europepmc.org/articles/pmc4110246?pdf=render	English	null	Association Between Contraceptive Use and Gestational Diabetes: Missouri Pregnancy Risk Assessment Monitoring System, 2007–2008	Preventing chronic disease	2,014	public-domain	4,113	Conclusion i di Findings suggest there may be a relationship between type of contraceptive method and GDM. More research is needed to verify contraception as a potential risk factor for GDM. Results Of the 2,741 women who completed the 2007–2008 PRAMS survey, 8.3% were diagnosed with gestational diabetes, and 17.9% of the respondents had used hormonal contraceptive methods. Women who used hormonal methods of birth control had higher odds for gestational diabetes (adjusted odds ratio [AOR] = 1.43; 95% confidence interval [CI], 1.32–1.55) than did women who used no contraception. A protective effect was also observed for women who had used barrier methods of contraception (AOR = 0.79; 95% CI, 0.72–0.86). Introduction Introduction The efficacy and safety of contraceptives have been questioned for decades; however, whether a relationship exists between hormonal contraceptives and gestational diabetes (GDM) is undetermined. The aim of this study was to investigate whether maternal risk for GDM was influenced by type of contraceptive method used before pregnancy. Methods Volume 11 — July 17, 2014 ORIGINAL RESEARCH Association Between Contraceptive Use and Gestational Diabetes: Missouri Pregnancy Risk Assessment Monitoring System, 2007–2008 Brittney A. Kramer; Jeremy Kintzel, MA; Venkata Garikapaty, PhD Suggested citation for this article: Kramer BA, Kintzel J, Garikapaty V. Association Between Contraceptive Use and Gestational Diabetes: Missouri Pregnancy Risk Assessment Monitoring System, 2007–2008. Prev Chronic Dis 2014;11:140059. DOI: http://dx.doi.org/10.5888/pcd11.140059 . Page 1 of 6 Preventing Chronic Disease \| Association Between Contraceptive Use and Gestational Di... Volume 11 — July 17, 2014 Methods Methods Data collected in 2007 and 2008 by the Missouri Pregnancy Risk Assessment Monitoring System (PRAMS) were analyzed to determine if type of contraception before pregnancy influenced maternal risk for GDM. We used a logistic regression model to determine the adjusted odds for GDM given exposure to hormonal forms of contraception. Preventing Chronic Disease \| Association Between Contraceptive Use and Gestat Preventing Chronic Disease \| Association Between Contraceptive Use and Gestat Association Between Contraceptive Use and Gestational Diabetes: Missouri Pregnancy Risk Assessment Monitoring System, 2007–2008 Brittney A. Kramer; Jeremy Kintzel, MA; Venkata Garikapaty, PhD Brittney A. Kramer; Jeremy Kintzel, MA; Venkata Garikapaty, PhD Suggested citation for this article: Kramer BA, Kintzel J, Garikapaty V. Association Between Contraceptive Use and Gestational Diabetes: Missouri Pregnancy Risk Assessment Monitoring System, 2007–2008. Prev Chronic Dis 2014;11:140059. DOI: http://dx.doi.org/10.5888/pcd11.140059 . Suggested citation for this article: Kramer BA, Kintzel J, Garikapaty V. Association Between Contraceptive Use and Gestational Diabetes: Missouri Pregnancy Risk Assessment Monitoring System, 2007–2008. Prev Chronic Dis 2014;11:140059. DOI: http://dx.doi.org/10.5888/pcd11.140059 . Suggested citation for this article: Kramer BA, Kintzel J, Garikapaty V. Association Between Contraceptive Use and Gestational Diabetes: Missouri Pregnancy Risk Assessment Monitoring System, 2007–2008. Prev Chronic Dis 2014;11:140059. DOI: http://dx.doi.org/10.5888/pcd11.140059 . PEER REVIEWED Introduction Over the years, concerns have been raised about the possible association between hormonal contraceptives and various chronic diseases, including cardiovascular disease, breast cancer, and metabolic dysfunction. However, little is known about hormonal contraceptive use and its role in the development of gestational diabetes (GDM). Research has established a relationship between oral, hormonal contraceptive use and increased levels of serum glucose, insulin, and altered lipid profiles (1–4); however, much of the research is still unable to reach consensus on the long-term effects of contraceptives and an increased risk for GDM. Studies have examined the effects of contraception on the metabolisms of women who were previously diagnosed with GDM and found that women using hormonal methods of contraception had a higher risk for type 2 diabetes than did women using nonhormonal contraceptive methods (4). Nevertheless, no studies have investigated the effects of hormonal contraceptive use before pregnancy and the risk for GDM. GDM is often diagnosed in weeks 24 through 28 of pregnancy and is characterized by no previous history of diabetes (type 1 or type 2) and high blood glucose levels (5). If left uncontrolled, GDM can result in macrosomia (“fat” baby), which can cause complications at birth and potentially force the mother to have a caesarian delivery. Other potential side effects involve preeclampsia and hypoglycemia (low blood glucose) in the mother, jaundice in the baby, and an increased risk for type 2 diabetes in both the mother and child later in life. GDM is estimated to affect 2% to 10% of all Page 2 of 6 Preventing Chronic Disease \| Association Between Contraceptive Use and Gestational Di... Page 2 of 6 Preventing Chronic Disease \| Association Between Contraceptive Use and Gestational Di... pregnancies, although US prevalence has increased since the late 1980s and has more than doubled since 1990 (6,7). Therefore, it is crucial to identify and understand risk factors for GDM to better tailor preventive and intervention measures. The Missouri Pregnancy Risk Assessment Monitoring System (PRAMS) is an ongoing, population-based survey designed to identify and monitor select maternal experiences, attitudes, and behaviors that occur before, during, and shortly after pregnancy among women delivering a live infant. Data from PRAMS can be used to determine whether a relationship exists between a woman’s contraceptive use before pregnancy and the development of GDM. Data collection PRAMS is conducted by the Missouri Department of Health and Senior Services as part of the national program sponsored by the Centers for Disease Control and Prevention (CDC). PRAMS is administered in 40 states and in New York City and represents approximately 78% of all US live births. Missouri began administering PRAMS in 2007. PRAMS collects information from a sample of women whose names are drawn from recent birth certificates (2 to 4 months after birth). A survey is mailed to each woman with telephone follow-up to those who do not respond. Responses are weighted to represent all live births in a given year. In Missouri, 2,741 women responded to the survey in 2007–2008, for an average weighted response rate of 63.9%. Study population Missouri has participated in PRAMS continuously since 2007, beginning with Phase V of the survey (2007–2008). Missouri is collecting Phase VII data (2012 to present); we used Phase V data because the Phase V questionnaire asked participating women to identify the type of contraceptive they were using prepregnancy and postpartum, if applicable. Women could respond that they or their partner were using tubal ligation, vasectomy, birth control pills, condoms, injections (monthly or every 3 months), contraceptive patches, a diaphragm, a cervical ring, an intrauterine device, the rhythm method, withdrawal, or another method. They could also respond that they were not sexually active. Women were identified as having been diagnosed with GDM if they reported that they had high blood glucose (diabetes) that started during the current pregnancy or if they had reported GDM on the matched birth certificate and did not respond in PRAMS that they had high blood glucose (diabetes) that started before the current pregnancy. This approach replicates GDM prevalence reported for Missouri by CDC’s PRAMS C-PONDER query tool for 2007; 2008 data are unavailable in C-PONDER because Missouri did not meet the minimum required weighted response rate threshold for inclusion of 70%. Methods of prepregnancy contraception were categorized as hormonal, barrier, fertility awareness, other, or none. Methods of hormonal contraception included the pill, injections (received monthly or every 3 months), contraceptive patches, cervical ring, or intrauterine devices (IUD). Barrier methods included condoms or diaphragm, and fertility awareness methods included the rhythm or withdrawal methods. Respondents who reported that they were not using birth control when they became pregnant or reported that they were not using any of these specified methods were categorized as not using any contraception. All others were categorized as “other.” These categories were selected on the basis of CDC categorization of birth control methods as either reversible birth control methods or permanent methods of birth control. All permanent methods (tubal ligation or vasectomy) were excluded because conception is next to impossible after these procedures, and the respondents were reporting this method as their contraceptive method before their most recent pregnancy. Introduction Research conducted by Visser et al compared hormonal and nonhormonal contraceptive use by diabetic women and found that high-dose oral contraceptives impaired glucose homeostasis (8). Likewise, Diab and Zaki found that fasting blood glucose was higher among users of oral contraceptives and Depo-Provera (depot-medroxyprogesterone acetate, DMPA), a progestogen-only reversible hormonal contraceptive injected every 3 months (9). After reviewing these studies, we hypothesized that an increased risk for GDM would be observed among women who had used hormonal methods of contraception. Our objective was to identify whether, after controlling for other factors, women who used hormonal methods of contraceptives before their pregnancy were more or less likely to develop GDM. Discussion This study provides evidence that hormonal contraceptive methods may increase a woman’s risk for GDM in her following pregnancy, even after adjusting for maternal age, race, education and income level, marital status, Medicaid status at delivery, and type of prenatal care received. Other variables that were associated with higher odds for GDM were women who had a high school education or more, were 30 years or older, were of nonwhite or nonblack race, and were overweight or obese before pregnancy. These findings are consistent with those of other studies, one of which found that risk for GDM was correlated with increasing maternal age, high prepregnancy BMI, and high weight gain during pregnancy (10). Similarly, another study found that woman from racial/ethnic minority groups had a higher prevalence of GDM and that age and high BMI were also independent risk factors (11). It has been long accepted in the medical field that higher BMI, increasing age (>25 y), and nonwhite race are risk factors for GDM (12). Our study, along with others, validates this consensus and suggests that intervention strategies should continue to focus on these demographic groups. Other variables associated with higher odds for GDM included adequate access to prenatal care and being enrolled in Medicaid at delivery. Women who are enrolled in Medicaid and are receiving adequate prenatal care are more likely to see a doctor more frequently during their pregnancy and therefore have any pregnancy complications, including GDM, observed and diagnosed. This observation was also determined by Alexander and Cornely, who found that women who received intensive prenatal care were more likely to have more pregnancy complications, but also the most preferable birth outcomes (13). Furthermore, Griffin and colleagues determined that Medicaid expansion programs allowed women to receive the same treatment and prevention services on par with adequate prenatal care (14). These studies explain the higher odds observed for women who are receiving adequate prenatal care or Medicaid services; they are more likely to be diagnosed with GDM because they are more likely to be receiving frequent adequate care and therefore to be screened and diagnosed with it as opposed to women who may not be able to receive adequate care and consequently miss the opportunity to be diagnosed with GDM. Protective effects were seen in women who had not intended or planned their pregnancy or who used barrier methods for contraception. Results Of the 2,741 women who completed the 2007–2008 PRAMS, 8.3% reported having been diagnosed with GDM in their most recent pregnancy. The most prevalent form of contraception used by Missouri PRAMS respondents was hormonal methods of contraception (17.9%), followed by barrier methods (17.2%), fertility awareness/rhythm method (6.8%), and other (2.3%). Of the total sample, 56% reported using no contraception. Results of multivariate logistic regression indicated that women who used hormonal birth control methods were 1.4 times more likely to develop GDM than were those who did not use any birth control method (95% confidence interval [CI], 1.32–1.55, P < .001) (Table). Several other variables were associated with higher odds for developing GDM: women aged 30 or older compared with those younger than 20 (AOR = 1.50; 95% CI, 1.34–1.67; P < .001), women who received adequate prenatal care compared with women who received inadequate or intermediate prenatal care (AOR = 2.36; 95% CI, 2.16–2.58), women enrolled in Medicaid at delivery compared with women who were not enrolled in Medicaid at delivery (AOR = 2.58; 95% CI, 2.36–2.81), women who identified as nonwhite or nonblack compared with white women (AOR = 5.54; 95% CI, 4.90–6.25), and women who were overweight or obese before pregnancy compared with women with a normal BMI before pregnancy (AOR = 3.04; 95% CI, 2.84–3.24) (Table). A protective effect for GDM was observed for women who had used barrier methods for contraception compared with women who did not use any contraceptive methods (AOR = 0.79; 95% CI, 0.72–0.86) (Table) and for women with an unintended pregnancy compared with women with an intended pregnancy (AOR = 0.39; 95% CI, 0.37–0.42) (Table). Data analysis Data were analyzed using SAS Enterprise Guide 4.3 (SAS Institute, Inc, Cary, North Carolina). A regression model was constructed with GDM as the outcome variable and other variables as predictor variables, representing respondents’ demographics and preconception health, pregnancy health, and other birth outcomes. These variables included the mother’s age, education level, race, Medicaid status (at delivery), marital status, contraceptive method, pregnancy intent, prepregnancy body mass index (BMI in kg/m , calculated from participants’ self-reported height and weight), pregnancy weight gain, income, folic acid multivitamin use, adequacy of prenatal care, number of prenatal stressors experienced, use of assisted reproductive technology or fertility drugs, preterm or low birth weight baby, WIC 2 Page 3 of 6 Preventing Chronic Disease \| Association Between Contraceptive Use and Gestational Di... participation, and a range of other prenatal maternal morbidities, including vaginal bleeding, high blood pressure, and preterm labor. participation, and a range of other prenatal maternal morbidities, including vaginal bleeding, high blood pressure, and preterm labor. All variables from the univariate model were selected as predictor variables for the final, multivariate model. A backward stepwise selection was performed on all variables, which included respondents’ age, education level, race, Medicaid status, marital status, pregnancy intent, contraceptive method, prepregnancy BMI, income, folic acid multivitamin use, and adequacy of prenatal care. For the adjusted multivariate model, methods of contraception were categorized and analyzed as hormonal, barrier, fertility awareness, other, or none. The final model was tested for collinearity, and all predictor variables were significant at P < .001. Acknowledgments The authors thank Mary Mosley, Missouri state PRAMS coordinator, for hosting this research project and making the data available to the authors and Leigh Tenkku for encouragement and continual help and support. No financial support was received for the analysis or support of this article. Page 4 of 6 Preventing Chronic Disease \| Association Between Contraceptive Use and Gestational Di... contraception are not increasing levels of hormones in their bodies and do not have the potential and undetermined side effects and, we propose, have a lower risk for GDM. PRAMS data are subject to several limitations. First, PRAMS is a self-reported survey administered 2 to 6 months after the birth of a child, and results may be subject to recall bias. Second, weighting may not completely adjust for bias resulting from nonresponse. Third, PRAMS surveys are sent to a sample of women who delivered live babies in Missouri, and data are not generalizable to pregnant women with other outcomes or in different states. This is the first study to evaluate the relationship between type of contraception used before pregnancy and risk for GDM. However, many studies have determined that hormonal methods of contraception have the potential to alter several homeostatic mechanisms including the hypothalamic pituitary axis as well as cortisol and carbohydrate metabolism. Burke suggested that prolonged exposure to high-dose estrogen treatment was associated with higher levels of unbound cortisol, which implied interference with cortisol homeostasis at the hypothalamic level (16). These higher levels of cortisol contribute to glucose intolerance and insulin resistance, which if left untreated can lead to diabetes (17). This study’s significant finding correlating contraceptive method and risk for GDM merits further exploration. The prevalence of GDM continues to increase worldwide, and definitive screening and preventive measures are needed to deter its chronic lifelong complications. Although researchers have not established a causal relationship between hormonal contraception use and GDM, results of our study suggest there may be an underlying correlating mechanism. More research is needed to assess hormonal contraception use as a potential risk factor for GDM. Author Information Corresponding Author: Venkata Garikapaty, PhD, Missouri Department of Health and Senior Services, Office of Epidemiology, Maternal and Child Health, 920 Wildwood Dr, P.O. Box 570, Jefferson City, MO 65102. Telephone: 573 -526-0452. E-mail: Venkata.Garikapaty@health.mo.gov. Author Affiliations: Brittney A. Kramer, Jeremy Kintzel, Missouri Department of Health and Senior Services, Jefferson City, Missouri. Author Affiliations: Brittney A. Kramer, Jeremy Kintzel, Missouri Department of Health and Senior Services, Jefferson City, Missouri. Discussion As described previously, women who do not plan their pregnancy often underuse prenatal care services and receive inadequate care (15). This effect is the opposite of what was described previously, since women with an unintended pregnancy often inadequately seek and use prenatal care, so they are less likely to be seen and diagnosed by a doctor for GDM. On the basis of our hypothesis, women who are not using any form of hormonal contraception should be at an observed lower risk for developing GDM. Women who practice barrier methods of Page 4 of 6 Preventing Chronic Disease \| Association Between Contraceptive Use and Gestational Di... Page 4 of 6 Preventing Chronic Disease \| Association Between Contraceptive Use and Gestational Di... References Unplanned pregnancy as a major determinant in inadequate use of prenatal care. Prev Med 1997;26(6):834–8. CrossRef PubMed 16. Burke CW. The effect of oral contraceptives on cortisol metabolism. J Clin Pathol 1969;1(1):11–8. CrossRef PubMed of prenatal care utilization in Rhode Island. Am J Public Health 1999;89(4):497–501. CrossRef PubMed 15. Delgado-Rodríguez M, Gómez-Olmedo M, Bueno-Cavanillas A, Gálvez-Vargas R. Unplanned pregnancy as a major determinant in inadequate use of prenatal care. Prev Med 1997;26(6):834–8. CrossRef PubMed 16. Burke CW. The effect of oral contraceptives on cortisol metabolism. J Clin Pathol 1969;1(1):11–8. CrossRef PubMed 17. Andrews RC, Walker BR. Glucocorticoids and insulin resistance: old hormones, new targets. Clin Sci 1999;96:513 –23. CrossRef PubMed References Page 5 of 6 Preventing Chronic Disease \| Association Between Contraceptive Use and Gestational Di... Page 5 of 6 Preventing Chronic Disease \| Association Between Contraceptive Use and Gestational Di... 10. Di Cianni G, Volpe L, Lencioni C, Miccoli R, Cuccuru I, Ghio A, et al. Prevalence and risk factors for gestational diabetes assessed by universal screening. Diabetes Res Clin Pract 2003;62(2):131–7. CrossRef PubMed 10. Di Cianni G, Volpe L, Lencioni C, Miccoli R, Cuccuru I, Ghio A, et al. Prevalence and risk factors for gestational diabetes assessed by universal screening. Diabetes Res Clin Pract 2003;62(2):131–7. CrossRef PubMed 11. Dornhorst A, Paterson CM, Nicholls JSD, Wadsworth J, Chiu DC, Elkeles RS, et al. High prevalence of gestational diabetes in women from ethnic minority groups. Diabet Med 1992;9(9):820–5. CrossRef PubMed 11. Dornhorst A, Paterson CM, Nicholls JSD, Wadsworth J, Chiu DC, Elkeles RS, et al. High prevalence of gestational diabetes in women from ethnic minority groups. Diabet Med 1992;9(9):820–5. CrossRef PubMed y g p 12. Diseases and conditions: risk factors: gestational diabetes. Mayo Clinic. http://www.mayoclinic.org/diseases- conditions/gestational-diabetes/basics/risk-factors/con-2001485413. Accessed January 3, 2014. 12. Diseases and conditions: risk factors: gestational diabetes. Mayo Clinic. http://www.mayoclinic.org/diseases- conditions/gestational-diabetes/basics/risk-factors/con-2001485413. Accessed January 3, 2014. 13. Alexander GR, Cornely DA. Prenatal care utilization: its measurement and relationship to pregnancy outcome. Am J Prev Med 1987;3(5):243–53. PubMed 13. Alexander GR, Cornely DA. Prenatal care utilization: its measurement and relationship to pregnancy outcome. Am J Prev Med 1987;3(5):243–53. PubMed 14. Griffin JF, Hogan JW, Buechner JS, Leddy TM. The effect of a Medicaid managed care program on the adequacy of prenatal care utilization in Rhode Island. Am J Public Health 1999;89(4):497–501. CrossRef PubMed 14. Griffin JF, Hogan JW, Buechner JS, Leddy TM. The effect of a Medicaid managed care program on the adequacy of prenatal care utilization in Rhode Island. Am J Public Health 1999;89(4):497–501. CrossRef PubMed 15. Delgado-Rodríguez M, Gómez-Olmedo M, Bueno-Cavanillas A, Gálvez-Vargas R. Unplanned pregnancy as a 14. Griffin JF, Hogan JW, Buechner JS, Leddy TM. The effect of a Medicaid managed care program on the adequacy of prenatal care utilization in Rhode Island. Am J Public Health 1999;89(4):497–501. CrossRef PubMed 14. Griffin JF, Hogan JW, Buechner JS, Leddy TM. The effect of a Medicaid managed care program on the adequacy of prenatal care utilization in Rhode Island. Am J Public Health 1999;89(4):497–501. CrossRef PubMed 15. Delgado-Rodríguez M, Gómez-Olmedo M, Bueno-Cavanillas A, Gálvez-Vargas R. References 1. Eschwege E, Fontbonne A, , Simon D, Thibult N, Balkau B, Saint-Paul M, et al. Oral contraceptives, insulin resistance and ischemic vascular disease. Int J Gynaecol Obstet 1990;31(3):263–9. CrossRef PubMed 1. Eschwege E, Fontbonne A, , Simon D, Thibult N, Balkau B, Saint-Paul M, et al. Oral contraceptives, insulin resistance and ischemic vascular disease. Int J Gynaecol Obstet 1990;31(3):263–9. CrossRef PubMed 2. Godsland IF, Crook D, Simpson R, Proudler T, Felton C, Lees B, et al. The effects of different formulations of oral contraceptive agents on lipid and carbohydrate metabolism. N Engl J Med 1990;323(20):1375–81. 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National diabetes statistics, 2011. National Diabetes Information Clearinghouse. http://diabetes.niddk.nih.gov/dm/pubs/statistics/#fast. Accessed January 16, 2014. 7. Getahun D, Nath C, Ananth CV, Chavez MR, Smulian JC. Gestational diabetes in the United States: temporal trends 1989 through 2004. Am J Obstet Gynecol 2008;198(5):525–e1–5. CrossRef PubMed 7. Getahun D, Nath C, Ananth CV, Chavez MR, Smulian JC. Gestational diabetes in the United States: temporal trends 1989 through 2004. Am J Obstet Gynecol 2008;198(5):525–e1–5. CrossRef PubMed 8. Visser J, Snel M, Van Vliet HA. Hormonal versus non-hormonal contraceptiv type 1 and 2. Cochrane Database Syst Rev 2006;(4):CD003990. PubMed 9. Diab KM, Zaki MM. Contraception in diabetic women: comparative metabolic study of Norplant, depot medroxyprogesterone acetate, low dose oral contraceptive pill and CuT380A. J Obstet Gynaecol Res 2000;26 (1):17–26. CrossRef PubMed 9. Diab KM, Zaki MM. Contraception in diabetic women: comparative metabolic study of Norplant, depot medroxyprogesterone acetate, low dose oral contraceptive pill and CuT380A. J Obstet Gynaecol Res 2000;26 (1):17–26. CrossRef PubMed Page 5 of 6 Preventing Chronic Disease \| Association Between Contraceptive Use and Gestational Di... Table Table. Maternal Characteristics and Odds of Developing Gestational Diabetes, Missouri Pregnancy Risk Assessment Monitoring System (N = 2,741), 2007–2008 Characteristic Prevalence Adjusted Odds Ratio (95% CI) Contraceptive method Hormonal 8.2 1.43 (1.32–1.55) Barrier 5.9 0.79 (0.72–0.86) Fertility awareness 10.0 1.38 (1.24–1.53) Other 12.0 0.59 (0.47–0.73) None 7.7 1 [Reference] Age, y <20 4.9 1 [Reference] 20–29 7.4 0.98 (0.89–1.08) ≥30 11.1 1.50 (1.34–1.67) Education level Less than high school 7.2 1 [Reference] High school or more 8.4 1.38 (1.27–1.50) Race White 7.9 1 [Reference] Black 7.7 1.32 (1.22–1.43) Other 18.0 5.54 (4.90–6.25) Medicaid status at delivery Medicaid 9.0 2.58 (2.36–2.81) Non-Medicaid 7.4 1 [Reference] Pregnancy intent a Page 6 of 6 Preventing Chronic Disease \| Association Between Contraceptive Use and Gestational Di... Characteristic Prevalence Adjusted Odds Ratio (95% CI) Unintended 6.3 0.39 (0.37–0.42) Intended 9.6 1 [Reference] Prepregnancy weight status (BMI, kg/m ) Underweight (<18.5) 2.3 0.79 (0.64–0.97) Normal weight (18.5–24.9) 4.8 1 [Reference] Overweight/obese (≥25.0) 11.5 3.04 (2.84–3.24) Income, $ <15,000 7.3 0.57 (0.51–0.64) 15,000–24,999 9.3 0.73 (0.65–0.82) 25,000–49,999 9.2 0.72 (0.66–0.80) ≥50,000 7.8 1 [Reference] Folic acid multivitamin use Use 4 times weekly 8.1 1 [Reference] No use 8.1 1.26 (1.16–1.37) Prenatal care status Adequate/plus 9.3 2.36 (2.16–2.58) Inadequate/intermediate 4.4 1 [Reference] Marital status Married 8.5 0.83 (0.77–0.89) Not married 7.7 1 [Reference] Abbreviation: CI, confidence interval; BMI, body mass index. All variables listed were included in final, multivariate model and found significant at P < .001 2 a The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Human Services, the Public Health Service, the Centers for Disease Control and Prevention, stitutions. For Questions About This Article Contact pcdeditor@cdc.gov Page last reviewed: July 17, 2014 Page last updated: July 17, 2014 Content source: National Center for Chronic Disease Prevention and Health Promotion Centers for Disease Control and Prevention 1600 Clifton Rd. Atlanta, GA 30333, USA 800-CDC-INFO (800-232-4636) TTY: (888) 232-6348 - Contact CDC–INFO
https://openalex.org/W4392476633	https://pubs.rsc.org/en/content/articlepdf/2024/cy/d3cy01488f	English	null	Investigations into the influence of nickel loading on MoO<sub>3</sub>-modified catalysts for the gas-phase hydrodeoxygenation of anisole	Catalysis science & technology	2,024	cc-by	15,126	Investigations into the influence of nickel loading on MoO3-modified catalysts for the gas-phase hydrodeoxygenation of anisole† ss Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: Catal. Sci. Technol., 2024 14, 2201 Received 25th October 2023, Accepted 28th February 2024 DOI: 10.1039/d3cy01488f rsc.li/catalysis ss Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence Cite this: Catal. Sci. Technol., 2024, 14, 2201 Simon Haida, Sebastian Löbner, Henrik Lund, Stephan Bartling, Carsten Kreyenschulte, Hanan Atia, Ali M. Abdel-Mageed, Christoph Kubis * and Angelika Brückner * Simon Haida, Sebastian Löbner, Henrik Lund, Stephan Bartling, Carsten Kreyenschulte, Hanan Atia, Ali M. Abdel-Mageed, Christoph Kubis * and Angelika Brückner * Molybdenum oxide-based catalysts are promising catalysts for the gas-phase hydrodeoxygenation (HDO) of lignocellulosic pyrolysis oils with high selectivities to arenes. The gas-phase HDO is conducted at temperatures between 300 °C and 400 °C at ambient pressure, resulting in a low hydrogen consumption and high energy efficiency. The loading of nickel forming a binary structure consisting of MoO3 and nickel molybdate (NiMoO4) during calcination results in a significant increase of the catalytic activity connected with a drastic shortening of the induction period observed for the unpromoted catalyst system. The promotional effect of nickel seems to be related to its enhanced reduction behaviour and hydrogen dissociation leading eventually to an improved formation of a molybdenum oxycarbohydride phase (MoOx- CyHz). The MoOxCyHz phase plays an important role in stabilizing active Mo5+ sites and prevents over- reduction to inactive MoO2. The activity and benzene selectivity are maximal when pure NiMoO4 is used as a catalyst. However, the selectivity towards undesired methane increases significantly indicating the decomposition of arenes. This effect is considerably reduced for catalysts with lower nickel contents (3– 5%) which still exhibit a highly improved activity compared to MoO3. In situ XRD studies revealed that the population of the MoOxCyHz phase is strongly affected by the nickel content, the structure of the hydrocarbon substrate and the hydrogen content during pre-reduction and catalysis. Received 25th October 2023, Accepted 28th February 2024 Received 25th October 2023, Accepted 28th February 2024 Leibniz-Institute for Catalysis e.V, Albert-Einstein Str. 29a, 18059 Rostock, Germany. E-mail: Christoph.Kubis@catalysis.de, Angelika.Brueckner@catalysis.de † Electronic supplementary information (ESI) available. See DOI: https://doi.org/ 10.1039/d3cy01488f Catalysis Science & Technology Catalysis Science & Technology Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal \| View Issue Leibniz-Institute for Catalysis e.V, Albert-Einstein Str. 29a, 18059 Rostock, † Electronic supplementary information (ESI) available. See DOI: https://doi.org/ 10.1039/d3cy01488f This journal is © The Royal Society of Chemistry 2024 f y Germany. E-mail: Christoph.Kubis@catalysis.de, Angelika.Brueckner@catalysis.de PAPER Investigations into the influence of nickel loading on MoO3-modified catalysts for the gas-phase hydrodeoxygenation of anisole† Simon Haida, Sebastian Löbner, Henrik Lund, Stephan Bartling, Carsten Kreyenschulte, Hanan Atia, Ali M. Abdel-Mageed, Christoph Kubis * and Angelika Brückner * Introduction reduce the oxygen content and thus gain a higher energy density. This can be achieved by the hydrodeoxygenation (HDO) reaction by using hydrogen. Apart from the utilization of respective products as renewable fuels, especially the production of BTX-aromatics (benzene, toluene and xylenes) Global warming and its consequences for our environment are one of the most challenging problems to date. For the development of a sustainable and circular economy a substitution of crude oil by renewable feedstocks is an important aspect.1 One alternative feedstock to oil is lignin, as it is produced naturally in large amounts and can provide a wide range of different possible products.2,3 Lignin is a complex polymer out of phenolic monomers of which the majority consists of sinapyl alcohol, coniferyl alcohol and p-coumaryl alcohol. For valorization, a pyrolysis step can be applied to convert biomass into its monomers, yielding a bio- oil. This can be performed by fast pyrolysis at T = 400–600 °C to obtain bio-oils in high yields.4 However, due to the acidic properties of these pyrolysis-oils, there is a tendency for repolymerization. Further, their oxygen content is too high for direct usage as a feedstock for the production of fuels or chemicals.5,6 Therefore, further process steps are required to Scheme 1 Hydrodeoxygenation of anisole to BTX-aromatics and selected possible by-products. Scheme 1 Hydrodeoxygenation of anisole to BTX-aromatics and selected possible by-products. Catal. Sci. Technol., 2024, 14, 2201–2217 \| 2201 This journal is © The Royal Society of Chemistry 2024 View Article Online Catalysis Science & Technology Paper Catalysis Science & Technology vacancies at the surface of the catalyst on which the oxygenate substrate adsorbs via its oxygen atoms (Scheme 2a). The catalytic cycle of the HDO reaction can be described as a reverse Mars–van Krevelen type mechanism.16–21 Several reports suggests also the in situ formation of a molybdenum oxycarbohydride phase (MoOxCyHz) which seems to stabilize the oxidation state (V) of the molybdenum ions (Scheme 2b).14,15,22,23 The mechanism of the formation of such a MoOxCyHz phase from MoO3 in a hydrogen/hydrocarbon feed consists of several steps: 1) formation of MoO3−x via partial reduction and generation of oxygen vacancies, 2) formation of molybdenum oxyhydride (MoOxHy) by insertion of dissociated hydrogen into the solid, and 3) incorporation of carbon into the MoOxHy intermediate to form MoOxCyHz.24,25 The dissociation of molecular hydrogen is catalyzed by MoO2 which is partly formed during reduction. Introduction The presence of a transition metal might promote the formation of a MoOxCyHz phase because of its enhanced activity in hydrogen dissociation.24 A significant over-reduction into MoO2 (Mo4+) leads to a significant decrease of catalytic activity. A MoOxCyHz phase cannot be formed from MoO2 because its precursor molybdenum oxyhydride (MoOxHy) species are not formed from MoO2. Coke formation is another important cause for the deactivation of HDO catalysts. A regeneration step consisting of in situ calcination followed by pre-reduction recovers the catalytic activity.14 seems to be an attractive route, as lignin is the most abundant polymer with aromatic rings in nature.7,8 In Scheme 1 a simplified reaction path for the HDO of anisole, which is often used as a model substrate, to BTX-aromatics is presented. p The HDO reaction can be conducted in the liquid phase at moderate temperatures (200–400 °C) and higher partial hydrogen pressures (40–200 bar). The early generation of HDO catalysts consisted of typical hydroprocessing catalysts such as sulfided NiMo/Al2O3 and CoMo/Al2O3 or transition metal based catalysts.9,10 Meanwhile, a large variety of catalyst types, e.g. supported mono- and bimetallic catalysts (noble and base metal based), supported and unsupported metal oxide catalysts (e.g. MoO3) and nitrides and phosphides have been developed and tested.11,12 Transition metal catalysts often show a high activity, but lower arene selectivity. In contrast, Mo based catalysts (NiMo, CoMo, MoO3) tend to possess lower activities, but higher arene selectivities. Alternatively, the process can be operated at moderate temperatures (300–400 °C) but low hydrogen pressures (p(H2) ≤1 atm) in the gas-phase. This not only has the advantage of higher arene selectivity, but also the gas-phase HDO as a downstream process is an opportunity to use the energy of the previous pyrolysis step. Thus, a process design with an optimized energy balance is possible, as no cooldown and compression are necessary. Respective economic and ecological benefits should increase the overall process efficiency. Prominent catalysts which are investigated for gas- phase HDO are supported transition metal catalysts as well as pristine and promoted MoO3. Due to the tendency of monometallic transition metal catalysts for ring hydrogenation, the arene selectivity is often relatively low. MoO3 is an interesting alternative, as it shows good activity in the HDO reaction with comparatively high selectivity towards arenes.12,13 Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Due to the importance of oxygen vacancies for substrate adsorption and activation, it is expected that changes in the electronic properties of the catalyst have a significant impact on the catalytic performance. To achieve this, oxide materials can be modified by the introduction of secondary metal atoms. For hydrodenitrogenation (HDN) and hydrodesulfurization (HDS), it was demonstrated that NiMoO4 and CoMoO4 have altered catalytic properties compared to bulk MoO3. Also, it is known that the It is widely accepted that the catalytically active sites for MoO3-based catalysts are Mo5+ ions, which are generated via partial reduction accompanied by the formation of oxygen Scheme 2 a) Catalytic cycle of the hydrodeoxygenation of anisole based on the reverse Mars–van Krevelen type mechanism; b) sequence of phase transformation steps for the generation of a molybdenum oxycarbohydride phase (adapted and slightly modified from Richard et al. (a)16 and Ledoux et al. (b)24). Scheme 2 a) Catalytic cycle of the hydrodeoxygenation of anisole based on the reverse Mars–van Krevelen type mechanism; b) sequence of phase transformation steps for the generation of a molybdenum oxycarbohydride phase (adapted and slightly modified from Richard et al. (a)16 and Ledoux et al. (b)24). This journal is © The Royal Society of Chemistry 2024 This journal is © The Royal Society of Chemistry 2024 2202 \| Catal. Sci. Catalyst characterization PXRD. Powder X-ray diffraction (PXRD) patterns were recorded on a PANalytical X'Pert θ/2θ-diffractometer equipped with an Xcelerator detector using automatic divergence slits and Cu Kα1/α2 radiation (40 kV, 40 mA; λ = 0.15406 nm, 0.154443 nm). Cu beta-radiation was excluded using a nickel filter foil. Finely pestled samples were mounted on silicon zero background holders. After data collection, obtained intensities were converted from automatic to fixed divergence slits (0.25°) for further analysis. Additionally, XRD powder patterns were recorded on a Stoe Stadi P transmission diffractometer equipped with a DECTRIs Mythen2 1K detector applying Ge(111) monochromatized Mo Kα1 radiation (50 kV, 40 mA, 0.70930 Å). The samples were ground to a fine powder and placed between two acetate foils before the measurement. Based on the discussed aspects vide supra, in this study we focus on the behavior of pure metal molybdates NiMoO4 and CoMoO4 as well as mixtures with minor amounts of these molybdate phases (1–5 wt% metal content) in a MoO3 matrix in the gas-phase hydrodeoxygenation of anisole. The influence of the content of molybdate phases in MoO3 on the catalytic performance and phase composition are investigated. A specific focus is laid on the formation and decomposition of a molybdenum oxycarbohydride phase (MoOxCyHz) under the variation of reaction conditions for different catalysts. Structure–performance relationships are derived based on the results from in situ XRD, XPS and DRIFTS investigations leading to a comprehensive understanding of the promoting effect of metal molybdates on the catalytic HDO reaction. In situ XRD studies were performed on a Stoe Stadi P equipped with a Stoe ht2-in situ oven and a Mythen 1K detector in Debye–Scherrer geometry using monochromatized Mo Kα1 radiation (50 kV, 40 mA, 0.70930 Å). The sample investigated was ground, pressed to pellets at 10 tons, crushed, and sieved to fractions of 100–150 μm. A specimen was filled into a quartz glass capillary (approx. 2 mm outer diameter, 1 mm inner diameter, opened on both sides) until a height of approx. 6 mm was achieved and fixed using quartz glass wool. After mounting the capillary into the oven, the capillary was flushed with He (10 mL min−1) and the sample was heated to the desired temperature (350 °C or 325 °C). Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This journal is © The Royal Society of Chemistry 2024 Catalyst characterization After equilibration, the gas feed was changed to the reaction gas mixture (details in the corresponding description) with a total flow of 10 mL min−1 and the reaction was monitored using static data collection over a 17° angular region (Mo-radiation). The gas dosage was determined using a set of Bronkhorst mass flow controller units. The applied temperature correction function was obtained by observation of well-known phase transitions (AgNO3, KClO4, Ag2SO4, SiO2, K2SO4, K2CrO4, WO3, BaCO3). Peak positions and profiles were fitted with the pseudo-Voigt function using the HighScore Plus software package (PANalytical). Phase identification was performed by using the PDF-2 database of the International Center of Diffraction Data (ICDD). Because different X-ray sources were used (Mo, Cu, Ag), the diffraction data were converted to the Q-vector for comparison and representation purposes by using eqn (1).37 Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Technol., 2024, 14, 2201–2217 View Article Online Table 1 Catalyst denotation Description ICP (M/wt%) Denotation Pure MoO3 — MoO3 Pure NiMoO4 27.34 NiMoO4 Pure CoMoO4 26.73 CoMoO4 5 wt% Ni in MoO3 4.91 Ni(5)MoO3 3 wt% Ni in MoO3 2.79 Ni(3)MoO3 1 wt% Ni in MoO3 1.02 Ni(1)MoO3 5 wt% Co in MoO3 4.73 Co(5)MoO3 Paper Catalysis Science & Technology Table 1 Catalyst denotation substitution of lattice metal atoms by low-valence dopants (LVD) does influence the redox properties of metal oxides and turn the oxide surface into a Lewis acid which in turn lowers the energy required for the formation of oxygen vacancies.26,27 For HDO, there are several reports in which molybdenum oxide-based materials doped or modified by transition metals such as Ni, Co or Pt showed improved activities compared to pure MoO3.28–33 When nickel and cobalt are used as modifiers most often the respective metal molybdate phases (NiMoO4 and CoMoO4) are obtained after catalyst preparation and calcination.29,32,34,35 Interestingly, no comparative studies have been performed to elucidate the impact of metal molybdate phases on catalyst performance and phase composition depending on their content. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4 This article is licensed under a Creative Commons Attribution 3.0 U The specimen was dry deposited onto a Si wafer. The accelerating voltage was set to 10 kV for imaging and spectroscopy, with spot size selected for optimum resolution or current, respectively. Elemental maps were calculated from the spectral imaging data set using the net counts fitting method provided in the Pathfinder software (Thermo Fisher). XPS. The XPS (X-ray Photoelectron Spectroscopy) measurements were performed on an ESCALAB 220iXL (Thermo Fisher Scientific) with monochromated Al Kα radiation (E = 1486.6 eV). Samples are prepared on a stainless-steel holder with conductive double-sided adhesive carbon tape. The measurements are performed with charge compensation using a flood electron system combining low energy electrons and Ar+ ions (pAr = 1 × 10−7 mbar). The electron binding energies are referenced to the C 1s core level of carbon at 284.8 eV (C–C and C–H bonds). For quantitative analysis, the peaks were deconvoluted with Gaussian– Lorentzian curves using the software Unifit 2023. The peak areas were normalized by the transmission function of the spectrometer and the element specific sensitivity factor of Scofield. Details on the pseudo in situ XPS measurements are given in the ESI† (SI-A). TG-MS. Thermogravimetric (TG) measurements were performed on a Sensys TG-DSC (Setaram, Caluire) coupled with an OmniStar quadrupole mass spectrometer (Pfeiffer Vacuum), scanning in the multiple ion detection (MID) mode. The MS ionization source was electron impact (EI) with an ionization energy of 70 eV. The samples were weighed (m ≈20 mg) in open Al2O3 crucibles (100 μL) and heated from 25 °C to 600 °C in syn. air (20 mL min−1) with a heating rate of 10 °C min−1. Catalyst preparation Commercial MoO3 (Carl Roth, ≥98%) was used as a catalyst, as well as for the synthesis. For the synthesis of NiMoO4 and CoMoO4, 13.303 g (45.7 mmol) Ni(NO3)2·6H2O (Sigma Aldrich, 99.1%) or 13.297 g (45.7 mmol) Co(NO3)2·6H2O (Carl Roth, ≥98%), respectively, and 8.076 g (6.5 mmol) (NH4)6- Mo7O24·4H2O (Carl Roth, ≥99%) were dissolved in water. After complete solvation, the respective solution was stirred for 0.5 h and the water was evaporated at T = 80 °C afterwards. The sample was dried at 100 °C overnight and calcined at 450 °C for 5 h in syn. air. Samples with 1, 3 and 5 wt% content of Co or Ni were prepared by the same procedure using respective amounts of the components to achieve the desired metal content. An overview about the catalyst materials used in this study is given in Table 1. According to the protocol of Glemser et al.,36 MoOxHy was prepared by forming a suspension of 5.00 g MoO3 and adding 1.8 g (27.5 mmol) zinc granules (Thermo Scientific, d = 1–5 mm, 99.999%). Hydrochloric acid was added 3.5 mL (4 N) and the mixture was stirred overnight. Afterwards, the suspension was filtered, the remaining zinc has been removed and the slurry was washed with deionized water until no chloride was detected with AgNO3. The product was dried at 60 °C. This journal is © The Royal Society of Chemistry 2024 Catal. Sci. Technol., 2024, 14, 2201–2217 \| 2203 2203 View Article Online Paper Paper Catalysis Science & Technology Catalysis Science & Technology Q‐vector ¼ 4π· sin θ ð Þ λ (1) Q‐vector ¼ 4π· sin θ ð Þ λ (1) particle size of 250–315 μm. All samples were pre-reduced in pure H2 (25 mL min−1) for 2 h at 325 °C. For adsorption experiments of vaporized liquid substrates, they were pumped into an evaporator by a syringe pump (Harvard Apparatus, PHD ULTRA 4400) at a flow rate of 2.75 μL min−1 with a carrier gas (He) flow rate of 25 mL min−1. The DRIFT spectra were registered with an optical resolution of 4 cm−1 with 64 scans per measurement. As a background spectrum, the spectrum of the pre-reduced catalyst at the experimental temperature under helium was used. particle size of 250–315 μm. All samples were pre-reduced in pure H2 (25 mL min−1) for 2 h at 325 °C. s Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. SEM. Scanning electron microscopy (SEM) data were acquired using a Quattro S (Thermo Fisher) equipped with a Schottky emitter, a 60 mm2 SDD detector (Thermo Fisher) for energy dispersive X-ray spectroscopy (EDS), a secondary electron (SE) detector (Everhart–Thornley type), and a back- scattered electron (BSE) detector (ring semiconductor type). This journal is © The Royal Society of Chemistry 2024 Catalytic testing The catalytic test experiments have been conducted in a gas- phase packed-bed flow reactor. The tube reactor had an inner diameter of 11.9 mm which was fixed in a furnace. A thermocouple was used for temperature regulation and placed inside the catalyst bed. The catalyst material was pressed and sieved to achieve particle sizes between 600 and 800 μm. Before the catalyst was placed in the reactor held with quartz wool, 200 mg of the material was mixed with approximately 2.3 g of SiO2 for diluting to a fixed volume. The SiO2 had a particle size of 500–800 μm. The anisole was transferred into the reactor using a saturator where the H2– N2-mixture was flushed through. The saturator temperature was kept at 20 °C which results in a total anisole flow of 0.236 mL min−1 in the gas-phase and a WHSV = 0.31 ganisole gcat −1 h−1. During the reaction, 40 mL min−1 N2 and 20 mL min−1 H2 at ambient pressure were used. Behind the reactor, 6.5 mL min−1 ethane was added as an internal standard. All gas flows have been controlled by Bronkhorst mass flow controller units. Prior to the addition of the substrate, the catalysts were heated up in N2 to 325 °C and pre-reduced in 100% H2 for 2 h at ambient pressure afterwards. After the pre-reduction step the catalysts were flushed with N2 and bypass measurements were performed for feed analysis. The organic reactants and products were monitored continuously during the reaction on stream with an online GC (see technical details below). Catalyst preparation For adsorption experiments of vaporized liquid substrates, they were pumped into an evaporator by a syringe pump (Harvard Apparatus, PHD ULTRA 4400) at a flow rate of 2.75 μL min−1 with a carrier gas (He) flow rate of 25 mL min−1. The DRIFT spectra were registered with an optical resolution of 4 cm−1 with 64 scans per measurement. As a background spectrum, the spectrum of the pre-reduced catalyst at the experimental temperature under helium was used. (1) This procedure gives diffractograms, which are independent of the used X-ray wavelength λ. The weighted average wavelengths were used for the Cu (1.54187 Å) X-ray source. Raman spectroscopy. Raman spectra were recorded with a Renishaw inVia Raman microscope, equipped with a 633 nm laser and a Leica 50× objective. The used laser power was 0.085 mW with an exposure time of 10 s, with one accumulation. Catalytic results Prior to the catalytic testing a pre-reduction step was conducted (325 °C, 1 bar H2) for 2 h. The catalytic performance over 17 h time-on-stream (TOS) has been tested under atmospheric pressure at 325 °C for each catalyst revealing noticeable differences in their activity and product distribution (Table 2 and Fig. 2). Ni(5)MoO3 and Ni(3)MoO3 are comparatively more active with conversions of 80.6% and 80.3% after 10 h TOS, respectively, without showing a distinct induction period and with only a low degree of deactivation. Conversely, for Ni(1) MoO3 the maximum conversion drops to 59.0% (6 h TOS) after passing through a noticeable induction period. Results and discussion Selected material properties of fresh catalysts Selected material properties of fresh catalysts Powder X-ray diffraction and Raman spectroscopy measurements were conducted to analyze the phase composition of fresh calcined catalyst materials (Fig. 1). The intense reflections of pure phase metal molybdates at 11 nm−1 and 21 nm−1 indicate the presence of NiMoO4, while reflections at 17 nm−1 and 20.5 nm−1 prove the existence of the CoMoO4 phase (Fig. 1a). For the nickel loaded catalysts (Ni(5)MoO3, Ni(3)MoO3 and Ni(1)MoO3), the respective reflections assigned to NiMoO4 can be observed at the same positions besides the major reflections from MoO3. This observation indicates that a binary structure consisting of NiMoO4 and MoO3 is present. The same result was observed for the cobalt containing sample (Co(5)MoO3) for which the CoMoO4 phase has been identified. These results were confirmed by Raman spectroscopy (Fig. 1b). The intense band for NiMoO4 observed at 961 cm−1 is characteristic of this phase and is attributed to the symmetric stretching mode of the terminal MO group.39 At the same time, bands at 818 cm−1 and 993 cm−1 belonging to the MoO3 phase are visible in the nickel loaded catalysts. The characteristic bands for the Mo–O–Co stretching vibration at ca. 937 cm−1 of CoMoO4 are visible together with respective band contributions for MoO3 in the cobalt containing catalyst.40 Fig. 1 Characterization of the fresh calcined samples. a) Normalized PXRD patterns; b) Raman spectra measured with a Renishaw inVia Raman microscope with λ = 633 nm and a laser power of 0.085 mW. its maximum activity (equivalent to 37.7% conversion) followed by a moderate deactivation with time on stream. This type of catalytic behaviour is in accordance with previously reported results.14 In contrast to this observation, the NiMoO4 catalyst shows already in the beginning a very high activity with conversions of about 99% which only decreased slightly to 98% during the first 4 h. Surprisingly, no further deactivation is observed after reaching a steady- state within 6 h TOS until 17 h TOS. Compared to this behaviour, CoMoO4 only reaches 41.6% conversion after an induction period of ca. 5 h TOS. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Conversion X ¼ 1 −AECN;i AECN;i;0 ·AECN;IS;0 AECN;IS ·100% (3) (3) Selectivity Sj ¼ AECN; j −AECN; j;0 AECN;i;0 −AECN;i ·C‐atoms; j C‐atoms;i ·100% (4) (4) This journal is © The Royal Society of Chemistry 2024 Product analysis by online GC and calculation of performance data The transfer line to the online GC system (Agilent 7890A) was kept at a temperature of 200 °C and the valve box of the GC was tempered at 210 °C. The sample loop had a volume of 250 μL. The GC column was a Quadrex 624 30 m × 250 μm × 2 μm. Helium was used as a carrier gas. The temperature program started at 40 °C for 2 min followed by heating up to 160 °C with 10 °C min−1 and a hold time of 5 min. Afterwards the oven was heated to 240 °C with 20 °C min−1 keeping the target temperature for additional 20 min. A FID was used as a detector. DRIFTS. In situ DRIFTS experiments were carried out with a Nicolet 6700 (Thermo Scientific) FTIR spectrometer equipped with an MCT detector and a Praying Mantis (Harrick Scientific) DRIFTS reaction cell. The dome of the reaction cell was mounted with CaF2 windows. The setup was coupled with an online MS system (Omnistar GSD 320, Pfeiffer Vacuum) with a secondary electron multiplier for gas analysis. The samples were pressed and sieved to obtain a The calculation of the conversion and selectivity is based on the corrected integral values of respective GC peaks using the “Effective Carbon Number Concept”.38 Thus, the integrals of the compounds were normalized by its corresponding response factor ECN (2). This journal is © The Royal Society of Chemistry 2024 2204 \| Catal. Sci. Technol., 2024, 14, 2201–2217 Fig. 1 Characterization of the fresh calcined samples. a) Normalized PXRD patterns; b) Raman spectra measured with a Renishaw inVia Raman microscope with λ = 633 nm and a laser power of 0.085 mW. Paper View Article Online View Article Online View Article Online Catalysis Science & Technology Catalysis Science & Technology Catalysis Science & Technology Paper AECN ¼ A ECN (2) (2) The conversion (3) was determined using the area of anisole (i) and the average of bypass measurements. The area of the internal standard (IS) is included to account for possible volume changes during the reaction. The selectivity for the products ( j) are given as carbon fractions (4). Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Thereafter, a subsequent deactivation has been observed (55.9%, 10 h TOS). MoOxCyHz formation proposed as an active intermediate (see discussion vide infra).24 In this context, it seems likely that MoO3 portions are reduced to a certain extent during the pre- reduction and the second step of partial carburisation is accomplished at such a short time scale, and that the effect of formation is not visible in the catalytic data. The induction period for Co(5)MoO3 is significantly longer (10 h) after which a conversion value of 50.0% is achieved. Interestingly, with ongoing time-on-stream the conversion increases slightly. The induction period of the cobalt containing catalysts might be caused by a much more complex reduction process of CoMoO4 which can consist of several intermediate steps (e.g. Co2Mo3O8 and CoMoO3), which are not observed for NiMoO4.41 Possibly, the reduction temperature might not be high enough to reduce CoMoO4 to Co0, which could act as a hydrogen activator. In addition, the hydrogenation capability of Co is lower compared to Ni, which will influence the activation of hydrocarbons. Which one of these properties dominates with respect to the catalytic performance is difficult to determine. The pronounced induction periods for MoO3, Ni(1)MoO3, CoMoO4 and Co(5)MoO3 might indicate complex phase transformations which follow different individual time dependencies. For the unpromoted MoO3 catalyst it is known from the literature that such an induction period is related to the formation of a MoOxCyHz phase.14 It is assumed that the lattice carbon in the oxycarbohydride phase stabilizes the active Mo5+ oxidation state and hinders the over-reduction into less active Mo4+ sites. Further aspects regarding the dynamics of the induction period are the pre-reduction step prior to substrate addition and the hydrogen to substrate ratio during the HDO reaction which effects the reducibility in the presence of the oxygenate substrate. The generation of MoOxCyHz is significantly improved when MoO3 is pre-reduced in pure hydrogen at elevated temperatures (300–400 °C).14,24,25 This journal is © The Royal Society of Chemistry 2024 Catalyst activity and induction periods For the unpromoted MoO3 catalyst an induction period for ca. 5 h TOS was observed until the catalyst system reaches Catal. Sci. Technol., 2024, 14, 2201–2217 \| 2205 This journal is © The Royal Society of Chemistry 2024 2205 Table 2 Anisole conversion and product selectivities (C-mol%) at 325 °C, WHSV = 0.31 ganisole gcat −1 h−1 and TOS = 10 h. Selectivity ratio: S(arenes)/ S(phenols) = R(A/P). Further data regarding the approximated overall carbon balance based on GC analysis (C yield) and the selectivity of uncalibrated unknown products are given in the ESI† (SI-B) Catalysis Science & Technology Paper View Article Online View Article Online Catalysis Science & Technology Catalysis Science & Technology Paper Table 2 Anisole conversion and product selectivities (C-mol%) at 325 °C, WHSV = 0.31 ganisole gcat −1 h−1 and TOS = 10 h. Selectivity ratio: S(arenes)/ S(phenols) = R(A/P). Further data regarding the approximated overall carbon balance based on GC analysis (C yield) and the selectivity of uncalibrated unknown products are given in the ESI† (SI-B) X(anisole) S(methane) S(benzene) S(toluene) S(xylenes) S(phenol) S(cresols) S(arenes)/S(phenols) MoO3 35.1 3.4 27.7 6.1 4.9 18.9 8.9 1.36 NiMoO4 97.9 20.2 41.8 10.1 3.0 4.7 1.5 8.85 Ni(5)MoO3 80.6 4.2 32.1 8.7 1.0 17.7 13.1 1.36 Ni(3)MoO3 80.3 3.7 32.7 9.6 3.5 15.4 12.0 1.67 Ni(1)MoO3 55.9 3.7 31.6 7.8 2.7 18.5 12.0 1.38 CoMoO4 44.6 3.2 25.3 5.7 1.6 27.9 14.5 0.77 Co(5)MoO3 49.3 3.6 27.8 5.6 2.0 28.9 16.1 0.79 Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The respective reflections for this MoOxCyHz phase are clearly observable at 10.1 nm−1, 22.9 nm−1, 27.1 nm−1, 31.5 nm−1 and 44.2 nm−1.24,25,48,49 This was expected for the unmodified MoO3 catalyst, as such an oxycarbohydride phase was already identified for the HDO of oxygenates.14,22 The reflections assignable to MoO2 were identified as well. Interestingly, while no MoO3 was found in all spent catalysts, the NiMoO4 phase for the nickel containing systems is present. Also, for the pure nickel molybdate catalyst only the respective reflections for NiMoO4 were observed in the spent specimen. The reflections for NiMoO4 and MoOxCyHz phases tend to overlap and are not easily distinguishable. For NiMoO4, no indication for the presence of a MoOxCyHz phase has been found by TG-MS as shown later. This together confirms that the formation of a MoOxCyHz phase occurred only for Ni(5)MoO3, Ni(3)MoO3, Ni(1)MoO3 and MoO3 in measurable amounts. Such a high methane selectivity for NiMoO4 cannot be explained solely by the HDO of anisole, as it would provide approximately 14.2% methane for the selective reaction towards benzene. In addition, transmethylated products, e.g. xylenes, were obtained, which are formed by the C-transfer from the methoxy group followed by oxygen removal. From this it can be concluded that the aromatic ring must be decomposed to a significant extent. It can be assumed that besides the MoOxCyHz phase, other effects have an influence on the selectivity. To test whether nickel has a dominant impact on the product selectivities, a catalytic control experiment was conducted with 3 wt% Ni on α-Al2O3. This catalyst showed, after 10 h TOS, a conversion of 16.7% with a methane selectivity of 44.2% and benzene selectivity of 25.8% (SI-B, Table SI-8†). In comparison to the performance data obtained for Ni(3)MoO3 (Table 2), it is shown that the selectivity and activity is not dominated by Ni but by a molybdenum species. Further, for the Ni/α-Al2O3 catalyst a strong deactivation was observed starting with a conversion of 55.0% at 1 h TOS decreasing to 11.0% after 17 h TOS. Such a deactivation behaviour was not observed for the Ni(X)MoO3 catalysts. Product selectivity For the unmodified molybdenum trioxide catalyst (MoO3), the selectivity after 10 h TOS (Table 2) towards aromatic hydrocarbons is moderate with 27.7% (benzene), 6.1% (toluene) and 4.9% (xylenes). Phenolic intermediates were formed with selectivities of 18.9% (phenol) and 8.9% (cresol), which results in an S(arenes)/S(phenols) ratio (R(A/P)) of 1.36. The selectivity to methane of 3.4% is at a low level. Hydrogenation products such as cyclohexane or cyclohexenes were not detected. It is noted that product selectivities strongly depend on the exact reaction conditions such as temperature, hydrogen : substrate : catalyst ratios and residence times. A systematic study of the impact of respective parameters goes beyond the scope of this study. The product selectivity profiles over time are given in the ESI† (SI-B). Based on these considerations, one hypothesis for the absent induction period for NiMoO4, Ni(5)MoO3 and Ni(3) MoO3 is the improved reduction behaviour, when nickel is present in significant amounts, which can promote the Fig. 2 Conversion of anisole HDO over 17 h TOS at 325 °C and P = 1 bar. Pre-reduced at 325 °C in 100% H2 for 2 h. The cobalt containing catalysts CoMoO4 and Co(5)MoO3 showed similar selectivities in comparison to MoO3, except the phenol and cresol selectivities which increased, resulting in a R(A/P) of ≈0.78 for both. For Ni(5)MoO3 and Ni(3)MoO3, the content of aromatic products increased to ca. 32–33% (benzene), ca. 9–10% (toluene) and ca. 1–3% (xylene). However, as the selectivities to cresols increased, the R(A/P) for Ni(5)MoO3 remained at the same level compared to MoO3. Ni(3)MoO3 showed a Fig. 2 Conversion of anisole HDO over 17 h TOS at 325 °C and P = 1 bar. Pre-reduced at 325 °C in 100% H2 for 2 h. 2206 \| Catal. Sci. Technol., 2024, 14, 2201–2217 This journal is © The Royal Society of Chemistry 2024 View Article Online View Article Online Catalysis Science & Technology Paper slightly increased R(A/P) of 1.67. For Ni(1)MoO3, the selectivity towards aromatic compounds dropped slightly while the selectivity to phenolic products increased compared to Ni(3)MoO3 (R(A/P) = 1.38). The selectivity to methane for these three nickel containing catalysts was ca. 4%. NiMoO4 phase is distinguished by a significantly rough surface. The analysis of spent Ni(5)MoO3 showed that after the reaction the microstructure was not changed and has a comparable structure as the fresh catalyst. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. There are several possible reasons for the detection of the NiMoO4 phase in the spent catalysts after 17 h TOS, although a considerable amount of reduced nickel species was expected. One option is the potential incomplete reduction of the bulk core. Another possibility is the lower reduction degree of the catalyst in the presence of anisole at 30 vol% hydrogen during the reaction compared to the pre-reduction step performed in pure hydrogen. Also, the cooling phase after the reaction with the remaining anisole inside the reactor could lead to reoxidation. The exposition of the This journal is © The Royal Society of Chemistry 2024 Product selectivity XRD measurements were conducted after the reaction only for the unpromoted and the nickel systems because it is hardly possible to distinguish between all possible phases for the cobalt systems on the basis of their complex diffractograms.41 The MoOxCyHz phase could be identified in each catalyst, except the NiMoO4 sample (Fig. 3). Utilization of nickel molybdate NiMoO4 leads to even higher selectivities towards benzene (41.8%) and toluene (10.1%) accompanied by lower values for the phenolic compounds. The low selectivity towards oxygenates leads to a high R(A/P) ratio of 8.85. This could be explained by the high activity of NiMoO4. Surprisingly, a very high value for the selectivity towards methane of 20.2% was obtained using this nickel molybdate catalyst. Characterization of spent catalysts The spent catalysts were analysed by XRD, TG-MS, SEM and XPS to gain a better understanding about the respective phase compositions. BET surface areas (SA) have been determined as well for selected spent catalyst materials and no significant increase was observed compared to the fresh ones (SI-C†). For MoO3 the SA increased from 3 m2 g−1 to 7 m2 g−1. For Ni(5)MoO3 the SA was increased during the reaction only from 15 m3 g−1 to 18 m2 g−1, while for NiMoO4 a decrease from 40 m2 g−1 to 32 m2 g−1 was found. This indicates that SA effects are perhaps not crucial for the altered catalytic performance between unpromoted and promoted catalysts. Fig. 3 Normalized XRD patterns of the spent catalysts after the HDO reaction of anisole for 17 h at 325 °C. Nevertheless, for Ni(5)MoO3, scanning electron microscopy (SEM) gave evidence for the increased SA for nickel containing systems (SI-D, Fig. SI 8 and 9†). For the fresh sample, it was observed that a binary structure of small scale NiMoO4 combined with larger MoO3 crystallites is present. While the MoO3 crystallites form planar surfaces with sharp edges, the Fig. 3 Normalized XRD patterns of the spent catalysts after the HDO reaction of anisole for 17 h at 325 °C. Catal. Sci. Technol., 2024, 14, 2201–2217 \| 2207 This journal is © The Royal Society of Chemistry 2024 View Article Online Catalysis Science & Technology Paper Fig. 5 Mo 3d XP spectra of the fresh and used catalysts after TOS = 17 h. a) Fresh MoO3; b) spent MoO3; c) fresh Ni(5)MoO3; d) spent Ni(5) MoO3; e) fresh NiMoO4; f) spent NiMoO4. y gy catalyst to air after demounting the reactor at elevated temperatures probably has an impact due to the pyrophoric properties of nickel. To support our assignments with respect to the oxycarbohydride phase, TG-MS measurements have been conducted to check for deposited coke and the decomposition of the MoOxCyHz phase. For the spent NiMoO4 catalyst the evolution of CO2 starts at 350 °C with a small peak and a second one starting at 400 °C with its maximum at ≈440 °C but at the same time no correlated water formation can be observed (Fig. 4). It can be concluded that only graphitic coke is decomposed during the calcination and no or marginal amounts of MoOxCyHz are present in the sample. This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. XP spectra of selected catalysts (NiMoO4, Ni(5)MoO3 and MoO3) were recorded to elucidate surface properties and Mo oxidation states. The Mo 3d region was deconvoluted using three components Mo6+, Mo5+ and Mo4+ using a similar fitting model to Murugappan et al.15 For fresh calcined catalysts after preparation, almost exclusively, Mo6+ could be detected. This was concluded from the Mo 3d5/2 and Mo 3d1/2 doublet signals at E ≈232.6 and 235.9 eV, respectively (Fig. 5).42 Only for MoO3 a minor fraction of approximately 2% Mo5+ (E ≈231.3 and 234.4 eV) was found. Fig. 5 Mo 3d XP spectra of the fresh and used catalysts after TOS = 17 h. a) Fresh MoO3; b) spent MoO3; c) fresh Ni(5)MoO3; d) spent Ni(5) MoO3; e) fresh NiMoO4; f) spent NiMoO4. after 17 h TOS, while Mo5+ and Mo4+ were present with 10% and 57%, respectively. The fraction of Mo5+ is of special relevance since it is ascribed to be involved in the substrate activation. As already discussed, the formation of an oxycarbohydride phase can lead to the stabilization of Mo5+ sites. Ni(5)MoO3 contained the largest fraction of Mo5+ (26%), and 54% Mo6+ and 20% Mo4+are also observed. NiMoO4 contained ca. 11% Mo5+, 85% Mo6+ and 4% Mo4+. The spent catalysts showed significant differences with respect to their molybdenum oxidation states. The unmodified MoO3 catalyst surface contains ca. 33% Mo6+ Fig. 4 Normalized TG-MS profiles of the spent catalysts treated in syn. air, 10 °C min−1. The increased amount of Mo5+ in Ni(5)MoO3 is consistent with the results from the literature, in which for Ni and Co modified molybdenum oxide an enhanced accumulation of Mo5+ was reported.28,29,32 It is surprising that the spent NiMoO4 catalyst is reduced only to a small degree and the majority of the used material consists of Mo6+, while it is expected that the reducibility is higher compared to pure MoO3.43 Nevertheless, the presented XPS data are in agreement with the XRD results, which showed the presence of NiMoO4 as the major phase. To study the reduction behaviour of the fresh catalyst materials in the absence of the anisole substrate, pseudo in situ XPS experiments were performed. The samples were treated at 325 °C and 1 bar of hydrogen for 2 h. The Fig. 4 Normalized TG-MS profiles of the spent catalysts treated in syn. air, 10 °C min−1. 2208 \| Catal. Sci. Characterization of spent catalysts In comparison to this, the decomposition of a MoOxCyHz phase starting with fast CO2 and water evolution at 380 °C and 400 °C is shown for MoO3 and Ni(5)MoO3, respectively. The decomposition of soft coke also takes place for Ni(1)MoO3 and Ni(3)MoO3 (SI-E, Fig. SI 10†). The amount of formed water should be correlated with the amount of the oxycarbohydride phase in the respective temperature regime. However, due to several experimental uncertainties regarding the possible lability when exposed to air, the quantification of the MoOxCyHz phase is hardly possible. Overall, these obtained results agree qualitatively with those obtained from XRD and indicate that Ni might have an influence on the formation and decomposition of the MoOxCyHz phase. Adsorption behaviour of anisole and benzene The adsorption behaviour of anisole (substrate) and benzene (product) was investigated by DRIFTS experiments. After heating up in a He flow to the reaction temperature (325 °C), the catalyst sample was reduced for 2 h in 100% H2. Afterwards, the liquid component was injected into an evaporator (230 °C) using a syringe pump (2.5 μL min−1) and the vapor was then introduced via gas-phase transport with He (25 mL min−1) as a carrier into the DRIFTS cell. The flow was stopped after no intensity changes of the vibrational bands in the DRIFT spectra were observed. In the next step, the system was first flushed with He (10 min), and then the atmosphere was switched to H2. After 5 min TOS, no major differences regarding respective band positions between the different catalysts were observed (Fig. 6). For the NiMoO4 catalyst the comparatively fast desorption of benzene was also observed (Fig. 7c). After four minutes of purging with He only very weak intensity bands at 3083 cm−1 and 3057 cm−1 (ν(CH) vibrational bands) were visible. However, after switching to H2 the roto-vibrational band of CH4 centered at 3016 cm−1 was observed after 2 min (Fig. 7d). The band intensity increased further within an additional 2 min and after passing through a maximum it decreased slowly. A comparable behaviour was obtained for the CoMoO4 catalyst. However, the evolution of methane started a few minutes later and a lower intensity for the band of methane was observed compared to the NiMoO4 catalyst (SI-I, Fig. SI 19†). In each system, the strong ν(Ph–O–CH3) vibrational band at 1251 cm−1, the bands for ν(C=C) at 1495 and 1600 cm−1, and for ν(CH3) at 2842 cm−1 as well as bands for ν(C–H) at around 3000–2850 cm−1 are visible.44 In addition, each catalyst shows the formation of methane with a typical roto- vibrational band centered at 3016 cm−1. The formation of methane is expected, because from a thermodynamic point of view, the hydrogenation of the O–CH3 bond is favoured due to a lower binding energy compared to the Ph–OCH3 bond. The bond dissociation energy (BDE) of the O–CH3 bond is at about 424 kJ mol−1.12,45 However, the adsorption Similar trends were also found in the online MS signals (m/z = 15) registered simultaneously during the DRIFTS measurements (Fig. 8). In the case of the NiMoO4 catalysts, the formation of methane started immediately after H2 was introduced into the system. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 6 DRIFT spectra of the catalysts with adsorbed anisole at 325 °C and H2 (25 mL min−1). of anisole on each catalyst seems to be quite strong, as the decrease of the corresponding signals were slow (>40 min), see SI-I (Fig. SI 21†). The most plausible explanation of the discrepancy between the XPS results of the spent and reduced catalysts is the reoxidation process which might take place due to the presence of an oxygenate substrate under a lower partial pressure of hydrogen during the reaction and/or the exposition to air in the course of disassembling the reactor. Further, water as a coupling product can cause a reoxidation. This effect might be especially relevant for nickel, which is known for its pyrophoric properties if being present in its metallic state with small particle size. The relative integral values for the ν(Ph–O–CH3) vibrational band decreased to ca. 50% after ca. 20 min. Because the molybdenum oxide and metal molybdates form oxygen vacancies under reducing conditions, this strong adsorption behaviour can be expected for oxygen containing substrates (e.g. ethers, alcohols etc.).16,46 Besides the chemisorption of the educts, fast product desorption is crucial for active catalysts. Thus, the adsorption behaviour of benzene was tested following the same experimental procedure. Once the addition of benzene vapour was stopped after reaching stationarity in the DRIFT spectra, the respective vibrational bands of benzene started to immediately decrease for the MoO3 catalyst (Fig. 7a). After four minutes of flushing with He, the vibrational bands nearly completely vanished, thus the majority of benzene seems to be desorbed. After the gas was switched to H2, no changes in the DRIFT spectra could be observed (Fig. 7b). This journal is © The Royal Society of Chemistry 2024 Catal. Sci. Technol., 2024, 14, 2201–2217 \| 2209 Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Technol., 2024, 14, 2201–2217 This journal is © The Royal Society of Chemistry 2024 Fig. 6 DRIFT spectra of the catalysts with adsorbed anisole at 325 °C and H2 (25 mL min−1). Paper View Article Online View Article Online View Article Online Catalysis Science & Technology Paper reduction degree of Ni and Mo on the surface, of all three materials, was significantly higher compared with the spent catalysts in the expected order NiMoO4 > Ni(5)MoO3 > MoO3 (SI-G, Fig. SI 13–17†). ( ) The observed behaviour based on the molybdenum XP spectra is also reflected in the Ni 2p spectra of the spent and in situ treated nickel containing catalysts (Ni(5)MoO3, NiMoO4). The spectroscopic data are given in the ESI† (SI-F and G). For the spent catalysts, Ni2+ was identified as the major nickel species for each catalyst. Only for NiMoO4 a small amount of Ni0 was detected. For both catalysts, during the pseudo in situ reduction the formation of mainly Ni0 was observed (SI-G, Fig. SI 16 and 17†). Additionally, H2-TPR- experiments were carried out, where the results agree with the observed reduction behaviour based on in situ XPS in the absence of oxygenates where the reduction degree increases with the nickel content. Further details are given in the ESI† (SI-H, Fig. SI 18). Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4 This article is licensed under a Creative Commons Attribution 3.0 U following the same protocol used for the HDO of anisole. The only products were methane and toluene and minor amounts of xylenes (SI-B, Table SI-9†). Note that the conversion is lower because of the increased WHSV compared to the experiments with anisole. However, the selectivity to methane of 21.0% after 10 h TOS is the same as found for the anisole HDO. With Ni(5) MoO3 as a catalyst methane with a selectivity of 2.8% is also formed in the reaction with benzene under the applied reaction conditions (SI-B, Table SI-10†). One hypothesis for this behaviour could be the formation of a molybdenum oxycarbohydride phase (MoOxCyHz) on a pre-reduced catalyst in the presence of benzene followed by the hydrogenolytic decomposition of this phase after the addition of hydrogen. As already mentioned, the appearance of a MoOxCyHz phase was described by Román-Leshkov et al. in the context of molybdenum oxide based HDO for the first time.14 However, its formation was already published before by Ledoux and co- workers for alkane isomerization using MoO3 as a catalyst.47–49 This phase is formed at temperatures between 300 and 380 °C in a H2/HC stream. To the best of our knowledge, there are no reports about such a phase in pure metal molybdates and only one for a cobalt promoted MoO3 system.32 appeared within 12 min. After going through the maximum, the methane evolution decreased over a longer period of more than 60 min. The CoMoO4 catalyst behaved similarly with some minor differences. The first plateau was reached also after 3 min, but the intensity of methane evolution of this plateau is significantly lower compared to the NiMoO4 catalyst. However, after ca. 20 min the maximum of methane evolution has reached a comparable intensity as for the NiMoO4 catalyst, but its decrease appeared to be slower. Contrary to the DRIFTS results, also for the MoO3 catalyst, methane formation could be observed, but to a significantly less extent compared to the metal molybdate systems. The maximum of the IC signal was reached after approximately 20 min and its intensity decreased slowly afterwards. Besides the molybdenum based catalysts, a control experiment with silica was performed to exclude other unknown methane sources. As expected, no methane formation was detected, and the signal remained very low. This journal is © The Royal Society of Chemistry 2024 Adsorption behaviour of anisole and benzene The signal increased very fast and reached a short temporary plateau after about 3 min. The signal intensity increased further until a maximum This journal is © The Royal Society of Chemistry 2024 Catal. Sci. Technol., 2024, 14, 2201–2217 \| 2209 2209 View Article Online Fig. 7 a) Adsorption experiment of benzene in He on MoO3 at 325 °C; b) switch to H2 after benzene adsorption on MoO3 at 325 °C; c) adsorption experiment of benzene in He on NiMoO4 at 325 °C; d) switch to H2 after benzene adsorption on NiMoO4 at 325 °C. Catalysis Science & Technology Paper Catalysis Science & Technology Ca Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 7 a) Adsorption experiment of benzene in He on MoO3 at 325 °C; b) switch to H2 after benzene adsorption on MoO3 at 325 °C; c) adsorption experiment of benzene in He on NiMoO4 at 325 °C; d) switch to H2 after benzene adsorption on NiMoO4 at 325 °C. Acidity properties and their relevance to the HDO reaction The acidity properties of MoO3 and NiMoO4 were determined by NH3-adsorption FTIR measurements. The samples have been pre-reduced in H2 at 325 °C for 2 h to prevent differences between the analysis and HDO reaction. At room temperature, 5% NH3 in He was pulsed on the samples until saturation. Afterwards, they were heated up to 300 °C and with 50 °C steps a spectrum was recorded. To prove the assignment of the evolved new reflections to a MoOxCyHz phase, molybdenum blue was treated with H2 : CH4 = 9 :1 at 325 °C for 1200 min. Molybdenum blue is a pre- reduced MoO3 which might consist of several MoOxHy intermediate phases and tend to selectively form a MoOxCyHz phase when treated with hydrocarbons. The material was synthesized as described by Glemser et al.36 The obtained reflections assigned to the molybdenum oxycarbohydride phase for the in situ treated MoO3 material are in agreement those obtained for the treated molybdenum blue (Fig. 9). Noticeable differences in the nature of the acidic sides between MoO3 and NiMoO4 have been observed (the spectroscopic data are presented in SI-J, Fig. SI 22–25†). MoO3 shows a strong vibrational band at 1425 cm−1 which is assigned to Brønsted-acid (BA) sites.55,56 Vibrational bands assignable to Lewis acid (LA) sites could not be detected. For NiMoO4 the vibrational band at 1425 cm−1 associated with BA sites can be observed as well, however it is significantly less intense compared to MoO3. Additionally, vibrational bands related to LA sites at 3350 cm−1 and 3270 cm−1 can be observed up to 300 °C. These LA sites could play a role in the improved catalytic properties, as they can act as adsorption sites for the oxygen containing substrates. BA sites are involved in the hydrogenolysis of the substrate. NiMoO4 combines both properties, while MoO3 only has BA sites. In addition, the influence of LA sites on the formation of MoOxCyHz has been discussed in the literature before and could be beneficial for its formation.32 When the feed ratio was altered to H2 : CH4 = 1 : 1, the observed trends were the same and the MoOxCyHz phase was formed after approximately 200 min. However, for the experiment with a feed ratio of H2 : CH4 = 1 : 9 a completely Fig. In situ XRD studies on the formation of MoOxCyHz phases In situ XRD experiments were carried out to gain a deeper understanding of the MoOxCyHz phase formation and the effect of nickel on this formation. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4 This article is licensed under a Creative Commons Attribution 3.0 U The formation of methane after the adsorption of benzene is quite interesting as it seems that benzene is not just simply adsorbed on the catalyst surface but underwent some chemical conversion. To further prove the decomposition of aromatic rings with NiMoO4 as a catalyst, a catalytic experiment in the fixed-bed reactor was performed with benzene as a substrate Several aspects with respect to the observed methane formation need to be considered. It is known that during the reduction of NiMoO4 and CoMoO4 the formation of a mixed- phase material occurs.41,50,51 When NiMoO4 is used, one fraction of metallic Ni and a second fraction of MoO3/MoO2 might be formed during the pre-reduction step. The molybdenum oxide phase could be transformed into the oxycarbohydride phase by the benzene/hydrogen mixture. Subsequently, hydrogen could be activated at the Ni center, which then diffuses to MoOxCyHz by hydrogen spillover.25,29,52–54 This activated hydrogen could lead to the hydrogenation of carbon atoms and formation of methane (Scheme 3). However, the catalytic experiment under HDO conditions with Ni/α-Al2O3 as a catalyst and anisole as a substrate showed that nickel alone could decompose aromatic rings accompanied by the formation of methane. This was also proven by using benzene as a substrate in another catalytic control experiment (SI-B, Table SI-11†). It Fig. 8 MS signal of methane evolution (m/z = 15) after benzene adsorption and switch to a H2 atmosphere at 325 °C. Fig. 8 MS signal of methane evolution (m/z = 15) after benzene adsorption and switch to a H2 atmosphere at 325 °C. This journal is © The Royal Society of Chemistry 2024 2210 \| Catal. Sci. Technol., 2024, 14, 2201–2217 2210 View Article Online Scheme 3 Simplified scheme for a possible pathway of the methane formation on a nickel molybdate catalyst. a) CH4 formation by the HDO reaction and b) CH4 formation by MoOxCyHz decomposition. Catalysis Science & Technology Catalysis Science & Technology Paper temperature used in the catalysis (325 °C) is sufficient for the formation of the MoOxCyHz phase from a less reactive substrate such as methane. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. can be concluded that both discussed events can occur at the same time and contribute to the high CH4 selectivity with NiMoO4. The series was started with a ratio of H2 : CH4 = 9 : 1 and after the introduction of the reactive gas, the transformation of MoO3 to MoO2 was initiated. With some time-shift the appearance of reflections which can be duly assigned to a MoOxCyHz phase could be clearly observed at Q ≈27 nm−1 and 31.5 nm−1 after ca. 200 min (Fig. 9). Those signals increased further over time within the entire investigated time span of 1200 min (SI-K, Fig. SI 26†). Acidity properties and their relevance to the HDO reaction 9 Normalized in situ XRD patterns for the MoO3 treatment at 350 °C in a H2/CH4 mixture (flow: 10 mL min−1) at TOS = 300 min for H2:CH4 = 9:1 and 1:9, TOS = 305 min for H2:CH4 = 1:1. Note that the broad reflection at Q ≈15 nm−1 belongs to the SiO2 capillary reactor. Ex situ diffractogram for MoOxHy treated in H2: CH4 = 9:1 at 325 °C for 1200 min forming a MoOxCyHz phase (▲) for comparison with the in situ data. This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4 This article is licensed under a Creative Commons Attribution 3.0 U However, it was expected that the generation of a molybdenum oxycarbohydride phase should be feasible, as it is reported in previous works that MoOxCyHz formation is possible with a variety of hydrocarbon substrates including n-alkanes and several oxygenates.14,24,25,49,57 To understand the importance of the pre-reduction step for this phase formation and the influence of the H2/CH4 ratio, the ratio was systematically varied in the following order H2/CH4 = 9 : 1, 1 : 1, 1 : 9. Scheme 3 Simplified scheme for a possible pathway of the methane formation on a nickel molybdate catalyst. a) CH4 formation by the HDO reaction and b) CH4 formation by MoOxCyHz decomposition. Influence of the feed composition on the MoOxCyHz phase formation starting from MoO3 To acquire profound knowledge on the reaction behaviour, the experiment series was started with the generation of MoOxCyHz in MoO3 with a H2/CH4 gas mixture. Methane was chosen because it represents a well-defined gaseous hydrocarbon, suitable for long-term in situ XRD experiments. The temperature for this experiment was set to 350 °C, because it was initially not clear, whereas the lower Fig. 9 Normalized in situ XRD patterns for the MoO3 treatment at 350 °C in a H2/CH4 mixture (flow: 10 mL min−1) at TOS = 300 min for H2:CH4 = 9:1 and 1:9, TOS = 305 min for H2:CH4 = 1:1. Note that the broad reflection at Q ≈15 nm−1 belongs to the SiO2 capillary reactor. Ex situ diffractogram for MoOxHy treated in H2: CH4 = 9:1 at 325 °C for 1200 min forming a MoOxCyHz phase (▲) for comparison with the in situ data. Fig. 9 Normalized in situ XRD patterns for the MoO3 treatment at 350 °C in a H2/CH4 mixture (flow: 10 mL min−1) at TOS = 300 min for H2:CH4 = 9:1 and 1:9, TOS = 305 min for H2:CH4 = 1:1. Note that the broad reflection at Q ≈15 nm−1 belongs to the SiO2 capillary reactor. Ex situ diffractogram for MoOxHy treated in H2: CH4 = 9:1 at 325 °C for 1200 min forming a MoOxCyHz phase (▲) for comparison with the in situ data. Fig. 9 Normalized in situ XRD patterns for the MoO3 treatment at 350 °C in a H2/CH4 mixture (flow: 10 mL min−1) at TOS = 300 min for H2:CH4 = 9:1 and 1:9, TOS = 305 min for H2:CH4 = 1:1. Note that the broad reflection at Q ≈15 nm−1 belongs to the SiO2 capillary reactor. Ex situ diffractogram for MoOxHy treated in H2: CH4 = 9:1 at 325 °C for 1200 min forming a MoOxCyHz phase (▲) for comparison with the in situ data. Catal. Sci. Technol., 2024, 14, 2201–2217 \| 2211 This journal is © The Royal Society of Chemistry 2024 View Article Online View Article Online Paper Catalysis Science & Technology different behaviour was found. In this case, the starting material MoO3 was practically not reduced with no changes in the diffractograms between the starting and end point (SI- K, Fig. SI 28 and 29†). s Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Formation of the MoOxCyHz phase with NiMoO4 and Ni(5) MoO3 as catalysts In the next step, an in situ XRD experiment with NiMoO4 was performed. The temperature was slightly lowered to 325 °C because a higher activity with respect to catalyst reduction was expected. Considering the methane evolution, observed in the DRIFTS experiments, a question was raised, whether the MoOxCyHz phase can be populated, is directly decomposed, or reached equilibrium when the treatment is not carried out stepwise. At the beginning, a reduction step with pure hydrogen was performed to get more insight into the reduction behaviour of NiMoO4. For this sample after the reduction step for 120 min the dominant reflections are assigned to Ni and NiO (Fig. 10). The results from pseudo in situ XPS measurement of the reduction with hydrogen suggested that Ni0 is the dominant nickel species on the surface (SI-G, Fig. SI 16†). Reflections with a lower intensity of MoO2, which overlap in part with those of Ni, are also visible at Q ≈18.4 nm−1, 26.0 nm−1 and 36.7 nm−1 (Fig. 10). Even in the case when reflections from nickel species contribute to the overall diffractogram and overlap with those of the MoOxCyHz phase, the major contribution of the three most intense reflections should stem from the MoOxCyHz phase, since the Ni content is only 5 wt%. Besides these reflections, those for MoO2 can also be observed at Q ≈26 nm−1 and 37 nm−1. Compared with the synthesis of the MoOxCyHz phase starting from MoO3, the Ni(5)MoO3 catalyst seems to form a significantly larger amount of the oxycarbohydride phase in agreement with an improved ratio between the integral values for MoOxCyHz/MoO2. When NiMoO4 was treated with the H2/CH4 (1 : 1) gas mixture, the result is nearly identical. The only difference is the absence of the reflections which belong to the MoO2 phase. Possible reasons for this absence could be a decrease in particle size and amorphousness compared to the nickel phases. It remains unclear whether a certain amount of a MoOxCyHz phase has been formed, for which also only weak and broad reflections (Fig. 9) are obtained. Influence of the feed composition on the MoOxCyHz phase formation starting from MoO3 It can be concluded that a certain lower limiting amount of hydrogen in the feed is required for the reduction of Mo6+ to Mo5+ and the succeeding formation of the oxycarbohydride phase. In the next step, an in situ XRD measurement was carried out with the Ni(5)MoO3 catalyst. Due to the low Ni loading of 5 wt% nickel phases (Ni, NiO) should not dominate the diffractogram. This system is of specific interest, because the material was much more efficient with respect to the catalytic performance regarding the HDO of anisole compared to MoO3. The experiment was conducted in a modified stepwise manner. After a pre-reduction step for 2 h in hydrogen, the system was treated with methane for several hours. The reflections for the MoOxCyHz phase were visible after 150 min TOS directly after the switch to the CH4 atmosphere (SI- K, Fig. SI 30†). It is noted that the respective reflection signals registered during the in situ measurements in the capillary were quite low in intensity. Therefore, a long-time scan measurement was performed afterwards (Fig. 11). The reflections at Q ≈27 nm−1 and 31.5 nm−1 and 44.2 nm−1 also fully match with those acquired for the reference sample obtained from the molybdenum blue material (Fig. 9). Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. During the experiment with CH4 at 350 °C (Fig. 12b) the formation began noticeably later after 190 min. Quasi- stationarity was reached after 230 min. Apparently, significantly more MoO2 was formed compared to the experiment with propene. A sequential in situ XRD experiment was conducted with the Ni(1)MoO3 catalyst material in which an intermittent reoxidation step was included. The sample was treated with a H2/C3H6 = 9 : 1 mixture at 325 °C and after the formation of the MoOxCyHz phase, a gas mixture consisting of O2/He = 1 : 9 was added for 4 h and then switched back to the initial hydrogen/propene mixture. The diffractogram of the starting material Ni(1)MoO3 shows the reflections of MoO3 and low intensity signals, related to the NiMoO4 phase (Fig. 13). After the treatment with H2/C3H6 = 9 : 1, pronounced broad reflections for the MoOxCyHz phase can be observed besides low intensity reflections for MoO2. The reflection signals for NiMoO4 and MoO3 are not visible anymore. For the interpretation of these results two aspects need to be considered. First, the slightly increased temperature during the experiment with methane could enhance the rate for the reduction of MoO3 to MoO2 to such an extent that the rate of carburisation is much lower in relation to this reduction step. Once the catalytically inactive MoO2 is formed, it cannot be transferred into MoOxCyHz.24 This is because MoO2 does not form molybdenum oxyhydride species (MoOxHy) which represents the precursor intermediate for the generation of MoOxCyHz. Additionally, the lower reactivity of methane could be one reason for the significant over-reduction to MoO2 because the carburisation of the partially reduced MoOxHy intermediate phase(s) might be slower with less reactive substrates. As already discussed for the nickel containing system Ni(5)MoO3, improved reduction properties could be responsible for the enhanced formation of the MoOxCyHz phase, which is also feasible when using propene.58 Thus, an activation treatment with a reactive hydrocarbon/hydrogen mixture could be promising to form significant amounts of After 4 h of oxidation, the reflections of MoO3 and NiMoO4 were detected again. However, tiny reflections of MoO2 are still observable. Presumably, the temperature of Fig. 13 Normalized in situ XRD-results from the monitoring of a stepwise active phase formation from Ni(1)MoO3 with an intermittent reoxidation step at 325 °C for 4 h with O2/He (1 : 9) and an H2/C3H6 (9 : 1) mixture. Fig. Effect of using more reactive propene instead of methane The oxycarbohydride formation starting from MoO3 was also investigated with propene to elucidate the effect of using a more reactive hydrocarbon compound compared to methane. It was found that not only the formation of the MoOxCyHz phase starts earlier, also a bigger portion of the MoOxCyHz phase was built up, indicated by a larger ratio between Fig. 10 Normalized long scan time ex situ XRD patterns for NiMoO4 after in situ reduction in pure H2 for 120 min and treatment in H2/CH4 (1 : 1) at 325 °C for 1200 min after no further changes in the diffractograms over time were observed. phase was built up, indicated by a larger ratio between Fig. 10 Normalized long scan time ex situ XRD patterns for NiMoO4 after in situ reduction in pure H2 for 120 min and treatment in H2/CH4 (1 : 1) at 325 °C for 1200 min after no further changes in the diffractograms over time were observed. Fig. 11 Normalized XRD results from the stepwise treatment of Ni(5) MoO3 with H2 and CH4 at 325 °C compared with MoO3. Fig. 11 Normalized XRD results from the stepwise treatment of Ni(5) MoO3 with H2 and CH4 at 325 °C compared with MoO3. Fig. 10 Normalized long scan time ex situ XRD patterns for NiMoO4 after in situ reduction in pure H2 for 120 min and treatment in H2/CH4 (1 : 1) at 325 °C for 1200 min after no further changes in the diffractograms over time were observed. Fig. 11 Normalized XRD results from the stepwise treatment of Ni(5) MoO3 with H2 and CH4 at 325 °C compared with MoO3. This journal is © The Royal Society of Chemistry 2024 2212 \| Catal. Sci. Technol., 2024, 14, 2201–2217 2212 View Article Online Catalysis Science & Technology Paper Paper such an oxycarbohydride phase to generate a catalyst with enhanced activity and stability. integral values for MoOxCyHz/MoO2. For comparison, Fig. 12 presents the in situ collected diffraction data of both experiments where MoO3 is exposed to H2 : C3H6 = 9 : 1 at 325 °C and H2 : CH4 = 9 : 1 at 350 °C. Note the unequal time scales, as the important changes occurred after different time intervals and previous diffractograms are not shown for better clarity. This journal is © The Royal Society of Chemistry 2024 Catalyst regeneration by oxidative treatment Another important aspect is catalyst regeneration by in situ oxidative treatment. It is known from the literature that the unpromoted MoO3 catalyst possesses good regeneration properties when a calcination step is conducted.14,46,48 Thus, we were interested to study the reoxidation behaviour of a nickel molybdate modified catalyst. During the reduction, the formation of a mixed-phase composition of the NiMoO4 fraction occurs and the Ni0 phase forms particles at the surface, a reformation of the nickel molybdate phase is possible in the course of oxidative treatment according to some reports.39 During the in situ XRD experiment, in which C3H6 was used at 325 °C, after 120 min the reflections at Q ≈31 nm−1 and Q ≈27 nm−1 which belong to the oxycarbohydride phase can be identified (Fig. 12a). After 150 min a steady state seems to have been reached and comparatively strong intensity reflections of the MoOxCyHz phase, besides the reflections for MoO2, were observable. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Promotion of the MoOxCyHz phase formation by nickel Fig. 14 In situ XRD experiments of different samples at 325 °C in a gas mixture of H2/C3H6 = 9 : 1 at TOS = 310 min. a) Ni(5)MoO3; b) two layers of NiO/MoO3; c) phys. mix of NiO/MoO3; d) MoO3. To acquire more insights about the beneficial role of lower nickel contents in the formation of the oxycarbohydride phase, a series of in situ XRD experiments were performed using Ni(5)MoO3 and MoO3 as reference materials in comparison to a physical mixture of NiO/MoO3 and layers of NiO and MoO3. To carry out this experimental approach, we would like to discuss the following points. The obtained results with Ni(5) MoO3, which showed the enhanced formation of the MoOxCyHz phase, could be related to the improved reducibility caused by nickel. It is important to mention that in the case of pure NiMoO4 the population of the oxycarbohydride phase declined, presumably because of the significantly higher nickel content which could lead to the fast hydrogenolysis of the MoOxCyHz phase. Thus, moderately enhanced reduction properties caused by a lower nickel content seem to be beneficial, as this improves the formation of a molybdenum bronze (MoOxHy) in which the carbon is intercalated from the feed gas. For the potential case, activated hydrogen provided by a spillover effect would be responsible for the improved MoOxCyHz phase formation; also a local separation of MoO3 from the H2 activator (nickel) could result in a better formation of the oxycarbohydride phase.24,52 To address this hypothesis, the first in situ XRD experiment was conducted with the following procedure: a two-layer catalyst bed consisting of NiO (first layer) and MoO3 (second layer) was prepared in the quartz capillary, in which the activated hydrogen created in the first layer could possibly lead to reduction and MoOxCyHz phase formation in the second layer by H2-spillover. As a second control experiment, a physical mixture (MoO3 + 5 wt% NiO) was also tested. Additionally, the reference catalysts Ni(5)MoO3 and MoO3 were investigated. The samples were treated for 5 h in a H2/C3H6 mixture (9 : 1) at 325 °C. For the sake of better comparison, the ratios of integral values for reflections of the target MoOxCyHz phase and the MoO2 phase, which results from over-reduction, were calculated. In all samples, the formation of the oxycarbohydride phase began with the start of the reduction, resulting eventually in a mixture of MoOxCyHz with Fig. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4 This article is licensed under a Creative Commons Attribution 3.0 U Interestingly, when the nickel oxide was added as a layer above the MoO3, the amount of the oxycarbohydride phase Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 12 Results from in situ XRD investigations on MoOxCyHz formation on MoO3 with a) H2 : C3H6 = 9 : 1 at 325 °C and b) H2 : CH4 = 9 : 1 at 350 °C. Fig. 13 Normalized in situ XRD-results from the monitoring of a stepwise active phase formation from Ni(1)MoO3 with an intermittent reoxidation step at 325 °C for 4 h with O2/He (1 : 9) and an H2/C3H6 (9 : 1) mixture. Fig. 12 Results from in situ XRD investigations on MoOxCyHz formation on MoO3 with a) H2 : C3H6 = 9 : 1 at 325 °C and b) H2 : CH4 = 9 : 1 at 350 °C. This journal is © The Royal Society of Chemistry 2024 Catal. Sci. Technol., 2024, 14, 2201–2217 \| 2213 View Article Online Paper Paper Catalysis Science & Technology 325 °C is not high enough for complete oxidation. Also, the reflections are less intense and sharp, which could be caused by an alteration in the microstructure during the reduction and oxidation, respectively. Nonetheless, by the second activation step the MoOxCyHz phase with similar large reflection intensities was formed. This is a good indication that regeneration of these catalysts is possible at least for a second cycle. Further experiments are required to study the impact of the temperature on the number of reoxidation cycles and the possible accumulation of inactive MoO2. Also, the influence of the nickel content on catalyst recycling by reoxidation needs to be studied in more detail within the framework of succeeding research studies.49,58 reflections at Q ≈26.7 nm−1 and 30.7 nm−1, and for MoO2 at Q ≈25.7 nm−1 and 36.5 nm−1, respectively (Fig. 14). As already discussed for the MoO3 system, when C3H6 is used instead of CH4, a significantly larger amount of the oxycarbohydride phase seems to be formed and the ratio of integral values for reflections at Q ≈26.7 nm−1 denoted as A(MoOxCyHz) and at Q ≈25.7 nm−1 denoted as A(MoO2), R = A(MoOxCyHz)/A(MoO2) increases to R = 1.5 (for CH4 this ratio was R = 0.1, see Table 3). When Ni(5)MoO3 was used, considerably more MoOxCyHz phase is formed and a ratio of R = 4.4 was determined (Fig. 14a). Also, with methane the formation of MoOxCyHz with Ni(5)MoO3 was promoted, resulting in a ratio of R = 2.8. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. remained constant (R = 1.5), compared to the pure MoO3 in H2/C3H6 (Fig. 14b vs. 14d). Because the H* migration could be hindered and not last until the end of the catalyst bed, the physical mixture of NiO and MoO3 was tested. For this purpose, MoO3 and 5 wt% of NiO have been mixed and pressed together to a particle size of 100–150 μm. From this experiment an integral ratio of R = 1.7 was obtained (Fig. 14c). In addition to the signals of MoOxCyHz and MoO2, a reflection of metallic Ni was observed at Q = 35.4 nm−1. It can be concluded that for both the layered system and the physical mixture, an improved oxycarbohydride phase formation could not be observed in these XRD experiments. Promotion of the MoOxCyHz phase formation by nickel 14 In situ XRD experiments of different samples at 325 °C in a gas mixture of H2/C3H6 = 9 : 1 at TOS = 310 min. a) Ni(5)MoO3; b) two layers of NiO/MoO3; c) phys. mix of NiO/MoO3; d) MoO3. This journal is © The Royal Society of Chemistry 2024 2214 \| Catal. Sci. Technol., 2024, 14, 2201–2217 View Article Online Catalysis Science & Technology Paper Table 3 Ratio of integral values for reflections at Q = 26.7 nm−1 (MoOx- CyHz) and Q = 25.7 nm−1 (MoO2) of several in situ XRD experiments Catalyst Gas mixture Ratio A(MoOxCyHz)/A(MoO2) MoO3 H2/CH4 (9 : 1) 0.1 MoO3 H2/C3H6 (9 : 1) 1.5 MoO3 + NiO H2/C3H6 (9 : 1) 1.5 MoO3 + NiO (phys. mix) H2/C3H6 (9 : 1) 1.7 Ni(5)MoO3 H2/C3H6 (9 : 1) 4.4 Ni(5)MoO3 H2/CH4 (9 : 1) 2.8 Table 3 Ratio of integral values for reflections at Q = 26.7 nm−1 (MoOx- CyHz) and Q = 25.7 nm−1 (MoO2) of several in situ XRD experiments mesoporous defective MoO3−x structure after the reduction, which could induce the formation of mixed-phases, increasing the tendency for an improved MoOxCyHz formation.25,50 These results reveal that improved H2 activation by nickel is probably not the only reason for the enhancement of the oxycarbohydride phase formation. They rather indicate that the effects of different catalyst compositions (modification by NiO or NiMoO4) and corresponding structural properties together with substrate properties, even though being different in nature, are most likely cumulative. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 6 G. W. Huber, S. Iborra and A. Corma, Chem. Rev., 2006, 106, 4044–4098. Catalytic control experiments in a fixed-bed reactor with NiMoO4, Ni(5)MoO3 and Ni(3)/α-Al2O3 as catalysts and anisole and benzene as substrates revealed that the decomposition of arenes and the formation of methane could take place on a molybdenum and a nickel species simultaneously. 7 C. Li, X. Zhao, A. Wang, G. W. Huber and T. Zhang, Chem. Rev., 2015, 115, 11559–11624. 8 R. G. Ramirez Brenes, E. M. Alhadeff, N. Bojorge, L. E. M. Trales and G. A. D. Pazos, Biofuels, Bioprod. Biorefin., 2023, 17, 664–681. The formation of the molybdenum oxycarbohydride phase is effected by the substrate : hydrogen : molybdenum molar ratios as well as the reactivity of the substrate. The moderate reduction properties caused by a lower nickel content seem to balance the interplay between the enhanced reduction of molybdenum MoO3 to MoOxHy phases – from which the MoOxCyHz phase is readily generated and stabilizes the active Mo5+ sites – and hydrogenolysis activity without leading to excessive over-reduction. 9 J. L. Brito, A. L. Barbosa, A. Albornoz, F. Severino and J. Laine, Catal. Lett., 1994, 26, 329–337. 10 E. Furimsky, Appl. Catal., A, 2000, 199, 147–190. 11 W. Jin, L. Pastor-Perez, D. K. Shen, A. Sepulveda-Escribano, S. Gu and T. R. Reina, ChemCatChem, 2019, 11, 924–960. 12 J. H. Zhang, J. M. Sun and Y. Wang, Green Chem., 2020, 22, 1072–1098. 13 M. W. Nolte and B. H. Shanks, Energy Technol., 2017, 5, 7–18. In Scheme 4, an extended catalytic cycle of the HDO of anisole with MoO3 (a) and the formation steps of the oxycarbohydride phase MoOxCyHz starting from a nickel molybdate modified catalyst (b) are proposed. The established catalytic cycle can be complemented by the hydrogenation of incorporated carbon of MoOxCyHz in the formation of methane to MoOxHy which can be carburized again. In addition, the HDO via the reverse Mars–van Krevelen mechanism takes place at the Mo5+ sites which are distributed over MoOxHy/MoOxCyHz and are stabilized by the molybdenum oxycarbohydride phase. 14 T. Prasomsri, M. Shetty, K. Murugappan and Y. Román- Leshkov, Energy Environ. Sci., 2014, 7, 2660–2669. 15 K. Murugappan, E. M. Anderson, D. Teschner, T. E. Jones, K. Skorupska and Y. Román-Leshkov, Nat. Catal., 2018, 1, 960–967. 16 V. O. O. Gonçalves, C. Ciotonea, S. Arrii-Clacens, N. Guignard, C. Roudaut, J. Rousseau, J.-M. Clacens, S. Royer and F. Conflicts of interest 19 M. Rellán-Piñeiro and N. López, ACS Sustainable Chem. Eng., 2018, 6, 16169–16178. The authors declare no conflict of interest. 20 D. Mei, A. M. Karim and Y. Wang, J. Phys. Chem. C, 2011, 115, 8155–8164. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Richard, Appl. Catal., B, 2017, 214, 57–66. 17 D. R. Moberg, T. J. Thibodeau, F. G. Amar and B. G. Frederick, J. Phys. Chem. C, 2010, 114, 13782–13795. 18 M. Shetty, B. Buesser, Y. Román-Leshkov and W. H. Green, J. Phys. Chem. C, 2017, 121, 17848–17855. Acknowledgements 21 M. Shetty, E. M. Anderson, W. H. Green and Y. Román- Leshkov, J. Catal., 2019, 376, 248–257. This research study was funded by the Deutsche Forschungsgemeinschaft (DFG, project number: 442613239). We thank Christine Rautenberg for the transmission-FTIR measurements, Anja Simula for the ICP measurements, Reinhard Eckelt for the BET measurements and Carl Julius Mussweiler for his support in the catalyst preparation and Raman measurements. 22 G. B. Báfero, G. B. Strapasson, D. S. Leite and D. Zanchet, ChemCatChem, 2023, 15, e202300663. 23 A. J. Kohler, C. H. Walter and B. H. Shanks, ACS Catal., 2023, 13, 14813–14827. 24 C. Bouchy, C. Pham-huu and M. J. Ledoux, J. Mol. Catal. A: Chem., 2000, 162, 317–334. 25 P. Delporte, F. d. r. Meunier, C. Pham-Huu, P. Vennegues, M. J. Ledoux and J. Guille, Catal. Today, 1995, 23, 251–267. Conclusion In molybdenum oxide based catalysts loaded with nickel or cobalt the transition metals are present as a binary structure of NiMoO4 and CoMoO4 combined with MoO3, respectively. Catalysts with lower amounts of nickel up to a metal content of 5 wt% show higher activities towards the hydrodeoxygenation (HDO) of anisole with a slightly improved selectivity towards the aromatic products compared to pure phase MoO3. The pronounced induction period observed for the unpromoted MoO3 catalyst was increasingly shortened with increasing nickel content. Pure phase NiMoO4 is the most active catalyst system and showed a further improvement of the selectivity for aromatic products. However, for NiMoO4 a significantly high methane selectivity was also observed which indicates the decomposition of aromatic rings. It is noteworthy that a reflection signal of metallic nickel was only observed for the physical mixture of NiO and MoO3. Interestingly, such a nickel reflection was absent in the Ni(5) MoO3 system, even though a reduction of the NiMoO4 phase might lead to the formation of metallic nickel.41 One reason for the absence of observable Ni reflections could be the formation of very small nickel particles which cannot be detected by XRD. A high dispersion of very small particles of Ni could be an explanation for the improved MoOxCyHz formation for nickel molybdate modified samples. One further effect could be the formation of an intermediate In situ XRD experiments showed that the formation of a molybdenum oxycarbohydride phase (MoOxCyHz), which is found to stabilize the active Mo5+ sites preventing the over- reduction to inactive Mo4+, is improved for the Ni modified catalysts with lower nickel contents (up to 5 wt%). For the pure nickel molybdate, the population of a MoOxCyHz phase Scheme 4 Conclusive overall scheme for the HDO of anisole with MoO3 (a) and a nickel molybdate modified catalyst (b). nclusive overall scheme for the HDO of anisole with MoO3 (a) and a nickel molybdate modified catalyst (b). MoO3 (a) and a nickel molybdate modified catalyst (b). Scheme 4 Conclusive overall scheme for the HDO of anisole with MoO3 (a) and a nickel molybdate modified catalyst (b). Catal. Sci. Technol., 2024, 14, 2201–2217 \| 2215 Catal. Sci. Technol., 2024, 14, 2201–2217 \| 2215 This journal is © The Royal Society of Chemistry 2024 This journal is © The Royal Society of Chemistry 2024 View Article Online Catalysis Science & Technology Paper Syamani, P. Karungamye, A. Sohail, D. S. Nawawi, A. H. Prianto, A. H. Iswanto, M. Ghozali, W. K. Restu, I. Juliana, P. Antov, L. Kristak, W. Fatriasari and A. Fudholi, Polym. Compos., 2022, 43, 4848–4865. could not be observed for samples treated in the presence of a hydrocarbon/oxygenate substrate and molecular hydrogen. Stepwise in situ DRIFTS-MS adsorption/hydrogenation experiments on NiMoO4 indicated the decomposition of anisole and benzene over the pre-reduced nickel molybdate which could lead to a respective carbon containing phase such as MoOxCyHz, which is hydrogenated to methane when exposed to pure hydrogen. Antov, L. Kristak, W. Fatriasari and A. Fudholi, Polym. Compos., 2022, 43, 4848–4865. 4 C. Liu, H. Wang, A. M. Karim, J. Sun and Y. Wang, Chem. Soc. Rev., 2014, 43, 7594–7623. 5 C. O. Tuck, E. Perez, I. T. Horvath, R. A. Sheldon and M. Poliakoff, Science, 2012, 337, 695–699. Open Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. en Access Article. Published on 05 March 2024. Downloaded on 10/24/2024 4:57:36 AM. 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https://openalex.org/W2730311787	https://europepmc.org/articles/pmc5741365?pdf=render	English	null	Effect of the spider toxin Tx3-3 on spinal processing of sensory information in naive and neuropathic rats: an in vivo electrophysiological study	Pain reports	2,017	cc-by	7,964	Abstract Abstract Introduction: Drugs that counteract nociceptive transmission in the spinal dorsal horn preferentially after nerve injury are being pursued as possible neuropathic pain treatments. In a previous behavioural study, the peptide toxin Tx3-3, which blocks P/Q- and R-type voltage-gated calcium channels, was effective in neuropathic pain models. yp g g p p Objectives: In the present study, we aimed to investigate the effect of Tx3-3 on dorsal horn neuronal responses in rats under physiological conditions and neuropathic pain condition induced by spinal nerve ligation (SNL). p y g p p y p g ( ) Methods: In vivo electrophysiological recordings of dorsal horn neuronal response to electrical and natural (mechanical and thermal) stimuli were made in rats under normal physiological state (naive rats) or after the SNL model of neuropathic pain. Methods: In vivo electrophysiological recordings of dorsal horn neuronal response to electrical and natural (mechanical and thermal) stimuli were made in rats under normal physiological state (naive rats) or after the SNL model of neuropathic pain. Results: Tx3-3 (0.3–100 pmol/site) exhibited greater inhibitory effect on electrical-evoked neuronal response of SNL rats than naive rats, inhibiting nociceptive C-fibre and Ad-fibre responses only in SNL rats. The wind-up of neurones, a measurement of spinal cord hyperexcitability, was also more susceptible to a dose-related inhibition by Tx3-3 after nerve injury. Moreover, Tx3-3 exhibited higher potency to inhibit mechanical- and thermal-evoked neuronal response in conditions of neuropathy. C l i T 3 3 di t d diff ti l i hibit ff t d h i l i l d thi diti hibiti t t Results: Tx3-3 (0.3–100 pmol/site) exhibited greater inhibitory effect on electrical-evoked neuronal response of SNL rats than naive rats, inhibiting nociceptive C-fibre and Ad-fibre responses only in SNL rats. The wind-up of neurones, a measurement of spinal cord hyperexcitability, was also more susceptible to a dose-related inhibition by Tx3-3 after nerve injury. Moreover, Tx3-3 exhibited higher potency to inhibit mechanical- and thermal-evoked neuronal response in conditions of neuropathy. Conclusion: Tx3-3 mediated differential inhibitory effect under physiological and neuropathic conditions, exhibiting greater potency in conditions of neuropathic pain. oltage-gated calcium channel, Neuropathic pain, Spinal cord, Peptide toxin, In vivo electrophysiology Keywords: Voltage-gated calcium channel, Neuropathic pain, Spinal cord, Peptide toxin, In vivo elec Effect of the spider toxin Tx3-3 on spinal processing of sensory information in naive and neuropathic rats: an in vivo electrophysiological study Gerusa D. Dalmolina,, Kirsty Bannistera, Leonor Gonc¸ alvesa, Shafaq Sikandara, Ryan Patela, Marta do Nascimento Cordeirob, Marcus Vin´ıcius Gomezc, Juliano Ferreirad, Anthony H. Dickensona Corresponding author. Address: Department of Pharmacology, Federal University of Santa Catarina, Campus Universit ´ario Reitor Joa˜o David Ferreira Lima, Centro de Ciˆencia Biol ´ogicas, BlocoD Trindade, Florian´opolis, SC,Brazil 88040-900. Tel.: 155 48 37212466. E-mail address: gerusadalmolin@yahoo.com.br (G.D. Dalmolin). PR9 2 (2017) e610 Neuropathic Neuropathic Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction structural classes and mechanisms of actions. Voltage-gated calcium channels (VGCCs) emerged as potential targets to treat severe pain conditions with the advent of a peptide toxin extracted from the venom of the cone snail Conus magus, the v-conotoxin MVIIA, as a treatment for severe pain refractory to other treatments.31,44,46 The v-conotoxin MVIIA (synthetic form known as ziconotide, Prialt) produces pain relief by blocking N- type VGCC.35 Natural products have been historically employed in the alleviation of pain. In recent years, the identification of drugs with analgesic properties from animal venoms has resulted in novel Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. yp Voltage-gated calcium channels comprise a family of ion channels divided into low-threshold (T-type) and high-threshold (L-, N-, P/Q-, and R-type) voltage-gated channels. Based on the sequence homology of the main pore-formed a1 subunit, VGCCs are divided into 3 families: Cav1, Cav2, and Cav3.16 The Cav2 family includes VGCCs that carry P/Q- (Cav2.1), N- (Cav2.2), and R-type (Cav2.3) currents. These currents are distinguished by their sensitivity to peptide toxins from snail and spider venoms18,36,41 and are present primarily in neurones, being involved in neurotransmitter release and synaptic plasticity.7,8 In the spinal cord, VGCCs are distributed among various types of neurones and have been implicated in the spinal processing of nociceptive transmission.57 Voltage-gated calcium channels comprise a family of ion channels divided into low-threshold (T-type) and high-threshold (L-, N-, P/Q-, and R-type) voltage-gated channels. Based on the sequence homology of the main pore-formed a1 subunit, VGCCs are divided into 3 families: Cav1, Cav2, and Cav3.16 The Cav2 family includes VGCCs that carry P/Q- (Cav2.1), N- (Cav2.2), and R-type (Cav2.3) currents. These currents are distinguished by their sensitivity to peptide toxins from snail and spider venoms18,36,41 a Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom. Dalmolin is now with Department of Pharmacology, Federal University of Santa Catarina, Florian ´opolis, SC, Brazil. Sikandar is now with Wolfson Institute of Biomedical Research, University College London, London, United Kingdom, b Ezequiel Dias Foundation, Belo Horizonte, MG, Brazil, c Research Institute of Santa Casa de Belo Horizonte, Belo Horizonte, MG, Brazil, d Department of Pharmacology, Federal University of Santa Catarina, Florian ´opolis, SC, Brazil a Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom. www.painreportsonline.com Corresponding author. Address: Department of Pharmacology, Federal University of Santa Catarina, Campus Universit ´ario Reitor Joa˜o David Ferreira Lima, Centro de Ciˆencia Biol ´ogicas, BlocoD Trindade, Florian´opolis, SC,Brazil 88040-900. Tel.: 155 48 37212466. E-mail address: gerusadalmolin@yahoo.com.br (G.D. Dalmolin). Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://dx.doi.org/10.1097/PR9.0000000000000610 Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. 2.3. Spinal nerve ligation Selective tight ligation of spinal nerves L5 and L6 was performed as first described by Kim and Chung.23 Briefly, under gaseous anaesthesia (isoflurane 1.5%–1.7%, delivered in a gaseous mix of 66% of N2O and 33% of O2), the rat was placed in a prone position and a midline incision was made from L4–S2. The left paraspinal muscles were separated from the spinous processes. Part of the L6 transverse process was removed to expose the L4 and L5 spinal nerves, and L6 was identified lying just under the sacrum. The left L5 and L6 spinal nerves were isolated using glass nerve hooks (Ski-ry Ltd, London, United Kingdom) and tightly ligated, using 6-0 silk thread, distal to their dorsal root ganglion and proximal to their conjunction to form the sciatic nerve. Haemostasis was confirmed, the wound was sutured, and the animal recovered from anaesthesia. For 2 weeks after surgery, the rats were housed in groups of 4 in plastic cages under a 12/12-hour day/night cycle, and their general health was monitored. The electrophysiological experiments were made at postoperative days 14 to 17, when neuropathic pain behaviours have been shown to be evident.11,19,30 Recently, the peptide toxin Tx3-3, extracted from the venom of the spider Phoneutria nigriventer was tested in a behavioural study in rodents and showed antinociceptive effect in experi- mental neuropathic pain rather than inflammatory pain.13 Tx3-3 blocks P/Q- and R-type VGCCs24 but induces a different profile of behavioural effects when compared to the P/Q-blocker v-conotoxin MVIIC, exhibiting better therapeutic window. Although v-conotoxin MVIIC elicited serious motor dysfunctions when injected by intrathecal (i.t.) or intracerebroventricular (i.c.v) routes, injection of Tx3-3 induced minimal motor effects by i.c.v route, only at doses of an order of magnitude higher than the antinociceptive dose.13 The therapeutic window exhibited by i.t. Tx3-3 in previous behavioural experiments (no side effects were elicited with a dose .10 times the median effective dose)13 is interesting in the search of clinically useful agents, given that most of VGCC blockers studied have a narrow therapeutic win- dow.28,39 However, whether this peptide toxin directly affects nociceptive transmission in dorsal horn spinal cord is not known. Furthermore, drugs that counteract the nociceptive transmission in the spinal dorsal horn preferentially after nerve injury are being pursued as possible neuropathic pain treatments. 2.3. Spinal nerve ligation Therefore, the aim of the present study was to investigate the effects of Tx3-3 on the evoked dorsal horn neuronal responses in normal physiolog- ical conditions and in neuropathic conditions induced by spinal nerve ligation (SNL) in rats. 1. Introduction Dalmolin is now with Department of Pharmacology, Federal University of Santa Catarina, Florian ´opolis, SC, Brazil. Sikandar is now with Wolfson Institute of Biomedical Research, University College London, London, United Kingdom, b Ezequiel Dias Foundation, Belo Horizonte, MG, Brazil, c Research Institute of Santa Casa de Belo Horizonte, Belo Horizonte, MG, Brazil, d Department of Pharmacology, Federal University of Santa Catarina, Florian ´opolis, SC, Brazil Corresponding author. Address: Department of Pharmacology, Federal University of Santa Catarina, Campus Universit ´ario Reitor Joa˜o David Ferreira Lima, Centro de Ciˆencia Biol ´ogicas, BlocoD Trindade, Florian´opolis, SC,Brazil 88040-900. Tel.: 155 48 37212466. E-mail address: gerusadalmolin@yahoo.com.br (G.D. Dalmolin). Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Different subtypes of VGCC blockers have been tested in animal models of pain, and the responsiveness of each subtype of VGCC blockers often depends on the nature of the pain state.10,28,45,50,54 Notably, under neuropathic pain conditions, spinal N- and R-type http://dx.doi.org/10.1097/PR9.0000000000000610 1 2 (2017) e610 1 G.D. Dalmolin et al.·2 (2017) e610 PAIN Reports® 2 calcium currents seem to play an important role.29,30 On the other hand, spinal P/Q-type VGCCs are less implicated in dorsal horn neuronal response after nerve damage30 but are related to central sensitization secondary to inflammatory pain.32,33 In line with electrophysiological studies, knockout animals have highlighted the role of the different VGCC subtypes in transmission and sensitization of pain.27,42,43 2.1. Animals Thirty nine male Sprague Dawley rats (Central Biologic Services, University College London, London, United Kingdom), weighing 200–250 g, were used in this study. They were housed at a maximum of 5 per cage on a 12-hour day/night cycle. Food and water were available ad libitum. All experimental procedures were approved by the UK Home Office and follow the guidelines of the International Association for the Study of Pain for the care and use of laboratory animals.59 The number of animals used was the minimum necessary to demonstrate consistent effects of drug treatments. 2.4. In vivo electrophysiology In vivo electrophysiology recordings were performed as pre- viously described.11 Briefly, anaesthesia was induced (isoflurane 5%; 66% N2O, and 33% O2) and a cannula was inserted into the trachea, to allow continuous anesthetic delivery along the experiment (isoflurane 1.5%; 66% N2O, and 33% O2). This level of anaesthesia was maintained throughout experiments that lasted up to 9 hours. During this time the core body temperature of the rat was monitored and maintained (36·5–37˚C) by means of a heating blanket connected to a rectal thermal probe via an automatic feedback control unit. The rats breathed spontane- ously throughout the experiment and therefore were able to regulate their acid–base balance. A laminectomy was performed (vertebrae L1–L3) to expose segments L4-L5 of the spinal cord. Extracellular recordings of single deep wide dynamic range dorsal horn neurones (500–1000 mm depth; laminae V–VI) were made using a parylene-coated tungsten electrode (125-mm diameter, 2 MV; A-M systems). Positive wide dynamic range neurones were identified by their ability to respond to a range of von Frey filaments and thermal stimuli applied to the receptive field. Data were captured and analysed by a CED 1401 interface coupled to Spike 2 software (Cambridge Electronic Design, Cambridge, United Kingdom). 2.2. Toxin purification The testing protocol consisted of an electrical test followed by the natural stimuli. Electrical stimulation consisted of a train of 16 transcutaneous electrical stimuli (2 milliseconds pulse width, 0.5 Hz) applied to the receptive field at 3 times the threshold current for C-fibre activation, which generated a post-stimulus histogram in which responses evoked by Ab-fibre (0–20 milliseconds), Ad-fibre (20–90 milliseconds), C-fibre (90–300 milliseconds), and postdischarge (300–800 milliseconds) were separated and quantified on the basis of latency. The number of action potentials in the C-fibre and postdischarge range evoked by the first stimulus of the train multiplied by the total number of electrical stimuli (16) is referred to as input (non potentiated response). Wind-up (potentiated response) was calculated as the total number of action potentials in the C-fibre and postdischarge range produced by the train of 16 stimuli minus the input. Phoneutria nigriventer venom was obtained by electrical stimu- lation of anesthetized spiders. The peptide toxin Tx3-3 was purified from the venom by a combination of chromatographic steps, as described previously.12 The criteria for the purity and identity of the Tx3-3 are based on the shape/morphology of the toxin peak in the final reverse phase high pressure liquid chromatography (RP-HPLC), the results of electrospray ioniza- tion quadrupole time-of-fly mass spectrometry (ESI/TOF MS) mass spectroscopy, and the determination of the N-terminal sequence of 20–20 amino acids of every sample. All of the samples used presented a purity of better than 95%. Tx3-3 has a molecular weight of 5100.00 Da and its aminoacid sequence is GKCADAWESCDNYPCCVVNGYSRTCMCSANRCNCDDTKTL- REHFG.6 Tx3-3 was stored at 220˚C and freshly diluted to experimental doses with saline solution (NaCl 0.9%). 2 (2017) e610 3 www.painreportsonline.com Following electrical stimuli, natural stimuli, comprising dynamic brush, mechanical punctate (application of von Frey filaments: 8, 15 and 60 g), and thermal stimuli (application of a constant water jet at 40, 45, and 48˚C), were applied to the neuronal receptive field for 10 seconds, and the total number of evoked spikes was recorded. Sufficient intervals (1–2 minutes) were allowed within and between stimuli to avoid sensitization of receptors. After 3 consecutive stable control trials (,20% variation for all param- eters), neuronal responses were averaged to give predrug control values with which subsequent responses were compared. Spinal applications of Tx3-3 (0.03–100 pmol/site, in a volume of 50 mL) were made directly onto the exposed surface of the spinal cord. 3.2. Tx3-3 exhibits higher potency to inhibit naturally evoked neuronal response in spinal nerve ligated than naive rats The effects of Tx3-3 applied directly onto the spinal cord on the electrically, mechanically and thermally evoked response of deep dorsal horn neurones were tested in naive and SNL rats. No significant difference was found between the 2 experimental groups in the mean cell depth of recorded neurones and the mean baseline neuronal responses evoked by electrical, me- chanical, and noxious thermal stimulation (Table 1), allowing direct comparison of the effects of Tx3-3 between the 2 experimental groups. When tested on a range of mechanical stimuli, Tx3-3 exhibited higher potency to inhibit dorsal horn neuronal responses in SNL than naive rats. Tx3-3 inhibited to the same extent neuronal responses evoked by noxious punctate mechanical stimulation (von Frey 26 and 60 g) in SNL and naive rats (Fig. 3A, B), but the same level of inhibition was achieved by a 100-fold lower dose of Tx3-3 in SNL rats (inhibition of 46.7 6 10.8% was achieved by Tx3-3 at 0.3 pmol/site; F(4,33) 5 9.781, P , 0.0001) in comparison to naive rats (42.2 6 3.9% was achieved by Tx3-3 at 30 pmol/site; F(3,26) 5 4.547, P 5 0.0109) (Fig. 3C). A leftward shift in the dose–response curve of Tx3-3 in SNL rats in comparison to naive rats was also seen in neuronal response evoked by nonnoxious mechanical stimuli (von Frey 8 g). In SNL rats, a significant inhibitory effect on neuronal response evoked by nonnoxious punctate mechanical stimulus (von Frey 8 g) was reached by application of 0.3 pmol/site of Tx3-3 (inhibition of 54.5 6 8.6%; F(4,33) 5 4.294, P 5 0.0066), while 30 pmol/site of Tx3-3 was needed to inhibit neuronal response in naive rats (inhibition of 41.7 6 13.7%; F(3,26) 5 3.493, P 5 0.0297) (Fig. 3C). Moreover, Tx3-3 exhibited inhibitory effect on nonnoxious dynamic me- chanical stimulation (brush) in SNL rats (inhibition of 52.2 6 8.7; F(4,33) 5 2.840, P 5 0.0404), but not in naive rats (F(3,26) 5 2.739, P 5 0.0637) (Fig. 3A, B). 2.6. Statistical analysis increasing stimuli number) in both naive (2-way ANOVA, F(3,240) 5 22.06, P , 0.0001) and SNL rats (2-way ANOVA, F(3,240) 5 28.47, P , 0.0001), but SNL rats were more susceptible than naive rats to the dose-dependent inhibition by Tx3-3 (Fig. 2A, B), which was particularly evident by the flattening of the curve of spikes/ stimulus x electrical pulses induced by application of 30 pmol/site of Tx3-3 (Fig. 2B). Tx3-3 (30 pmol/site) also inhibited input (number of spikes evoked by the first stimulus of the electrical train) in SNL rats (50.5 6 14.1% of inhibition; Paired t test, P 5 0.0064), but no difference was found in input of naive rats treated with Tx3-3, at any dose tested (Fig. 2Aa, Bb). Statistical analysis was performed using GraphPad Prism Version 5.01. One-way analysis of variance (ANOVA) with Dunnett’s post hoc test was used in dose–response curve experiments; 2-way ANOVA was used to access the effect of Tx3-3 on wind-up response; Student’s paired t test was used to access the effect of Tx3-3 on input responses; Student’s unpaired t test was used to compare neuronal characteristics and evoked responses be- tween naive and SNL rats. F and P values are given at maximum peak of each significant interval; P , 0.05 were considered significant. 2.2. Toxin purification The doses of Tx3-3 used were chosen based on previous behavioural study.13 The effect of each dose was followed for an hour, with tests carried out at 10, 30, and 60 minutes post-drug. One neurone per rat was recorded in each experiment. In SNL rats, all neurones recorded had the receptive field over the left hindpaw, ipsilateral to surgery. In naive rats, either left or right side of spinal cord was analysed, in a counterbalanced manner. Table 1 Comparison of dorsal horn neuronal characteristics between naive and SNL rats. Measure Naive rats SNL rats Cell depth, mm 619 6 13.5 675 6 38.3 C-fibre threshold, mA 1.2 6 0.1 1.1 6 0.09 C-fibre response (APs) 558.5 6 64.3 506.2 6 32.8 Ab-response (APs) 139.7 6 14.6 144 6 13.1 Ad-response (APs) 176.7 6 27.1 205.9 6 15.7 Post-discharge (APs) 446.7 6 38.0 545.9 6 64.5 von Frey 8 g (APs) 325.8 6 44.7 292.8 6 38.6 von Frey 26 g (APs) 918.6 6 80.8 792.9 6 55.5 von Frey 60 g (APs) 1070 6 91.8 974.3 6 54.6 48˚C heat (APs) 1152 6 81.8 1216 6 70 Data for the electrically evoked responses are expressed as the mean number of action potentials (APs) 6 SEM evoked by a train of 16 electrical stimuli at 3 times the threshold for C-fibres. Data of mechanical and thermal stimuli are expressed as the number of APs evoked during the 10-second period for which the stimulus was applied to the cell’s peripheral receptive field. Data were analysed using Student unpaired t test. SNL, spinal nerve ligated. Table 1 Comparison of dorsal horn neuronal characteristics between naive and SNL rats. Data for the electrically evoked responses are expressed as the mean number of action potentials (APs) 6 SEM evoked by a train of 16 electrical stimuli at 3 times the threshold for C-fibres. Data of mechanical and thermal stimuli are expressed as the number of APs evoked during the 10-second period for which the stimulus was applied to the cell’s peripheral receptive field. Data were analysed using Student unpaired t test. SNL, spinal nerve ligated. 3.1. Tx3-3 shows greater inhibition profile on electrically evoked neuronal responses in spinal nerve ligated than naive rats Under neuropathic conditions, Tx3-3 inhibited Ad, C-fibre, and input responses as well as postdischarge response, while only postdischarge was inhibited in normal animals. Moreover, Tx3-3 produced a greater inhibition of neuronal wind-up in SNL than naive rats. The VGCCs targeted by Tx3-3 are differentially expressed in dorsal horn spinal neurones. P/Q- type VGCCs are expressed on nerve terminals of nonpeptidergic IB4-positive C-fibres, while R-type VGCCs are predominantly localized to somatodendritic compartments51 but are also expressed on nerve terminals of peptidergic C-fibres and Ad- fibres.17 Subcellular localization functionally link VGCC subtypes with specific neuronal processes. P/Q-type VGCCs are involved in initiating the release of neurotransmitters at presynaptic sites,7,9 while R-type VGCCs play minor role in basal neurotrans- mitter release, being involved in presynaptic mechanisms of plasticity.5,14,53 The inhibition of synaptic transmission through inhibition of neurotransmitter release may account for the effect of Tx3-3 on postdischarge and wind-up responses evoked by electrical stimulation in physiological conditions. In agreement, the blockade of spinal P/Q-type VGCCs by application of v-agatoxin-IVA in rats attenuated both postdischarge and wind-up neuronal responses.30 However, the inhibitory profile of v-agatoxin-IVA remained unchanged after SNL.30 Keeping this in mind, the differential effect mediated by Tx3-3 in neuropathic conditions is thus unlikely to be mediated by its action on P/Q- type VGCCs. Furthermore, classically, P/Q-type VGCC blockers present modest effects against neuropathic pain.10,50,55 In- terestingly, the inhibitory profile of Tx3-3 on electrically evoked neuronal responses resemble those elicited by the R-type VGCC blocker SNX-482.29 Both Tx3-3 and SNX-482, a peptide isolated from the venom of the spider Hysterocrates gigas,34 inhibited postdischarge and wind-up responses under control conditions, but exhibited a greater inhibitory profile in neuropathic conditions induced by SNL, inhibiting also nociceptive C-fibre and Ad-fibre responses.29 Moreover, like SNX-482, the input and wind-up responses were rather affected by Tx3-3 after neuropathy. Figure 1. Dose–response curve of Tx3-3 on the electrically evoked neuronal responses recorded from naive (A) and SNL (B) rats. Ab-fibre (Ab), Ad-fibre (Ad), C-fibre (C) and postdischarge (PD) measurements are shown. Data are expressed as mean maximum inhibition response during the first hour after drug application onto spinal cord of rats (50 mL/site) 6 SEM. In spinal nerve ligation rats, the neuronal response was evaluated 14 to 17 days after surgery. Each column represents the mean response of 5 to 7 neurones. Vertical lines show the SEM. 3.1. Tx3-3 shows greater inhibition profile on electrically evoked neuronal responses in spinal nerve ligated than naive rats Tx3-3 caused an inhibitory effect on the dorsal horn neuronal responses to electrical stimulation in SNL rats and, to a far lesser extent, in naive animals. In naive rats, Tx3-3 (30 pmol/site) mediated statistically significant inhibitions of postdischarge response, compared to predrug values (45 6 10% inhibition; F(3,25) 5 3.704, P 5 0.0248), but no effect was seen on Ab, Ad, and C-fibre responses (Fig. 1A). In SNL rats, Tx3-3 caused a clear dose-dependent inhibition of postdischarge (50.6 6 16.08% inhibition; F(3,25) 5 3.081, P 5 0.0457), C-fibre (37.3 6 13.97% inhibition; F(3,25) 5 6.286, P 5 0.0025), and Ad- (27.9 6 8.1% inhibition; F(3,25) 5 3.011, P 5 0.049) response, but no effect was seen on Ab-fibre responses (F(3,25) 5 1.466, P 5 0.2477) (Fig. 1B). We also analysed the effects of Tx3-3 on wind-up of neurones, as a measure of hypersensitivity. Tx3-3 inhibited the wind-up (increases in number of spikes evoked by Neuronal responses to thermal stimulation (40, 45, and 48˚C) were also inhibited by spinal application of Tx3-3 in naive and SNL rats. Tx3-3 inhibited neuronal responses evoked by noxious thermal stimulus (48˚C) in both naive (inhibition of 41 6 15%; G.D. Dalmolin et al.·2 (2017) e610 PAIN Reports® 4 Figure 1. Dose–response curve of Tx3-3 on the electrically evoked neuronal responses recorded from naive (A) and SNL (B) rats. Ab-fibre (Ab), Ad-fibre (Ad), C-fibre (C) and postdischarge (PD) measurements are shown. Data are expressed as mean maximum inhibition response during the first hour after drug application onto spinal cord of rats (50 mL/site) 6 SEM. In spinal nerve ligation rats, the neuronal response was evaluated 14 to 17 days after surgery. Each column represents the mean response of 5 to 7 neurones. Vertical lines show the SEM. Statistical analysis was determined using 1-way analysis of variance followed by Dunnett’s post hoc test. Asterisks denote the significance levels in comparison to predrug value, P , 0.05, P , 0.01. physiological and neuropathic conditions. We showed that Tx3- 3 mediated differential inhibitory effect on deep dorsal horn neuronal responses under physiological and neuropathic con- ditions, exhibiting greater potency after nerve injury. ditions, exhibiting greater potency after nerve injury. When tested on neuronal responses evoked by electrical stimulation, Tx3-3 exhibited a greater inhibitory profile in SNL than in naive rats. 3.1. Tx3-3 shows greater inhibition profile on electrically evoked neuronal responses in spinal nerve ligated than naive rats Statistical analysis was determined using 1-way analysis of variance followed by Dunnett’s post hoc test. Asterisks denote the significance levels in comparison to predrug value, P , 0.05, P , 0.01. Thus the effect of Tx3-3 on electrically evoked dorsal horn neuronal responses in neuropathic conditions are consistent with a major blockade of R-type VGCCs. In the SNL animals, the wind- up profile differed from that in the control group as the initial response, an index of presynaptic mechanisms delivering the input onto the neurones, was markedly increased, perhaps indicative of enhanced presynaptic spinal mechanisms in keeping with the augmented effects of the toxin after neuropathy. F(3,23) 5 3.324, P 5 0.0375) and SNL rats (inhibition of 38.7% 6 7.6; F(3,25) 5 6.912, P 5 0.00015) (Fig. 3D, E). In SNL rats, Tx3-3 mediated a broader inhibition of neuronal response to thermal stimulus, inhibiting also the responses evoked by 40˚C (inhibition of 62.5% 6 11.6; F(3,25) 5 4.532; P 5 0.0118). Moreover, lower doses of Tx3-3 were required to achieve significant inhibition in SNL rats than in naive rats (Fig. 3D, E). g p y The antinociceptive properties of Tx3-3 were previously shown in neuropathic pain models of sciatic nerve ligation and diabetic neuropathy.13 In the present study, we have addressed the effect of Tx3-3 in in vivo electrophysiology recordings of dorsal horn neurones after the SNL model of chronic neuropathic pain.23 In line to the earlier behavioural study, in which Tx3-3 attenuated the tactile allodynia induced by traumatic neuropathy,13 here we show that Tx3-3 inhibits dorsal horn neuronal response evoked by innocuous mechanical stimulation in SNL rats. Despite the effect on innocuous mechanical response, Tx3-3 did not affect Ab-fibres responses. However, electrically evoked responses are suprathreshold and syncronious; by contrast, natural stimuli evoked by 8 g von Frey stimulation applied over 10 seconds are more amenable to inhibition and are likely to activate also Overall, the maximum inhibitions produced by Tx3-3 were established around 10 to 20 minutes, and the inhibitory effect lasted approximately 60 minutes (Appendix). No difference was found in timecourse effect of Tx3-3 between naive and SNL rats (data not shown). 4. Discussion In this study, we addressed the effect of the peptide toxin Tx3-3, a P/Q- and R-type VGCC blocker, in in vivo electrophysiological measurements of dorsal horn neuronal responses under 2 (2017) e610 www.painreportsonline.com 5 , which could explain this effect. Importantly, we show chanically evoked dorsal horn responses were more to Tx3 3 in conditions of neuropathy The higher potency high frequency of fiber activation produces a dynam the VGCC conformational state, switching betw activated and inactivated state 1 The access to the Effect of spinally applied Tx3-3 on the electrically evoked neuronal wind-up and input responses recorded from naive (A, Aa) and spina s. Data are expressed as mean number of spikes evoked per stimulus of the electrical train of 16 stimuli (wind-up; A, B) and mean nu the first stimulus of the electrical train 3 16 (input; Aa, Bb) 6 SEM. Each point or column represents the mean response of 5 to 7 neurone SEM. Statistical analysis was determined using 2-way analysis of variance for wind-up response (A, B) and paired t test for input resp enote the significance levels in comparison to predrug values, P , 0.01, *P , 0.001. Effect of spinally applied Tx3-3 on the electrically evoked neuronal wind-up and input responses recorded from naive (A, Aa) and spinal nerve ligation s. Data are expressed as mean number of spikes evoked per stimulus of the electrical train of 16 stimuli (wind-up; A, B) and mean number of spikes the first stimulus of the electrical train 3 16 (input; Aa, Bb) 6 SEM. Each point or column represents the mean response of 5 to 7 neurones. Vertical lines Figure 2. Effect of spinally applied Tx3-3 on the electrically evoked neuronal wind-up and input responses recorded from naive (A, Aa) and spinal nerve ligation (B, Bb) rats. Data are expressed as mean number of spikes evoked per stimulus of the electrical train of 16 stimuli (wind-up; A, B) and mean number of spikes evoked by the first stimulus of the electrical train 3 16 (input; Aa, Bb) 6 SEM. Each point or column represents the mean response of 5 to 7 neurones. Vertical lines show the SEM. Statistical analysis was determined using 2-way analysis of variance for wind-up response (A, B) and paired t test for input response (Aa, Bb). Asterisks denote the significance levels in comparison to predrug values, P , 0.01, **P , 0.001. 4. Discussion Ad-fibres, which could explain this effect. Importantly, we show that mechanically evoked dorsal horn responses were more sensitive to Tx3-3 in conditions of neuropathy. The higher potency of Tx3-3 in SNL rats suggests a higher affinity of Tx3-3 to VGCC after neuropathy. It has been shown that in neuropathic states the high frequency of fiber activation produces a dynamic change in the VGCC conformational state, switching between resting, activated, and inactivated state.1 The access to the inactivated channel is greater during high-frequency neuronal firing conditions, improving the affinity of certain VGCC blockers, as occurring with G.D. Dalmolin et al.·2 (2017) e610 PAIN Reports® 6 Figure 3. Dose–response curve of Tx3-3 on naturally evoked neuronal responses recorded from naive (A, D) and spinal nerve ligation (SNL) (B, E) rats. Evoked dorsal horn neuronal response to mechanical (A, B) and thermal (D, E) stimuli and dose–response curve of Tx3-3 in naive and SNL rats (C) are shown. Data are expressed as mean maximum inhibition response during the first hour after drug application onto spinal cord of rats (50 mL/site) 6 SEM. In SNL rats the neuronal response was evaluated 14 to 17 days after surgery. Each point represents the mean response of 5 to 7 neurones. Vertical lines show the SEM. Statistical analysis was determined using 1-way analysis of variance followed by Dunnett’s post hoc test for each mechanical and thermal stimulus. Asterisks denote the significance levels in comparison to predrug values, P , 0.05, P , 0.01, P , 0.001. Figure 3. Dose–response curve of Tx3-3 on naturally evoked neuronal responses recorded from naive (A, D) and spinal nerve ligation (SNL) (B, E) rats. Evoked dorsal horn neuronal response to mechanical (A, B) and thermal (D, E) stimuli and dose–response curve of Tx3-3 in naive and SNL rats (C) are shown. Data are expressed as mean maximum inhibition response during the first hour after drug application onto spinal cord of rats (50 mL/site) 6 SEM. In SNL rats the neuronal response was evaluated 14 to 17 days after surgery. Each point represents the mean response of 5 to 7 neurones. Vertical lines show the SEM. Statistical analysis was determined using 1-way analysis of variance followed by Dunnett’s post hoc test for each mechanical and thermal stimulus. Asterisks denote the significance levels in comparison to predrug values, P , 0.05, P , 0.01, *P , 0.001. 4. Discussion dorsal horn neuronal response evoked by nonnoxious punctate mechanical stimulus is more sensitive to the R-type VGCC blocker SNX-482 in neuropathic conditions.29 Furthermore, P/Q-type VGCCs are also found in the deeper laminae of the dorsal horn,51 suggesting its presence on touch-sensitive large fibres, and there are reports of increased levels of P/Q-type in DRG, especially in medium and large myelinated afferent fibers, in neuropathic pain models.49,56 Thus, nerve injury may establish a functional re- organization of neuronal phenotypes within the spinal cord, by changing the pattern of expression or activity of some ion channels, including the VGCCs target by Tx3-3, which explains the outcome effect of Tx3-3 on neuronal response evoked by nonnoxious stimuli in neuropathic rats. some v-conotoxins that exhibit higher potency when binding to the inactivated state of VGCC.3,47,52,58 Such state-dependent block- ade property could account for the more potent blockade of Tx3-3 during the higher frequency of spontaneous neuronal firing present after SNL.11 The higher magnitude of inhibition and dose-related effect of Tx3-3 on wind-up responses in neuropathic rats further support the notion that the reorganization of VGCCs kinetics properties following nerve damage favors the Tx3-3 binding. In this regard, it is noteworthy that neuronal responses evoked by nonnoxious thermal and mechanical stimuli were sensitive to Tx3-3 inhibition preferentially in neuropathic conditions, indicating an enhanced excitability of spinal transmission from afferents that expressed Tx3-3-sensitive VGCCs after neuropathy. In agreement, 2 (2017) e610 2 (2017) e610 7 www.painreportsonline.com It is also worth mentioning the possible role of the auxiliary a2d subunit of VGCCs in our current results. The a2d subunits of VGCCs (comprises a2d-1, a2d-2, a2d-3, and a2d-4) regulate VGCC biophysical properties, trafficking, and membrane expres- sion2,15,22 and are upregulated in the dorsal horn spinal cord, mediating spinal hyperexcitability, under neuropathic condi- tions.4,25,26 Recently it was shown that the disruption of the a2d1 gene expression leads to a delay in development of mechanical hypersensitivity in neuropathic mice, which correlates with a reduction in the response of deep dorsal horn neurones to a range of mechanical stimuli.37 Which subtype of Cav2 family is mainly affected by a2d regulation in neuropathic conditions remains to be clearly defined. Disclosures Supported by the Wellcome Trust London Pain Consortium strategic award. G. D. Dalmolin is supported by Capes/ Toxinologia (process 8849-11-0). There are not any financial or other relationships that might lead to a conflict of interest in this study. 4. Discussion However, most aspects of the R- type pore-forming a1 subunit (alpha 1E) are affected by a2d regulation, including the kinetics of activation-inactivation- deactivation of R-type VGCCs,40 and the trafficking of Cav2.1 (which conducts P/Q-type currents) to cell surface is prevented by mutation in the a2d2 subunits.20,21 Therefore, it is possible that the adaptations on VGCC function and expression carried out by a2d subunits in neuropathic conditions underlie the gain of effect of Tx3-3. References [1] Baccei ML, Kocsis JD. Voltage-gated calcium currents in axotomized adult rat cutaneous afferent neurons. J Neurophysiol 2000;83:2227–38. [2] Bauer CS, Nieto-Rostro M, Rahman W, Tran-Van-Minh A, Ferron L, Douglas L, Kadurin I, Sri Ranjan Y, Fernandez-Alacid L, Millar NS, Dickenson AH, Lujan R, Dolphin AC. The increased trafficking of the calcium channel subunit alpha2delta-1 to presynaptic terminals in neuropathic pain is inhibited by the alpha2delta ligand pregabalin. J Neurosci 2009;29:4076–88. [3] Berecki G, Motin L, Haythornthwaite A, Vink S, Bansal P, Drinkwater R, Wang CI, Moretta M, Lewis RJ, Alewood PF, Christie MJ, Adams DJ. Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage- dependently block recombinant and native N-type calcium channels. Mol Pharmacol 2010;77:139–48. This is the first electrophysiological study addressing the effects of Tx3-3 on sensory transmission in spinal cord of rats. The present data extend results from previous behavioural studies, showing a prevalent antinociceptive effect of Tx3-3 on neuropathic states and suggest the R-type VGCC as the main target of such action. [4] Boroujerdi A, Kim HK, Lyu YS, Kim DS, Figueroa KW, Chung JM, Luo ZD. Injury discharges regulate calcium channel alpha-2-delta-1 subunit upregulation in the dorsal horn that contributes to initiation of neuropathic pain. PAIN 2008;139:358–66. p p [5] Breustedt J, Vogt KE, Miller RJ, Nicoll RA, Schmitz D. Alpha1E-containing Ca21 channels are involved in synapticplasticity. Proc Natl Acad Sci USA 2003;100:12450–5. Appendix. Timecourse of cumulative doses of toxin Tx3-3 in mechanical evoked neuronal response (n 5 3). Article history: Received 21 December 2016 Received in revised form 20 May 2017 Accepted 24 May 2017 The Tx3-3 affected the neuronal responses evoked by thermal stimulation in naive and SNL rats. These results are in accordance with the previously reported inhibitory effect of Tx3- 3 in heat-induced nociceptive pain.13 Notably, the doses of Tx3- 3 used here are comparable to those used in the above- mentioned behavioral study. In the present study, however, the lower dose of Tx3-3 showed a tendency to increase the thermally evoked neuronal response in naive rats. This may result from the blockade of P/Q-type VGCCs presented on inhibitory interneurones.48,51 Likewise, spinal application of the P/Q-type VGCC blocker v-agatoxin IVA tended to facilitate neuronal responses at lower doses.30 However, this phenom- enon was not seen in SNL rats, possibly because in this neuropathic condition, R-type VGCCs come into play, as demonstrated by the increased sensitivity of thermally evoked neuronal response to the R-type VGCC blocker SNX-482 in SNL rats.29 Therefore, possibly, the neuropathy may cause a path- ophysiological upregulation of R-type VGCC activity (or promote a given conformational state) that favors the action of Tx3-3 on it, explained by the enhanced sensitivity of SNL rats to Tx3-3 inhibitory effect on thermally evoked neuronal responses (as lower doses were needed to achieve inhibitory effect in SNL rats compared to control rats). 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https://openalex.org/W4361881891	https://figshare.com/articles/journal_contribution/Supplementary_Figure_2_from_Cyclophosphamide_Induces_a_Type_I_Interferon_Associated_Sterile_Inflammatory_Response_Signature_in_Cancer_Patients_Blood_Cells_Implications_for_Cancer_Chemoimmunotherapy/22445391/1/files/39896433.pdf	English	null	Supplementary Figure 2 from Cyclophosphamide Induces a Type I Interferon–Associated Sterile Inflammatory Response Signature in Cancer Patients' Blood Cells: Implications for Cancer Chemoimmunotherapy	null	2,023	cc-by	116	CD14++CD16-IL8RA+ CD14+CD16+IL8RA+ HLA-DR in CD14++CD16- A B MFI (% of maximum) ents (% of maximum) CD8+IL8RA+ CD8+OX40+ C M Eve m) Events (% of maximum E Figure S2 CD14++CD16-IL8RA+ CD14+CD16+IL8RA+ HLA-DR in CD14++CD16- A B MFI (% of maximum) ents (% of maximum) CD8+IL8RA+ CD8+OX40+ C M Eve m) Events (% of maximum E Figure S2 CD14++CD16-IL8RA+ CD14+CD16+IL8RA+ HLA-DR in CD14++CD16- A B MFI (% of maximum) ents (% of maximum) M Eve CD14+CD16+IL8RA+ HLA-DR in CD14++CD16- B MFI (% of maximum) M HLA-DR in CD14++CD16- B MFI (% of maximum) M CD14++CD16-IL8RA+ A ents (% of maximum) Eve A B HLA-DR in CD14++CD16- CD8+IL8RA+ CD8+OX40+ C m) Events (% of maximum E C Figure S2
https://openalex.org/W2792130808	https://europepmc.org/articles/pmc6058571?pdf=render	English	null	An α-tocopheryl succinate enzyme-based nanoassembly for cancer imaging and therapy	Drug delivery	2,018	cc-by	10,417	Introduction Synthetic polymers [such as poly(lactic-co-glycolic acid) and polycaprolactone] and natural polymers (such as hyaluronic acid) are approved by the US Food and Drug Administration (FDA) for clinical applications (Danhier et al., 2012; Li & Tan, 2014; Zhang et al., 2014). Recently, a number of formulation strategies have been developed for cancer therapy (Yoon et al., 2015; Kemp et al., 2016; Kim et al., 2016; Tran et al., 2016; Song et al., 2017). Because of their toxicity-related properties, the tumor-select- ive delivery of anticancer agents is a crucial issue in the development of formulations for anticancer drugs. It is known that nanocarriers are capable of tumor-specific drug delivery based on the enhanced permeability and retention (EPR) effect, which may be considered as a passive tumor tar- geting strategy (Matsumura & Maeda, 1986; Maeda et al., 2000). To compensate for the drawbacks of passive tumor targeting based on the EPR effect, various active tumor tar- geting strategies (such as the introduction of tumor-targeting ligands to nanocarrier development) have been designed (Danhier et al., 2010; Lammers et al., 2012; Shim et al., 2017). Although remarkable anticancer activities of diverse nanocar- riers have been verified in cell culture and animal models, only a few of them have been approved for clinical use. One of the major obstacles in extending the application of the developed nanocarriers to human systems is the toxicity of the pharmaceutical excipients comprising the nanocarriers. To overcome these toxicities, biocompatibility and bio- degradability have been regarded as important requisites during the development of pharmaceutical formulations. g With the use of these polymers, intrinsic proteins (such as the human serum albumin [HSA]) have been also widely studied for the preparation of nanoformulations (Hawkins et al., 2008; Elsadek & Kratz, 2012). HSA is the most abundant protein in the plasma (35–50 g/L in human serum) and there- fore it has been used for the development of injection dos- age forms (Elzoghby et al., 2012; Zhang et al., 2016; Liu et al., 2017). HSA nanoparticles can be prepared by several prepar- ation methods, such as coacervation, emulsification, thermal gelation, nanospray drying, nanoparticle albumin-bound technology, & self-assembly (Elzoghby et al., 2012). The albu- min-bound paclitaxel (nab-paclitaxel) nanoparticle has been approved by the USFDA in 2005 and is available in the mar- kets (Abraxane, Abraxis BioScience; Gradishar, 2006; Elsadek & Kratz, 2012). ABSTRACT Nanoassembly (NA) based on a D-a-tocopherol succinate (aTS) conjugated lysozyme (Lys) (Lys-aTS) was fabricated for tumor-selective delivery of curcumin (CUR) for breast cancer therapy. Lys and aTS were used as a biocompatible enzyme and a hydrophobic residue, respectively, for the preparation of nanocarriers in this study. Compared with CUR-loaded cross-linked Lys (c-Lys/CUR) NA, Lys-aTS/CUR NA exhibited a smaller hydrodynamic size (213 nm mean diameter), a narrower size distribution, and a more spherical shape. Sustained drug release was observed from the Lys-aTS/CUR NA for five days at a normal physiological pH (pH 7.4). The developed Lys-aTS/CUR NA showed enhanced cellular accumula- tion, antiproliferative effects, and apoptotic efficacies in MDA-MB-231 human breast adenocarcinoma cells. According to the results of optical imaging test in the MDA-MB-231 tumor-bearing mouse mod- els, the Lys-aTS/CUR NA-injected group exhibited a more tumor-selective accumulation pattern, rather than being distributed in the normal tissues and organs. The observed tumor targetability of Lys-aTS/ CUR was further studied, which revealed improved in vivo anticancer activities (better inhibition of tumor growth and induction of apoptosis in the tumor tissue) after an intravenous administration in the MDA-MB-231 tumor-bearing mouse models. All these results indicate that the newly developed enzyme-based nanocarrier, the Lys-aTS NA, can be a promising candidate for the therapy of breast cancers. KEYWORDS a-tocopherol succinate; curcumin; lysozyme; nanoassembly; tumor targeting CONTACT Hyun-Jong Cho hjcho@kangwon.ac.kr College of Pharmacy, Kangwon National University, Chuncheon 24341, Republic of Korea Supplemental data for this article can be accessed here. 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Song Yi Lee and Hyun-Jong Cho College of Pharmacy, Kangwon National University, Chuncheon, Republic of Korea ARTICLE HISTORY Received 13 January 2018 Revised 22 February 2018 Accepted 26 February 2018 KEYWORDS a-tocopherol succinate; curcumin; lysozyme; nanoassembly; tumor targeting An a-tocopheryl succinate enzyme-based nanoassembly for cancer imaging and therapy Song Yi Lee and Hyun-Jong Cho DRUG DELIVERY, 2018 VOL. 25, NO. 1, 738–749 https://doi.org/10.1080/10717544.2018.1446476 DRUG DELIVERY, 2018 VOL. 25, NO. 1, 738–749 https://doi.org/10.1080/10717544.2018.1446476 DRUG DELIVERY, 2018 VOL. 25, NO. 1, 738–749 https://doi.org/10.1080/10717544.2018.1446476 DRUG DELIVERY, 2018 VOL. 25, NO. 1, 738–749 htt //d i /10 1080/10 ho@kangwon.ac.kr College of Pharmacy, Kangwon National University, Chuncheon 24341, Republic of Korea ma UK Limited, trading as Taylor & Francis Group. under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, um, provided the original work is properly cited. Introduction It reduces the immunological responses that can be induced by foreign materials contained in polymeric nanocarriers (Elzoghby et al., 2012). A number of modified HSA-based nanoparticles have been developed for anticancer drug delivery to the tumor region (Byeon et al., 2016; Liu et al., 2017). DRUG DELIVERY 739 group of Lys. aTS (21.2 mg, 0.04 mmol) was dissolved in dimethyl sulfoxide (DMSO, 12 mL) and the pH was adjusted to a value of 4.0 by adding 1 N HCl. EDC (11.5 mg, 0.06 mmol) and NHS (6.7 mg, 0.06 mmol) were dissolved in that solution by stirring and the pH value was adjusted to 7 by adding 1 N NaOH. Lys (288 mg, 0.02 mmol) dissolved in DMSO (12 mL) was slowly added to the aTS/EDC/NHS solution and the mix- ture was stirred for 24 h. The solution of the mixture was then dialyzed against distilled water (DW) for two days with a dialysis membrane (molecular weight cutoff [MWCO]: 6–8 kDa). The resultant was freeze-dried for future use, after adding sucrose (1%, w/v). As a control group, cross-linked Lys (c-Lys) was synthesized via an EDC/NHS-coupled reaction. c-Lys was synthesized by the same procedure, without the addition of aTS. Similar to albumin-based nanocarriers, several lysozyme (Lys) nanovehicles have been developed for the delivery of drug cargos (Li et al., 2015; Lin et al., 2015; Mahanta et al., 2015). Lys has 129 amino acids with a molecular weight of 14.4 kDa (Canfield, 1963). Chemically, it is known as N-acetyl- muramide glycanhydrolase and it can hydrolyze the 1,4-beta- linkages between the N-acetylmuramic acid and the N-acetyl- D-glucosamine residues of peptidoglycans. Lys is abundant in milk, mucus, saliva, and tears and it is recognized as a part of the immune system. Its antibacterial and anticancer activities have also been elucidated in recent years (Aminlari et al., 2014; Mahanta et al., 2015). Self-assembled Lys nanogels, alone or in combination with other materials, have been developed as drug delivery systems (Li et al., 2015; Lin et al., 2015). In this study, D-a-tocopherol succinate (aTS) was chemically conjugated to Lys as a hydrophobic residue. aTS was shown to inhibit cancer cell proliferation while inducing apoptosis in cancer cells via mitochondrial destabilization, without affecting the proliferation of normal cells (Prasad et al., 2003). Materials The fluorescence spectra of Lys, c-Lys, and Lys-aTS were obtained by using a fluorescence spectrometer (FP-6500, Jasco Corp., Tokyo, Japan). Lys, c-Lys, and Lys-aTS were dis- persed in PBS (5 mM, pH 7.2) at a Lys concentration of 0.1 mg/mL. Each emission spectrum (300–500 nm) was scanned at a fixed excitation wavelength of 280 nm. CUR, deuterium oxide (D2O), hexadeuterodimethyl sulfoxide (DMSO-d6), Lys (from chicken egg white, 70000 U/mg), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC), and poly(ethylene glycol) 400 (PEG 400) were purchased from Sigma-Aldrich (Saint Louis, MO). Sodium dodecyl sulfate (SDS), aTS, and the 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay kit were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Cy5.5-NHS was purchased from BioActs (DKC Corp., Incheon, Korea). Roswell Park Memorial Institute (RPMI) 1640 medium, penicillin, streptomycin, and heat-inactivated fetal bovine serum (FBS) were acquired from Gibco Life Technologies, Inc. (Grand Island, NY). All the other reagents were of analytical grade and purchased from commer- cial sources. The amine groups of Lys, c-Lys, and Lys-aTS were quanti- tatively determined by the TNBS assay. Lys, c-Lys, or Lys-aTS was dissolved in the reaction buffer (0.1 M sodium bicarbon- ate, pH 8.5) at 50 lg/mL. A 0.25 mL aliquot of the TNBS solu- tion (0.01%, w/v) was added to each of the protein solutions (0.5 mL). The mixtures were incubated at 37 C for 2 h. SDS (10%, w/v) solution (0.25 mL) and 1 N HCl (0.125 mL) were then added to each of the mixtures. The absorbance was measured at 335 nm with a multi-mode microplate reader (SpectraMax i3, Molecular Devices, Sunnyvale, CA), and the relative absorbance was calculated by comparing the absorb- ance of each group to that of the Lys group. Introduction Moreover, it can also enhance the inhibition of tumor growth as in case of ionizing radiation and hyperther- mia therapy (Prasad et al., 2003; Angulo-Molina et al., 2014). Because of the anticancer activities of aTS and its derivatives, they have been used as a component of nanoparticles for cancer therapy (Zeng et al., 2013; Tao et al., 2015; Zeng et al., 2015; Mallick et al., 2016; Muddineti et al., 2017; Palao- Suay et al., 2017). Herein, aTS-conjugated Lys (Lys-aTS) was synthesized and curcumin (CUR) was incorporated into the Lys-aTS nanoassembly (NA). The physicochemical properties, in vitro anticancer activities, in vivo tumor targetability, and the in vivo anticancer activities of the Lys-aTS/CUR NA were assessed. The synthesis of c-Lys and Lys-aTS was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). According to Laemmli’s discontinuous method (Laemmli, 1970), gel electrophoresis was performed with a running gel (20% acrylamide) and a stacking gel (5% acryl- amide). Lys, c-Lys, and Lys-aTS were blended with the Laemmli sample buffer and were heated at 95 C for 5 min to denature the proteins. A 20 mL aliquot of the sample and a protein marker (EzWay Protein-Multicolor Ladder, KOMA Biotech, Seoul, Korea) were loaded into the wells. The loaded amount of Lys, c-Lys, and Lys-aTS was 10 mg. The electrophor- esis was carried out for 90 min at 100 V. The gel was then stained with Coomassive R250 (Bio-Rad Laboratories, Inc., Hercules, CA) for 30 min and then was de-stained overnight. Lys, c-Lys, and Lys-aTS were dispersed in the phosphate buffered saline (PBS, 5 mM, pH 7.2) using a Lys concentration of 0.1 mg/mL. They were analyzed by the ChirascanTM-plus circular dichroism (CD) spectrometer (Applied Photophysics Ltd., Surrey, UK). The path length (1 mm) was fixed within the quartz cell. The step size was 1 nm, the bandwidth was 1 nm, and the wavelength ranged between 200 and 260 nm. Fabrication and characterizations of CUR-loaded NAs CUR was encapsulated as a hydrophobic model drug in the Lys-aTS NAs by the dialysis method (Jeong et al., 2017). Both CUR (6 mg) and Lys-aTS (96 mg) were dissolved in DMSO (6 mL). The solution was then transferred to a dialysis bag (MWCO: 6–8 kDa) and was dialyzed against DW for 6 h. The resultant was lyophilized after adding sucrose (1%, w/v). In case of the c-Lys/CUR NA, 96 mg of c-Lys was used instead of Lys-aTS for preparing the c-Lys/CUR NA using the same procedure. The CUR contents in the c-Lys/CUR NA and the Lys-aTS/ CUR NA were quantitatively determined by using a high-per- formance liquid chromatography (HPLC) system equipped with a pump (PU-2089 Plus; Jasco, Tokyo, Japan), an automatic injector (AS-2050 Plus), and an UV/Vis detector (UV-1575) as previously reported (Lee et al., 2016). A reverse phase C18 column (Gemini, 250 4.6 mm; Phenomenex, Torrance, CA) with a guard column (SecurityGuard Guard Cartridge kit, Phenomenex, Torrance, CA) was used for analyzing the CUR. For calculating the drug encapsulation efficiency, the CUR- loaded NA was dissolved in DMSO and further diluted with the mobile phase. The mobile phase was composed of aceto- nitrile, tetrahydrofuran, and water (35:20:45, v/v/v). The flow rate was 1 mL/min and the injection volume was 20 lL. The absorbance was monitored at 425 nm by an UV/Vis detector. The inter- and intra-day variances were within the accept- able range. The cellular distribution of the CUR-loaded NA was assessed by confocal laser scanning microscopy (CLSM). MDA- MB-231 cells, at a density of 1.0 105 cells per well (surface area of 1.7 cm2 per well), were seeded onto culture slides (BD Falcon, Bedford, MA) and were incubated for 24 h at 37 C. The CUR solution and the Lys-aTS/CUR NA (corresponding to a CUR concentration of 10 lg/mL) were added to the cells and they were incubated for 2 and 6 h, respectively. The cells were then washed with PBS (pH 7.4) at least thrice and fixed with a 4% (v/v) solution of formaldehyde. The liquid content was removed by drying and the VECTASHIELD mounting medium with 40,6-diamidino-2-phenylindole (DAPI) (H-1200; Vector Laboratories, Inc., Burlingame, CA) was added to the fixed cells for staining the nuclei and to prevent fading. The fluorescence signals of CUR and DAPI in the cells were observed by CLSM (LSM 880, Carl-Zeiss, Thornwood, NY). Synthesis and characterizations of c-Lys and Lys-aTS The molecular weights of c-Lys and Lys-aTS were assessed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The matrix used was 2,5-dihydroxybenzoic acid (DHB), and the Voyager DE-STR mass spectrometer (Applied Biosystems, Framingham, MA) To fabricate the structure of the Lys-based NA, aTS was cova- lently bonded as a hydrophobic residue to Lys. To synthesize the Lys-aTS, an EDC/NHS-coupled amide bond was formed between the carboxylic acid group of aTS and the amine S. Y. LEE AND H.-J. CHO 740 at 3, 6, 24, 48, 72, and 120 h and were put into the HPLC vials. Then, equivalent volumes of the fresh release media (PBS, pH 7.4) were added. The amounts of CUR released were quantitatively determined by the described HPLC method. was used for all the analyses. The instrument was operated in a linear mode with nitrogen lasers (337 nm) and the accel- erating voltage was 25 kV. The particle characteristics of c-Lys and Lys-aTS dispersion in DW were assessed by a dynamic light scattering (DLS) method (ELS-Z1000; Otsuka Electronics, Tokyo, Japan) accord- ing to manufacturer’s protocol. c-Lys or Lys-aTS was dis- persed in DW (5 mg/mL) by vortex-mixing for 5 min and their mean diameters and polydispersity index values were also measured. Cellular accumulation and distribution The cellular accumulation efficiency of the CUR-loaded NA developed in this study was evaluated in MDA-MB-231 cells using flow cytometry. The MDA-MB-231 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). The cells were cultured with RPMI 1640 supplemented with 10% (v/v) FBS, 1% (v/v) penicillin (100 U/mL), and streptomycin (0.1 mg/ mL) in a humidified atmosphere with 5% carbon dioxide at 37 C. The MDA-MB-231 cells were seeded onto six-well plates at a density of 6.0 105 cells per well and were incu- bated for 24 h at 37 C. The CUR solution and the Lys-aTS/ CUR NAs (corresponding to a CUR concentration of 10 lg/mL) were added to the cells and were incubated for 2 and 6 h, respectively. After washing with PBS (pH 7.4) at least thrice, the cells were collected after centrifugation at 16,100 g for 5 min. The cell pellets were resuspended with FBS solution (2%, v/v). The cellular accumulation efficiency, represented as the cell count according to the fluorescence intensity, was evaluated by a FACSCalibur Fluorescence-activated Cell Sorter (FACSTM) equipped with the CELLQuest software (Becton Dickinson Biosciences, San Jose, CA). V ¼ 0:5 longest diameter ðshortest diameterÞ2 (1) After the tumor volume reached 150–200 mm3, 0.1 mL ali- quots of the Cy5.5 solution or the Cy5.5-Lys-aTS/CUR NA dis- persion, containing 100 mg/kg dose of Cy5.5, were injected to the tail veins of the mice. Prior to a whole body scanning by the Spectral Lago X (Spectral Instruments Imaging; Tucsan, AZ), the mice were anesthetized by the inhalation of isoflur- ane (2.5%). The Cy5.5 filter (excitation: 640 nm; emission: 710 nm) was selected for the detection of NIRF in the body and the AMIView software (ver. 1.7.01) was used for image analyses. The whole body scan images were taken at 0 (pre), 4, and 24 h post-injection. The liver, lungs, heart, kidneys, spleen, and the tumor were dissected from the mice at 24 h post-injection for ex vivo NIRF imaging. The ex vivo images were obtained by using a fluorescence in vivo imaging sys- tem (FOBI; NeoScience Co., Ltd., Suwon, Korea) with a red laser source, according to the manufacturer’s protocol. The apoptotic effects of the CUR-loaded NA were assessed in MDA-MB-231 cells. MDA-MB-231 cells were cultured according to the aforementioned method, seeded on six-well plates at a density of 1.0 105 cells per well and were incu- bated for 24 h at 37 C. Lys, Lys-aTS, CUR, and the Lys-aTS/ CUR NA (corresponding to a CUR concentration of 2 lg/mL) was added to the cells and were incubated for 24 h. The cells were washed with PBS (pH 7.4) at least thrice and the cell pellets were collected by centrifugation at 16,100 g for 5 min. They were then suspended in the reaction buffer of the fluor- escein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (BD Pharmingen, BD Biosciences, San Jose) and the cells were stained with Annexin V-FITC and propidium iodide (PI), according to the manufacturer’s protocol. The fluorescence intensity values of both the reagents in the cells were ana- lyzed by a FACSTM equipped with the CellQuest software. In vivo antitumor efficacies The in vivo antitumor efficacies of the developed nanosys- tems were assessed in MDA-MB-231 tumor-xenografted mouse models prepared with BALB/c nude mice (female, five weeks old). The mice were reared in a light-controlled room at a temperature of 22 ± 2 C and a relative humidity of 55 ± 5%. The animal study was approved by the Animal Care and Use Committee of the Kangwon National University. The MDA-MB-231 cells were cultured according to the aforemen- tioned protocols and aliquots of the MDA-MB-231 cell sus- pension (2.0 106 cells in 0.1 mL) were injected into the dorsal regions of the mice. The tumor volume was calculated using the Equation (1). When the tumor volume was approxi- mately 150 mm3, the mice were randomly divided into four groups and the tumor volume was monitored. Lys-aTS and Lys-aTS/CUR NA were dispersed in water for injection. The CUR solution (1 mg/mL) was prepared by dissolving CUR in 37.5% (w/w) PEG400 solution. Lys-aTS, CUR or the Lys-aTS/ CUR NA was injected into the tail veins of the mice, at a dose of 5 mg/kg CUR, on days 5, 8, 12, 15, 19, and 22. The body weight of each mouse was monitored along with the tumor volume. On the final day, the tumor tissues were dis- sected and fixed in formaldehyde (4%, v/v) solution for histo- logical staining. The tumor tissues with a thickness of 6 lm were deparaffinized and hydrated with ethanol. The tumors Fabrication and characterizations of CUR-loaded NAs The particle characteristics of the c-Lys/CUR NA and the Lys-aTS/CUR NA were investigated. The mean diameter, poly- dispersity index, and the zeta potential values of the c-Lys/ CUR NA and the Lys-aTS/CUR NA dispersions in DW were measured using DLS and laser Doppler methods (ELS-Z1000; Otsuka Electronics, Tokyo, Japan), according to the man- ufacturer’s protocol. The mechanisms of cellular entry of the Lys-aTS/CUR NA were tested by treatment with endocytosis inhibitors (genis- tein and chlorpromazine) in MDA-MB-231 cells (Lee et al., 2017a). The fluorescence signal of encapsulated CUR was used as an indicator of the amount of cellular uptake. The MDA-MB-231 cells were seeded onto six-well plates at a density of 4.0 105 cells per well and were incubated for 1 day at 37 C. Genistein (200 mM) or chlorpromazine (10 mg/ mL) was co-incubated with the Lys-aTS/CUR NA (at a CUR concentration of 10 mg/mL) in MDA-MB-231 cells for 4 h. After removing the samples, the cells were washed with PBS at least thrice. The cells were detached from the bottom of the well-plate and were collected by centrifugation. The cell pel- lets were then suspended with PBS supplemented with FBS (2%, v/v) for flow cytometry analyses. The cell count, The morphological shapes of the c-Lys/CUR NA and the Lys-aTS/CUR NA were observed by transmission electron microscopy (TEM). An aliquot of dispersed NA was stained with 2% (w/v) phosphotungstic acid. It was loaded onto cop- per grids using films, dried for 10 min, and observed were by TEM (LEO 912AB OMEGA; Carl Zeiss, Oberkochen, Germany). An aliquot of the dispersed NAs (0.15 mL), including 30 lg CUR, was introduced into Mini-GeBAflex tubes (14 kDa MWCO; Gene Bio-Application Ltd., Kfar Hanagide, Israel). They were next transferred to the release media (10 mL PBS at pH 7.4) and incubated in a shaking water bath at 50 rpm at 37 C. Aliquots of the release media (0.2 mL) were collected The morphological shapes of the c-Lys/CUR NA and the Lys-aTS/CUR NA were observed by transmission electron microscopy (TEM). An aliquot of dispersed NA was stained with 2% (w/v) phosphotungstic acid. It was loaded onto cop- per grids using films, dried for 10 min, and observed were by TEM (LEO 912AB OMEGA; Carl Zeiss, Oberkochen, Germany). DRUG DELIVERY 741 measuring the absorbance at 675 nm using a multi-mode microplate reader (SpectraMax i3, Molecular Devices, Sunnyvale, CA). Fabrication and characterizations of CUR-loaded NAs indicated by the fluorescence intensity, was measured using a FACSTM equipped with the CellQuest software . The per- centage (%) of the mean fluorescence intensity value of the (Lys-aTS/CUR NA þ genistein) or the (Lys-aTS/CUR NA þ chlorpromazine)-treated groups relative to that of the Lys-aTS/CUR NA-treated group was calculated. Female BALB/c nude mice (five weeks old, Charles River, Wilmington, MA) were used for preparing the MDA-MB-231 tumor-xenografted mouse model. The mice were reared in a light-controlled room maintained at a temperature of 22 ± 2 C and a relative humidity of 55 ± 5%. The experimen- tal procedures were approved by the Animal Care and Use Committee of the Kangwon National University. Aliquots of the MDA-MB-231 cell suspension (2.0 106 cells in 0.1 mL) were injected into the dorsal regions of the mice. The tumor volume (V, mm3) was calculated using the Equation (1): In vitro anticancer activities The antiproliferative activity of the Lys-aTS/CUR NA was eval- uated in MDA-MB-231 cells by a colorimetric assay. MDA-MB- 231 cells, at a density of 5.0 103 cells per well, were seeded onto 96-well plate and incubated at 37 C for 24 h. Lys, Lys- aTS, CUR, and the Lys-aTS/CUR NA, corresponding to CUR concentrations of 0.5, 1, 2.5, 5, and 10 lg/mL, respectively, were treated for 48 and 72 h; aTS at concentrations of 0.1, 1, 10, 25, and 50 lg/mL was added to the cells and incubated for 72 h. After eliminating those samples, the cells were treated with the MTS-based CellTiter 96V R AQueous One Solution Cell Proliferation Assay Reagent (Promega Corp., Fitchburg, WI) at 37 C, according to the manufacturer’s protocol. The absorbance was measured at 490 nm with a multi-mode microplate reader (SpectraMax i3, Molecular Devices, Sunnyvale, CA) and the cell viability was presented by comparing with the absorbance value of the control (no treatment) group. (1) Near-infrared fluorescence (NIRF) imaging The biodistribution of the fabricated NA in the MDA-MB-231 tumor-xenografted mouse model was assessed by a real-time NIRF imaging. Cy5.5 was conjugated to Lys-aTS as a NIRF dye. Cy5.5-NHS was conjugated to the amine group of Lys via the formation of an amide bond. Lys-aTS (20 mg) was dis- persed in PBS (5 mL at pH 7.4), and the Cy5.5-NHS solution (0.2 mg in 0.02 mL DMSO) was added to that dispersion. This mixture was stirred for 4 h and dialyzed against DW with a dialysis membrane (MWCO: 6–8 kDa). It was lyophilized for further use. CUR was encapsulated in the Cy5.5-Lys-aTS NA using the dialysis method described in the Section “Fabrication and characterizations of CUR-loaded NAs”. The content of Cy5.5 in the NA was quantitatively analyzed by S. Y. LEE AND H.-J. CHO 742 were stained with hematoxylin and eosin (H&E) and further processed for a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay by standard procedures. For the TUNEL assay, 3,30-diaminobenzidine (DAB) was added and the tissues were incubated for the development of color for the detection of DNA fragmentation produced by the apoptotic signaling cascades. formation of an amide bond between the amine and car- boxylic acid groups of Lys itself. As a control group, c-Lys was synthesized without the addition of aTS in this study. Commonly, the use of both EDC and NHS reagents might contribute to the formation of a stable amide bond between the amine and the carboxylic acid groups without side reactions. The synthesis of c-Lys and Lys-aTS was verified by several experimental methods (Figure 2; Supplementary Figures S1 and S2). The change in the amino acid sequence, corre- sponding to the molecular weights of the synthesized Lys derivatives, was tested by a SDS PAGE assay (Figure 2(A)). In the Lys lane, the band corresponding to the monomer was primarily observed, while bands corresponding to dimers and multimers were hardly seen. However, the c-Lys group exhib- ited multiple bands, indicating the presence of monomers, dimers, and trimers. Contrary to the c-Lys group, the Lys-aTS group showed the formation of a smaller number of multi- mers, which was similar to the Lys group. During the process of Lys-aTS synthesis, aTS seemed to have successfully conju- gated to Lys without the formation of self-crosslinks within the structure of Lys. The secondary structure of Lys was investigated by CD analysis (Supplementary Figure S1). Statistical analyses The Student’s t-test was used for statistical analyses of the data. All experiments were performed at least thrice. All the data are presented as the mean ± standard deviation (SD). Near-infrared fluorescence (NIRF) imaging In the far UV region, the minimum peak was formed around 208 nm in this study as reported (Cho et al., 2011). It is known that minimum peaks at 208 and 222 nm imply the contribution of n ! p transfer to the peptide bond of a-heli- ces (Price, 2000; Cho et al., 2011). The CD values (at 208 nm) of c-Lys and Lys-aTS were significantly lower than that of Lys. The formation of crosslinks within the structure of Lys and the attachment of aTS to Lys seemed to alter the secondary structure of Lys, including a reduction in the a-helix content. The tertiary structure of Lys was studied by fluorescence intensity detection (Supplementary Figure S2). It is known Synthesis and characterization of Lys-aTS The CUR-loaded Lys-aTS NA was fabricated and its thera- peutic potential in breast cancer was evaluated in this study (Figure 1). To prepare the structure of the Lys-based NA, aTS was used as a hydrophobic residue, which was cova- lently bonded to Lys. Lys has 6 lysine and 11 arginine resi- dues and has amine groups in its structure (Masuda et al., 2005). The amine group of Lys was linked to the carboxylic acid group of aTS to produce an EDC/NHS-coupled amide bond (Figure 1). Due to the hydrophobic nature of aTS, Lys- aTS may show properties of self-assembly in aqueous envi- ronments, leading to the formation of the NA structure. It is reported that Lys also has 11 free carboxylic acid groups, although two residues (aspartate 52 and glutamic acid 35) are directly involved in the chemical reactions of Lys (Lin & Koshland, 1969). Therefore, the addition of EDC/NHS reagents in Lys could contribute to self-conjugation via the Figure 1. Schematic illustration of tumor-targeted therapy of Lys-aTS/CUR NA. Figure 1. Schematic illustration of tumor-targeted therapy of Lys-aTS/CUR NA. re 2. Synthesis of c-Lys and Lys-aTS and their characterizations. (A) The result of SDS-PAGE assay. Protein standard, Lys, c-Lys, and Lys-aTS were loaded onto wells for gel electrophoresis. (B) The result of TNBS assay. Each point represents the mean ± SD (n ¼ 3). p < .001, compared with Lys group. %%%p < .001, pared with c-Lys group. (C) MALDI-TOF choromatograms of c-Lys and Lys-aTS. Relative intenstiy (%) accroding to mass (m/z) is plotted. (D) Size distribution dia- ms of c-Lys and Lys-aTS. Differential intensity according to the diameter is plotted. DRUG DELIVERY 743 DRUG DELIVERY 743 743 Figure 2. Synthesis of c-Lys and Lys-aTS and their characterizations. (A) The result of SDS-PAGE assay. Protein standard, Lys, c-Lys, and Lys-aTS were loaded onto the wells for gel electrophoresis. (B) The result of TNBS assay. Each point represents the mean ± SD (n ¼ 3). p < .001, compared with Lys group. %%%p < .001, compared with c-Lys group. (C) MALDI-TOF choromatograms of c-Lys and Lys-aTS. Relative intenstiy (%) accroding to mass (m/z) is plotted. (D) Size distribution dia- grams of c-Lys and Lys-aTS. Differential intensity according to the diameter is plotted. The relative absorbance value of Lys-aTS was significantly lower than those of Lys and c-Lys (p < .001). Synthesis and characterization of Lys-aTS From the rela- tive absorbance values, approximately 20% of the amine groups in Lys seemed to participate in the reaction with the carboxylic acid groups of aTS and those of Lys itself. The lower number of amine groups quantified in Lys-aTS than in c-Lys, indicated the efficient covalent bonding of aTS to Lys in Lys-aTS compared with self-crosslinking of Lys in c-Lys. The molecular weights of the synthesized materials were determined by MALDI-TOF analysis (Figure 2(C)). The major peak of Lys was seen at an m/z value of 14325.77 and the presence of multimers were also observed in this study (data not shown; Farmer & Caprioli, 1991). In case of the c-Lys The relative absorbance value of Lys-aTS was significantly lower than those of Lys and c-Lys (p < .001). From the rela- tive absorbance values, approximately 20% of the amine groups in Lys seemed to participate in the reaction with the carboxylic acid groups of aTS and those of Lys itself. The lower number of amine groups quantified in Lys-aTS than in c-Lys, indicated the efficient covalent bonding of aTS to Lys in Lys-aTS compared with self-crosslinking of Lys in c-Lys. The molecular weights of the synthesized materials were determined by MALDI-TOF analysis (Figure 2(C)). The major peak of Lys was seen at an m/z value of 14325.77 and the presence of multimers were also observed in this study (data not shown; Farmer & Caprioli, 1991). In case of the c-Lys that an emission spectrum in the range of 330–345 nm may be due to the tryptophan residues in proteins (Zemser et al., 1994). The highest fluorescence intensity of Lys determined in this study was observed at an excitation wavelength of 341 nm (Supplementary Figure S2). The relative fluorescence intensity values of c-Lys and Lys-aTS at the tested excitation and emission wavelengths were 86.7 and 59.1% respectively, compared to that of Lys. The crosslinking of Lys itself and the Lys-aTS conjugation seemed to alter the tertiary structure of Lys. T lo ti g ca lo c- in T d that an emission spectrum in the range of 330–345 nm may be due to the tryptophan residues in proteins (Zemser et al., 1994). The highest fluorescence intensity of Lys determined in this study was observed at an excitation wavelength of 341 nm (Supplementary Figure S2). Synthesis and characterization of Lys-aTS The relative fluorescence intensity values of c-Lys and Lys-aTS at the tested excitation and emission wavelengths were 86.7 and 59.1% respectively, compared to that of Lys. The crosslinking of Lys itself and the Lys-aTS conjugation seemed to alter the tertiary structure of Lys. The chemical conjugation of aTS to Lys was also tested by using the TNBS assay (Figure 2(B)). The TNBS assay has been used for the quantitative analysis of the amine groups. 744 S. Y. LEE AND H.-J. CHO Figure 3. Particle characterizations of c-Lys/CUR NA and Lys-aTS/CUR NA. (A) Size distribution diagrams of c-Lys/CUR NA and Lys-aTS/CUR NA dispersion. The differ- ential intensity is plotted according to the mean diameter. (B) TEM images of c-Lys/CUR NA and Lys-aTS/CUR NA dispersion. The length of the scale bar is 200 nm. (C) Drug release profile of Lys-aTS/CUR NA. Released amounts of CUR (%) from Lys-aTS/CUR NA dispersion are presented. Each point represents the mean ± SD (n ¼ 3). 744 S. Y. LEE AND H.-J. CHO Figure 3. Particle characterizations of c-Lys/CUR NA and Lys-aTS/CUR NA. (A) Size distribution diagrams of c-Lys/CUR NA and Lys-aTS/CUR NA dispersion. The differ- ential intensity is plotted according to the mean diameter. (B) TEM images of c-Lys/CUR NA and Lys-aTS/CUR NA dispersion. The length of the scale bar is 200 nm. (C) Drug release profile of Lys-aTS/CUR NA. Released amounts of CUR (%) from Lys-aTS/CUR NA dispersion are presented. Each point represents the mean ± SD (n ¼ 3). hydrophobic moiety (aTS in this study) can produce smaller particles with narrower size distributions, than the self-crosslinking of Lys (as observed in the c-Lys group). group, the major peak was observed at m/z 14288.86 and the low intensity peaks of multimers were also observed (data not shown). Two major peaks at m/z values of 14352.95 and 14873.40 were present in the spectrum of Lys-aTS. While the peak at m/z 14352.95 may indicate Lys itself, the peak at m/z 14873.40 seems to be related to Lys-aTS. The particle characteristics of c-Lys and the Lys-aTS dispersion in aqueous media were also evaluated (Figure 2(D) and Supplementary Table S1). The dispersion of the c-Lys group exhibited a mean diameter of 833 nm and a polydispersity index of 0.49. Aggregates were observed in the aqueous environment as shown in the particle size distribution diagram (Figure 2(D)). Synthesis and characterization of Lys-aTS In comparison with these data, the Lys-aTS dispersion group showed a mean diameter of 286 nm and a polydispersity index of 0.24. A unimodal peak was observed in the size dis- tribution profile of the Lys-aTS group. The attachment of a Preparation and characterization of Lys-aTS/CUR NA The CUR-loaded NAs were prepared and their particle proper- ties were evaluated (Figure 3 and Supplementary Table S1). Upon loading the CUR onto the c-Lys NA and the Lys-aTS NA, their hydrodynamic size and polydispersity index were reduced. It indicated that more compact nanocarriers could be formed upon drug loading. The zeta potential values of both the c-Lys/CUR NA and the Lys-aTS/CUR NA were posi- tive. The drug encapsulation efficiency values of the c-Lys/ CUR NA and the Lys-aTS/CUR NA were 70 and 64%, respect- ively. However, the hydrodynamic size of the c-Lys/CUR NA DRUG DELIVERY 745 745 Figure 4. Cellular uptake and antiproliferation studies in MDA-MB-231 cells. (A) Cellular accumulated amounts of CUR quantitatively analyzed by flow cytometry. CUR or Lys-aTS/CUR NA (10 lg/mL CUR concentration) was incubated for 2 and 6 h. Black, green, and red colors indicate control, CUR, and Lys-aTS/CUR NA group, respectively. Each point represents the mean ± SD (n ¼ 3). ###p < .001, compared with CUR group. (B) Intracellular distribution of NPs monitored by CLSM imaging. CUR or Lys-aTS/CUR NA (10 lg/mL CUR concentration) was incubated for 2 and 6 h. Green and blue colors indicate CUR and DAPI, respectively. The length of the scale bar in the image is 20 lm. (C) Antiproliferation assay in MDA-MB-231 cells. Cell viability (%) values of Lys, Lys-aTS, CUR, and Lys-aTS/CUR NA, according to CUR concentrations, are presented after 48 and 72 h incubation. Each point represents the mean ± SD (n ¼ 3). #p < .05, compared with CUR group. ###p < .001, compared with CUR group. Figure 4. Cellular uptake and antiproliferation studies in MDA-MB-231 cells. (A) Cellular accumulated amounts of CUR quantitatively analyzed by flow cytometry. CUR or Lys-aTS/CUR NA (10 lg/mL CUR concentration) was incubated for 2 and 6 h. Black, green, and red colors indicate control, CUR, and Lys-aTS/CUR NA group, respectively. Each point represents the mean ± SD (n ¼ 3). ###p < .001, compared with CUR group. (B) Intracellular distribution of NPs monitored by CLSM imaging. CUR or Lys-aTS/CUR NA (10 lg/mL CUR concentration) was incubated for 2 and 6 h. Green and blue colors indicate CUR and DAPI, respectively. The length of the scale bar in the image is 20 lm. (C) Antiproliferation assay in MDA-MB-231 cells. release from the Lys-aTS/CUR NA may contribute to its improved antitumor efficacy. release from the Lys-aTS/CUR NA may contribute to its improved antitumor efficacy. (493 ± 82 nm) was higher than that of the Lys-aTS/CUR NA (213 ± 18 nm). In case of the c-Lys/CUR NA group, the mean diameter seemed to be less suitable for EPR effect (aimed at passive tumor targeting) rather than the Lys-aTS/CUR NA group, and the formation of aggregates was also observed (Figure 3(A); Bae & Park, 2011). Moreover, according to the TEM images, the c-Lys/CUR NA exhibited a fibril-like shape while the Lys-aTS/CUR NA showed a round morphology (Figure 3(B)). It is known that the hen egg white Lys can eas- ily form amyloid fibrils after treatment with several factors (such as heat, salt, and organic solvents; Arnaudov & de Vries, 2005; Ow & Dunstan, 2013). The formation of irrevers- ible fibrillary protein aggregates and their accumulation in the body may lead to amyloidosis, similar to Alzheimer’s and prion diseases (Arnaudov & de Vries, 2005). The short fibril aggregate shape together with the higher hydrodynamic size and the broad size distribution of the c-Lys/CUR NA seemed to be unsuitable for its in vivo application. Thus, only the Lys-aTS/CUR NA group was further tested in the follow- ing studies. (493 ± 82 nm) was higher than that of the Lys-aTS/CUR NA (213 ± 18 nm). In case of the c-Lys/CUR NA group, the mean diameter seemed to be less suitable for EPR effect (aimed at passive tumor targeting) rather than the Lys-aTS/CUR NA group, and the formation of aggregates was also observed (Figure 3(A); Bae & Park, 2011). Moreover, according to the TEM images, the c-Lys/CUR NA exhibited a fibril-like shape while the Lys-aTS/CUR NA showed a round morphology (Figure 3(B)). It is known that the hen egg white Lys can eas- ily form amyloid fibrils after treatment with several factors (such as heat, salt, and organic solvents; Arnaudov & de Vries, 2005; Ow & Dunstan, 2013). The formation of irrevers- ible fibrillary protein aggregates and their accumulation in the body may lead to amyloidosis, similar to Alzheimer’s and prion diseases (Arnaudov & de Vries, 2005). The short fibril aggregate shape together with the higher hydrodynamic size and the broad size distribution of the c-Lys/CUR NA seemed to be unsuitable for its in vivo application. Thus, only the Lys-aTS/CUR NA group was further tested in the follow- ing studies. Preparation and characterization of Lys-aTS/CUR NA Cell viability (%) values of Lys, Lys-aTS, CUR, and Lys-aTS/CUR NA, according to CUR concentrations, are presented after 48 and 72 h incubation. Each point represents the mean ± SD (n ¼ 3). #p < .05, compared with CUR group. ###p < .001, compared with CUR group Cellular accumulation and distribution The cellular accumulation and intracellular distribution of the developed NA were studied in MDA-MB-231 cells by flow cytometry and CLSM imaging analyses, respectively (Figures 4(A,B)). MDA-MB-231 cells are human breast adeno- carcinoma cell lines that are used for the evaluation of the anticancer activities of nanocarriers targeting breast cancers (Jeong et al., 2017; Lee et al., 2017b). The cellular accumulation efficiency of the Lys-aTS/CUR NA was evaluated in MDA-MB-231 cells (Figure 4(A)). In this study, the intrinsic fluorescence signal of CUR was used for detecting the cellular movement of the developed NAs in cancer cells. The mean fluorescence intensity values of the Lys-aTS/CUR NA at 2 and 6 h were 1.45- and 2.07-folds higher than those of the CUR group, respectively (p < .001). The mean fluorescence intensity of the Lys-aTS/CUR NA group at 6 h was 25.4% higher than the value at 2 h. On the contrary, the mean fluorescence intensity of CUR at 6 h was slightly reduced compared to the value at 2 h. A similar pattern was also observed in the results of CLSM imaging (Figure 4(B)). A strong intracellular fluorescence signal was observed for the Lys-aTS/CUR NA group rather than for the CUR group after 2 and 6 h of incubation. The Lys-aTS/CUR NA showed a higher cellular accumulation efficiency than that of the CUR solution, which may be due to the mechanism of endocytosis of nano- carriers. It seems that the endocytosis of nanocarriers can The release pattern of CUR from the Lys-aTS/CUR NA was tested at conditions of normal physiological pH (Figure 3(C)). The released amounts of CUR from the Lys-aTS/CUR NA on days 1 and 5 were 11.5 ± 1.4 and 26.5 ± 7.5%, respectively. The initial burst release (initial 24 h in this study) was not so severe and a sustained drug release was observed for 120 h at a pH of 7.4. Sustained drug release has been regarded as one of the ideal characteristics of injection formulations, as it can reduce the frequency of doses and enhance patient com- pliance. It is expected that the observed sustained drug S. Y. LEE AND H.-J. CHO 746 An apoptosis assay was performed to reveal the mecha- nisms of anticancer activities of the developed NAs (Figure S5). It is reported that CUR can induce apoptosis in MDA-MB- 231 cells and subsequently contribute to cancer cell death (Wang et al., 2016). Cellular accumulation and distribution Especially, aTS may have additional proa- poptotic activities in cancer cells via mitochondrial inhibition and the production of superoxide radicals (Gruber et al., 2014; Qu et al., 2016). The apoptotic effects in the Lys, Lys- aTS, CUR, and the Lys-aTS/CUR NA-treated groups in MDA- MB-231 cells were assessed by Annexin V-FITC and the PI staining method (Supplementary Figure S5). Lower right (LR; Annexin V-FITC positive and PI negative) and upper right (UR; Annexin V-FITC positive and PI positive) panels in the Supplementary Figure S5 indicate early apoptosis and late apoptosis/cell death, respectively. Therefore, the sum of population percentages in (LR þ UR) panels was used to evaluate the apoptotic effects in this study. Notably, the Lys- aTS/CUR NA group exhibited a higher percentage of apop- totic populations in (LR þ UR) panels than the CUR group. The more efficient induction of apoptosis in the Lys-aTS/CUR NA group may be due to its higher cellular accumulation effi- ciency than that in the CUR group. From the results of in vitro anticancer activities, the Lys-aTS NA can be used effi- ciently for the delivery of CUR to cancer cells. increase the cellular entry rate of the drug cargo rather than the passive diffusion of the drug itself. Moreover, the electro- static interaction between the positive zeta potential of the Lys-aTS/CUR NA and the negative charge of the cellular membrane might also contribute to its enhanced cellu- lar uptake. increase the cellular entry rate of the drug cargo rather than the passive diffusion of the drug itself. Moreover, the electro- static interaction between the positive zeta potential of the Lys-aTS/CUR NA and the negative charge of the cellular membrane might also contribute to its enhanced cellu- lar uptake. The mechanisms of endocytosis of the developed Lys-aTS/ CUR NA were investigated by flow cytometry analysis after treatment with endocytosis inhibitors (Supplementary Figure S3). Genistein or chlorpromazine has been used to elucidate the mechanisms of caveolae- or clathrin-mediated endocyto- sis (Lee et al., 2017a). In particular, the percentages of fluor- escence intensity of the (Lys-aTS/CUR NA þ genistein) and (Lys-aTS/CUR NA þ chlorpromazine) groups were 94.55 ± 2.29 (p < .05) and 67.05 ± 1.94% (p < .001), respectively, relative to that of the Lys-aTS/CUR NA group. Co-treatment of chlorpro- mazine and Lys-aTS/CUR NA significantly reduced the cellular uptake amount of CUR (p < .001). Cellular accumulation and distribution It is possible to conclude that clathrin-mediated endocytosis might be the principal route for endocytosis of the developed Lys-aTS/CUR NA in MDA-MB-231 cells. In vitro anticancer activities The in vitro anticancer activities of the Lys-aTS/CUR NAs were demonstrated in MDA-MB-231 cells (Figure 4(C); Supplementary Figures S4 and S5). The antiproliferative effi- cacies of Lys, Lys-aTS, CUR, and Lys-aTS/CUR NA were com- pared by MTS-based assays (Figure 4(C)). Lys did not show severe cytotoxicity in the tested CUR concentration range. Lys also exhibits negligible cytotoxicity in breast cancer cell lines (such as MCF-7; Mahanta et al., 2015). The half maximal inhibitory concentration (IC50) value of aTS after 72 h of incu- bation in MDA-MB-231 cells was 25.73 ± 1.19 lg/mL (Supplementary Figure S4). The anticancer activities of aTS, mediated through apoptosis and chemosensitivity, is already reported (Kanai et al., 2010). Due to the presence of aTS in Lys-aTS, Lys-aTS also exhibited slight antiproliferative effects. However, its antiproliferative efficacy was lower than those of CUR and the Lys-aTS/CUR NA. The Lys-aTS, CUR and the Lys- aTS/CUR groups exhibited higher cytotoxicity on longer peri- ods of incubation. Notably, after 72 h of incubation, the dif- ference between the IC50 values of CUR (10.33 ± 0.67 lg/mL) and Lys-aTS/CUR NA (7.95 ± 0.09 lg/mL) was significant (p < .01) (Supplementary Table S2). In both the incubation groups (48 and 72 h of incubation), the cell viability of the Lys-aTS/CUR NA was significantly lower than that of CUR at a CUR concentration of 10 lg/mL (p < .001 and .05, respect- ively). The antiproliferative efficacy of the Lys-aTS/CUR NA was more improved than CUR, which can be explained by the higher cellular accumulation efficiency of the Lys-aTS/ CUR NA in spite of the differences in various details of the experimental conditions (such as the incubation time and the CUR concentration) as shown in Figures 4(A,B). It seems that using a nanosized carrier can increase the endocytosis of CUR in cancer cells, subsequently leading to the enhanced antiproliferative efficiency NIRF imaging Tumor targetability of the fabricated Lys-aTS/CUR NA was verified by NIRF imaging in MDA-MB-231 tumor-xenografted mouse models (Figure 5). Cy5.5-NHS was conjugated as a NIRF dye to the amine group of Lys via an amide bond. As shown in Figure 5(A), the Cy5.5-Lys-aTS/CUR NA was distrib- uted in the body while free Cy5.5 was hardly observed in the body due to its immediate excretion at 4 h. Notably, the Cy5.5-Lys-aTS/CUR NA mainly accumulated in the tumor region at 24 h post-injection. According to the quantitative data analysis at 24 h (Figure 5(B)), the mean fluorescence intensity of the Cy5.5-Lys-aTS/CUR NA in the tumor region was 3.63-fold higher than that of the free Cy5.5 (p < .001). This was further confirmed by ex vivo NIRF imaging studies with the dissected tumor tissues (Figure 5(C)). A strong fluor- escence signal was detected in the Cy5.5-Lys-aTS/CUR NA group in comparison to the free Cy5.5 group. The biodistri- bution of the injected Cy5.5-Lys-aTS/CUR NA was also eval- uated by ex vivo NIRF imaging (Figure 5(D)). The fluorescence intensity values in the liver, lungs, heart, kidneys, spleen, and tumor were compared in the free Cy5.5 and the Cy5.5-Lys- aTS/CUR NA-treated groups. The fluorescence intensity in the tumor tissue of the Cy5.5-Lys-aTS/CUR NA-treated group was 6.67-fold higher than the free Cy5.5-treated group (p < .01). Foreign materials are prone to be trapped in the reticulo- endothelial system (RES), such as the liver and spleen, via phagocytosis. Avoiding the accumulation of nanoparticles in RES organs and a selective delivery to the tumor region have been considered as common objectives for the development of tumor-targeted nanosystems of anticancer agents. The relative fluorescence intensity ratios of tumor to (liver- þ spleen) in the free Cy5.5 and the Cy5.5-Lys-aTS/CUR NA DRUG DELIVERY 747 747 Figure 5. NIRF imaging test in MDA-MB-231 tumor-bearing mouse model. Free Cy5.5 or Cy5.5-conjugated Lys-aTS/CUR NA was injected into the tail vein of the mouse model. (A) Whole body-scanned images acquired at 0 (pre), 4, and 24 h post-injection. Red dashed circle indicates the tumor region. (B) Mean fluorescence intensity values in the tumor region. Each point represents the mean ± SD (n ¼ 3). þþþp < .001, compared with free Cy5.5 group. (C) Ex vivo NIRF image of dis- sected tumor tissues at 24 h post-injection. (D) Mean fluorescence intensity values in liver, lungs, heart, kidneys, spleen, and tumor are presented. The authors report no conflicts of interest. The authors report no conflicts of interest. In vivo anticancer activities The in vivo anticancer activities of the Lys-aTS/CUR NA were tested in MDA-MB-231 tumor-bearing mouse models after multiple dosing (Figure 6). For CUR delivery to the tumor region, the introduction of nanocarriers is necessary to over- come the drawbacks (that is, poor water-solubility). Following the in vivo and ex vivo biodistribution studies (Figure 5), the in vivo anticancer activities were verified by studying the inhibition of tumor growth, changes in body weight, and immunohistological staining of dissected tumor tissues. As shown in Figure 6(A), among all experimental groups, the highest inhibition of tumor growth was observed in the Lys- aTS/CUR NA group. It is noted that the tumor volume of the Lys-aTS/CUR NA group was significantly lower than those of all the other groups on the final day (p < .05). The tumor vol- ume on day 26 of the Lys-aTS/CUR NA group was 28.0, 32.8, and 32.9% of those of the control, CUR, and the Lys-aTS groups, respectively. Also, there was no significant alteration in the body weight during the entire monitoring period (Figure 6(B)). It may imply the absence of severe systemic toxicity after the intravenous administration of the developed CUR-loaded NA. The dissected tumor tissues from each group were examined by H&E and TUNEL staining (Figure 6(C)). In particular, the brown color in the staining results of TUNEL assay, indicative of apoptotic events in tumor tissues, of the Lys-aTS/CUR NA group, was stronger than those of all the other groups. As observed in the results of the cellular apop- tosis assay (Supplementary Figure S5), apoptosis seemed to be efficiently induced in the tumor region after the arrival of the CUR-loaded NA. The most efficient inhibition of tumor growth by the Lys-aTS/CUR NA is supported by this apop- totic efficacy. Conclusively, the aTS-linked enzymatic nanocar- rier can selectively deliver CUR to the tumor tissue, so that it can exert its efficient in vivo anticancer activities. References Aminlari L, Hashemi MM, Aminlari M. (2014). Modified lysozymes as novel broad spectrum natural antimicrobial agents in foods. J Food Sci 79:R1077–90. Angulo-Molina A, Reyes-Leyva J, Lopez-Malo A, et al. (2014). The role of alpha tocopheryl succinate (a-TOS) as a potential anticancer agent. Nutr Cancer 66:167–76. Arnaudov LN, de Vries R. (2005). Thermally induced fibrillar aggregation of hen egg white lysozyme. Biophys J 88:515–26. Bae YH, Park K. (2011). Targeted drug delivery to tumors: myths, reality and possibility. J Control Release 153:198–205. Byeon HJ, Thao le Q, Lee S, et al. (2016). Doxorubicin-loaded nanopar- ticles consisted of cationic- and mannose-modified-albumins for dual- targeting in brain tumors. J Control Release 225:301–13. Canfield RE. (1963). The amino acid sequence of egg white lysozyme. J Biol Chem 238:2698–707. Cho HJ, Balakrishnan P, Chung SJ, et al. (2011). Evaluation of protein sta- bility and in vitro permeation of lyophilized polysaccharides-based microparticles for intranasal protein delivery. Int J Pharm 416:77–84. Danhier F, Feron O, Preat V. (2010). To exploit the tumor microenviron- ment: passive and active tumor targeting of nanocarriers for anti-can- cer drug delivery. J Control Release 148:135–46. g y Danhier F, Ansorena E, Silva JM, et al. (2012). PLGA-based nanoparticles: an overview of biomedical applications. J Control Release 161:505–22. Elsadek B, Kratz F. (2012). Impact of albumin on drug delivery-new appli- Danhier F, Ansorena E, Silva JM, et al. (2012). PLGA-based nanoparticles: an overview of biomedical applications. J Control Release 161:505–22. p an overview of biomedical applications. J Control Release 161:505–22. Elsadek B, Kratz F. (2012). Impact of albumin on drug delivery-new appli- cations on the horizon. J Control Release 157:4–28. Funding This research was supported by the National Research Foundation of Korea (NRF), funded by the Korean government [MSIP; No. NRF- 2015R1A1A1A05027671]. NIRF imaging Y. LEE AND H.-J. CHO 748 significantly higher than those of CUR. According to the results of the NIRF imaging test in MDA-MB-231 tumor-bear- ing mouse models, the Lys-aTS/CUR NA showed a selective accumulation in the tumor, rather than in the other organs and tissues. Following intravenous injections in the MDA-MB- 231 tumor-xenografted mouse models, the Lys-aTS/CUR NA exhibited a higher inhibition of tumor growth and apoptotic events in the tumor tissue than the other groups. All these findings indicate that the Lys-aTS/CUR NA can be used as efficient and safe enzyme-based nanocarrier for the therapy of breast cancers. groups were 0.018 and 1.321, respectively. The values imply a higher tumor selectivity of the Lys-aTS/CUR NA after its intravenous injection. The observed in vivo tumor targetabil- ity of the Lys-aTS/CUR NA may reduce the unwanted effects of CUR and improve its anticancer activities. NIRF imaging Each point repre- sents the mean ± SD (n ¼ 3). þþp < .01, compared with free Cy5.5 group. Figure 5. NIRF imaging test in MDA-MB-231 tumor-bearing mouse model. Free Cy5.5 or Cy5.5-conjugated Lys-aTS/CUR NA was injected into the tail vein of the mouse model. (A) Whole body-scanned images acquired at 0 (pre), 4, and 24 h post-injection. Red dashed circle indicates the tumor region. (B) Mean fluorescence intensity values in the tumor region. Each point represents the mean ± SD (n ¼ 3). þþþp < .001, compared with free Cy5.5 group. (C) Ex vivo NIRF image of dis- sected tumor tissues at 24 h post-injection. (D) Mean fluorescence intensity values in liver, lungs, heart, kidneys, spleen, and tumor are presented. Each point repre- sents the mean ± SD (n ¼ 3). þþp < .01, compared with free Cy5.5 group. gure 6. In vivo anticancer activity tests in MDA-MB-231 tumor-bearing mouse models. (A) Tumor volume profiles of control, CUR, Lys-aTS, and Lys-aTS/CUR NA oups. CUR, Lys-aTS and Lys-aTS/CUR NA were injected intravenously on day 5, 8, 12, 15, 19, and 22, respectively. Each point indicates the mean ± SD (n 3). < .05, compared with the control group. #p < .05, compared with CUR group. $p < .05, compared with Lys-aTS group. (B) Profiles of body weight of control, R, Lys-aTS, and Lys-aTS/CUR NA groups. Each point indicates the mean ± SD (n 3). (C) Staining images of dissected tumor tissues. The microscopic images of &E staining (upper panel) and TUNEL assay (lower panel) are shown. The length of scale bar in the image is 100 lm. Figure 6. In vivo anticancer activity tests in MDA-MB-231 tumor-bearing mouse models. (A) Tumor volume profiles of control, CUR, Lys-aTS, and Lys-aTS/CUR NA groups. CUR, Lys-aTS and Lys-aTS/CUR NA were injected intravenously on day 5, 8, 12, 15, 19, and 22, respectively. Each point indicates the mean ± SD (n 3). &p < .05, compared with the control group. #p < .05, compared with CUR group. $p < .05, compared with Lys-aTS group. (B) Profiles of body weight of control, CUR, Lys-aTS, and Lys-aTS/CUR NA groups. Each point indicates the mean ± SD (n 3). (C) Staining images of dissected tumor tissues. The microscopic images of H&E staining (upper panel) and TUNEL assay (lower panel) are shown. The length of scale bar in the image is 100 lm. S. Conclusions d-a-Tocopheryl succin- ate/phosphatidyl ethanolamine conjugated amphiphilic polymer- based nanomicellar system for the efficient delivery of curcumin and to overcome multiple drug resistance in cancer. ACS Appl Mater Interfaces 9:16778–92. Kim HJ, Kim A, Miyata K, et al. (2016). Recent progress in development of siRNA delivery vehicles for cancer therapy. Adv Drug Deliv Rev 104:61–77. Ow SY, Dunstan DE. (2013). The effect of concentration, temperature and stirring on hen egg white lysozyme amyloid formation. Soft Matter 9:9692–701. Laemmli UK. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–5. Lammers T, Kiessling F, Hennink WE, et al. (2012). Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress. J Control Release 161:175–87. Palao-Suay R, Martın-Saavedra FM, Rosa Aguilar M, et al. (2017). Photothermal and photodynamic activity of polymeric nanoparticles based on a-tocopheryl succinate-RAFT block copolymers conjugated to IR-780. Acta Biomater 57:70–84. Lee JJ, Lee SY, Park JH, et al. (2016). Cholesterol-modified poly(lactide-co- glycolide) nanoparticles for tumor-targeted drug delivery. Int J Pharm 509:483–91. Prasad KN, Kumar B, Yan XD, et al. (2003). Alpha-tocopheryl succinate, the most effective form of vitamin E for adjuvant cancer treatment: a review. J Am Coll Nutr 22:108–17. Lee SY, Lee JJ, Nam SY, et al. (2017a). Fabrication of polymer matrix- free nanocomposites based on Angelica gigas Nakai extract and their application to breast cancer therapy. Colloids Surf B Biointerfaces 159:781–90. Price NC. (2000). Conformational issues in the characterization of pro- teins. Biotechnol Appl Biochem 31:29–40. Qu Q, Ma X, Zhao Y. (2016). Anticancer effect of a-tocopheryl succinate delivered by mitochondria-targeted mesoporous silica nanoparticles. ACS Appl Mater Interfaces 8:34261–9. Lee SY, Park JH, Ko SH, et al. (2017b). Mussel-inspired hyaluronic acid derivative nanostructures for improved tumor targeting and penetra- tion. ACS Appl Mater Interfaces 9:22308–20. Shim T, Lim C, Hoang NH, et al. (2017). Recent advance of pH-sensitive nanocarriers targeting solid tumors. J Pharm Invest 47:383–94. Li Z, Tan BH. (2014). Towards the development of polycaprolactone based amphiphilic block copolymers: molecular design, self-assembly and biomedical applications. Mater Sci Eng C Mater Biol Appl 45:620–34. Song J, Yang X, Yang Z, et al. (2017). Rational design of branched nano- porous gold nanoshells with enhanced physico-optical properties for optical imaging and cancer therapy. ACS Nano 11:6102–13. Li Z, Xu W, Zhang C, et al. (2015). Self-assembled lysozyme/carboxyme- thylcellulose nanogels for delivery of methotrexate. Int J Biol Macromol 75:166–72. Conclusions Elsadek B, Kratz F. (2012). Impact of albumin on drug delivery-new appli- cations on the horizon. J Control Release 157:4–28. The Lys-aTS/CUR NA was developed as an enzymatic nano- system for the imaging and therapy of breast cancers. Lys-aTS was synthesized by the formation of an amide bond between the amine group of Lys and the carboxylic acid group of aTS. CUR, as a poorly water-soluble drug, was incor- porated into the Lys-aTS nanostructure. Compared with the c-Lys/CUR NA, Lys-aTS/CUR NA exhibited a smaller hydro- dynamic size (213 nm mean diameter), a narrower size distri- bution, and a more spherical shape, which can be regarded as a safe and efficient tumor-targeting drug delivery system. The sustained drug release from the Lys-aTS/CUR NA was also observed for five days at normal physiological condi- tions. The cellular accumulation, antiproliferative effects, and the apoptotic efficiencies of the Lys-aTS/CUR NA were Elzoghby AO, Samy WM, Elgindy NA. (2012). Albumin-based nanopar- ticles as potential controlled release drug delivery systems. J Control Release 157:168–82. Farmer TB, Caprioli RM. (1991). Assessing the multimeric states of pro- teins: studies using laser desorption mass spectrometry. Biol Mass Spectrom 20:796–800. Gradishar WJ. (2006). Albumin-bound paclitaxel: a next-generation tax- ane. Expert Opin Pharmacother 7:1041–53. Gruber J, Staniek K, Krewenka C, et al. (2014). Tocopheramine succinate and tocopheryl succinate: mechanism of mitochondrial inhibition and superoxide radical production. Bioorg Med Chem 22:684–91. Hawkins MJ, Soon-Shiong P, Desai N. (2008). Protein nanoparticles as drug carriers in clinical medicine. Adv Drug Deliv Rev 60:876–85. Jeong JY, Hong EH, Lee SY, et al. (2017). Boronic acid-tethered amphi- philic hyaluronic acid derivative-based nanoassemblies for tumor tar- geting and penetration. Acta Biomater 53:414–26. 749 DRUG DELIVERY Kanai K, Kikuchi E, Mikami S, et al. (2010). Vitamin E succinate induced apoptosis and enhanced chemosensitivity to paclitaxel in human bladder cancer cells in vitro and in vivo. Cancer Sci 101:216–23. Matsumura Y, Maeda H. (1986). A new concept for macromolecular ther- apeutics in cancer chemotherapy: mechanism of tumoritropic accu- mulation of proteins and the antitumor agent SMANCS. Cancer Res 46:6387–92. Kemp JA, Shim MS, Heo CY, et al. (2016). “Combo” nanomedicine: co- delivery of multi-modal therapeutics for efficient, targeted, and safe cancer therapy. Adv Drug Deliv Rev 98:3–18. Muddineti OS, Kumari P, Ghosh B, et al. (2017). Conclusions Tao W, Zhang J, Zeng X, et al. (2015). Blended nanoparticle system based on miscible structurally similar polymers: A safe, simple, targeted, and surprisingly high efficiency vehicle for cancer therapy. Adv Health Mater 4:1203–14. Liu G, Tsai HI, Zeng X, et al. (2017). Phosphorylcholine-based stealthy nanocapsules enabling tumor microenvironment-responsive doxorubi- cin release for tumor suppression. Theranostics 7:1192–203. Tran TH, Thapa RK, Nguyen HT, et al. (2016). Combined phototherapy in anti-cancer treatment: therapeutics design and perspectives. J Pharm Invest. 46:505–17. Lin L, Xu W, Liang H, et al. (2015). Construction of pH-sensitive lyso- zyme/pectin nanogel for tumor methotrexate delivery. Colloids Surf B Biointerfaces 126:459–66. Wang K, Zhang C, Bao J, et al. (2016). Synergistic chemopreventive effects of curcumin and berberine on human breast cancer cells through induction of apoptosis and autophagic cell death. Sci Rep 6:26064. Lin TY, Koshland DE. Jr. (1969). Carboxyl group modification and the activity of lysozyme. J Biol Chem 244:505–8. Liu L, Bi Y, Zhou M, et al. (2017). Biomimetic human serum albumin nanoparticle for efficiently targeting therapy to metastatic breast can- cers. ACS Appl Mater Interfaces 9:7424–35. Yoon IS, Park JH, Kang HJ, et al. (2015). Poly(D,L-lactic acid)-glycerol- based nanoparticles for curcumin delivery. Int J Pharm 488:70–7. Zemser M, Friedman M, Katzhendler J, et al. (1994). Relationship between functional properties and structure of ovalbumin. J Protein Chem 13:261–74. Maeda H, Wu J, Sawa T, et al. (2000). Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release 65:271–84. Zeng X, Tao W, Mei L, et al. (2013). Cholic acid-functionalized nanopar- ticles of star-shaped PLGA-vitamin E TPGS copolymer for docetaxel delivery to cervical cancer. Biomaterials 34:6058–67. Mahanta S, Paul S, Srivastava A, et al. (2015). Stable self-assembled nano- structured hen egg white lysozyme exhibits strong anti- proliferative activity against breast cancer cells. Colloids Surf B Biointerfaces 130:237–45. Zeng X, Tao W, Wang Z, et al. (2015). Docetaxel-loaded nanoparticles of dendritic amphiphilic block copolymer H40-PLA-b-TPGS for cancer treatment. Part Part Syst Charact 32:112–22. Mallick A, More P, Syed MM, et al. (2016). Nanoparticle-mediated mito- chondrial damage induces apoptosis in cancer. ACS Appl Mater Interfaces 8:13218–31. Zhang H, Huang S, Yang X, et al. (2014). Current research on hyaluronic acid-drug bioconjugates. Eur J Med Chem 86:310–7. Masuda T, Ide N, Kitabatake N. (2005). Structure-sweetness relationship in egg white lysozyme: role of lysine and arginine residues on the elicitation of lysozyme sweetness. Chem Senses 30:667–81. Conclusions Zhang L, Liu Y, Liu G, et al. (2016). Prolonging the plasma circulation of proteins by nano-encapsulation with phosphorylcholine-based poly- mer. Nano Res 9:2424–32.
https://openalex.org/W2097530120	https://zenodo.org/records/2173316/files/article.pdf	English	null	Watching legislation for the public	National municipal review	1,914	public-domain	3,222	WATCHING LEGISLATION FOR THE PUBLIC T HROUGH years of continuous effort the Citizens Union of Kew York has developed a workable plan and an efficient organization T for keeping watch on law-making at the state capital of Albany, and for advocating the enactment of desirable measures and opposing the passage of vicious or ill-considered bills. Public-spiritedlawyers, engaged in a successful fight for the establishment of free transfers on the surface transit lines in New York, discovered that laws had been finding their way into the statute books through the expert lobbying of special interests and that the public seldom understood the purpose or effect of these laws until they had gone into operation. Studying the railroad law, they found innumerable special provisions ingeniously devised to meet particular cases, though couched in general language. In fact, the railroad law of the state was then and, because not yet revised, still is, largely a patchwork of such. special provisions. The importance of creating an organization that would watch the work of the legislature for the public as closely as it was watched for the rail- roads by counsel and paid legislative agents was apparent. The Citi- zens Union undertook to form such an organization. Its committee on legislation was thereupon organized. A lawyer under retainer was sent to the state capital to represent the committee and keep it informed of the progress of legislative events. Out of this beginning has grown the present thorough-going machinery of the legislative work of the Union. This work of the union was started in 1903. 360 360 NATIONAL MUNICIPAL REVIEW [April the ownership and use of land will be completely divorced, when the whole city will, in effect, stand in the relationship of a tenant to an absen- tee landlord. HERBERT S. SWAN.^ New York City. investigator for the Commission on New Sources of City Revenue. COMMITTEE Oh’ LEOISLATION The committee on legislation meets weekly in Xew York while the legislature is in session at Albany and considers annually an average of about six hundred bills relating to, or affecting conditions in, the city of New York. It is divided into sub-committees, to which it refers these bills for detailed examination and from which it receives reports at its weekly meetings. . Each sub-committee is composed of one or more members and is charged with responsibility for examining one class of proposed legis- lation. Thus there are sub-committees on electoral legislation, civil WATCHING LEGISLATION FOR T H E PUBLIC 361 19141 courts legislation, criminal courts legislation, tenement house legislation, railroad and franchise legislation, and a large sub-committee on charter legislation to which are referred charter amendments and bills affecting the structure of the city government that do not fall within the scope of other sub-committees. 1 Chapter 247, Laws of 1913. MEMBERS BECOME EXPERTS The personnel of the committee on legislation does not change rapidly from year to year, very few members being added at the beginning of each legislative session, and these selected with the greatest care. Some members have served on the committee since it was first organized and are still serving. The membership usually numbers between fifteen and twenty. Those who have been engaged for several years in the examination for the committee of a particular class of legislation have, of course, become expert in that branch. When a lawyer was sought to draft a home rule 1aw,1 the Municipal Government Association went to the Citizens Union com- mittee to secure the expert. The consolidated election law was edited by two members of the committee; the direct primary bill introduced in the 1913 legislature on behalf of the Progressive party was drafted by another member; and the Massachusetts ballot law enacted in Decem- ber, 1913, was largely the work of the Union’s sub-committee on electoral legislation. These men were selected for this work because of their expert ability. The most valuable criticisms of proposed franchise legislation have frequently come from the committee on legislation. The committee is responsible for numerous safeguarding provisions of the present rapid transit act and for the debt limit law under which rapid transit bonds have been excluded in computing the constitutional limitation upon the city’s borrowing capacity. Most of the members are lawyers. The work of the committee on legislation consists not only in criticising bills, but also in devising and drafting needed legislation. Its criticisms are frequently constructive. It not only points out the dangerous or undesirable features of bills, but also assists in preparing corrective amendments. Members of the committee are often called into confer- ence with legislative leaders and with representatives of groups seeking legislation on particular subjects. FUNCTIONS O F LEGISLATIVE BUFtEAU-PUBLICITY To make the action of the committee effective, theunion maintains a well-equipped bureau in Albany during each session. This legislative bureau is in charge of the secretary of the committee on legislation, assisted by a staff of legislative experts, clerks and stenographers. The activities of the bureau are more ext.ensive (in respect of the quantity. 1 Chapter 247, Laws of 1913. NATIONAL MUNICIPAL REVIEW [April of legislation in which it displays interest) and oftentimes more effective than those of any of the corporation lobbies. The agents of the Citizens Cnion have acquired expert knowledge of the many tricks and devices by which legislation may be advanced or retarded. Such knowledge is essential in order that the significance of each move may be fully understood. But also, the influence of the Union upon legislation is equally due to its facilities for securing publicity. The newspapers and their representatives are for the most part free from influence that might prevent their publishing accurate accounts of legislative events, and the results of Citizens Union investigations receive wide publication in the daily press. The importance of quick, accurate and complete publicity, cannot be over-estimated. The ((sneak bill,” as the phrase implies, is always “slipped through” the legislature with the greatest secrecy, ( I the ways” having been (‘greased” by the lobby agent of a special interest. If before the bill reaches h a 1 passage its purpose and the particular situa- tions to which its provisions apply, and their effect, become publicly known, it is no longer possible to pass it secretly. Public interest and attention are easily attracted to such a bill once it has been publicly exposed; and legislators hesitate to put themselves on record in favor of any vicious measure in the face of publicity in their home districts. Nothing better can be hoped for from a legislature of elected representa- tives than legislation truly representing the best sentiment of the com- munity; and no important reforms can be fully realized till they have the support of public sentiment. So, in building up an organization for the purpose of influencing legislation, the Union rightly puts its emphasis on the securing of the quickest and widest accurate publicity for all legislative proposals and the action of legislators thereon. “TICKLERS” ON PROGRESS OF BILLS Close watch must be kept upon the progress of each bill, and, because of the great number of measures, this can only be done with the aid of ‘ I tickler” card systems and “tickler” memorandum sheets prepared daily from the manuscript journals of the two houses. It not infre- quently happens that on a single legislative day the New York legisla- ture acts upon scores of bills affecting New York City, and no human being is capable of keeping constantly in mind the status of the hundreds of bills in which the Citizens Union is interested. Therefore, since there may be an occasion during the progress of a bill toward final passage when the objection of a single legislator will greatly delay its advance- ment or perhaps even bring about its defeat, the advantage of the “tick- 3 Xote.Summariea are made of the provisions of all bills, whether they particularly affect the city of New York and are of special interest to the Uuion or not; and copiea are mailed daily, not only to members of the committee on legislation, but also to a number of civic organizations throughout the state. The preparation of the summaries and certain other branches of the work that consist solely in furnishing information without criticism of proposed legislation, are being performed this year by the Vote- Legislative Association, formed several years ago largely through the instrumentality of the Citizens Union and with the i d of some other civic bodies and citizens to extend the benefits of the legislative information bureau so that it may be used by organizations in all parts of the state. Since the scope of the work of the Union is limited to New York City, it is clear that the function of furnishing information to civic worken in all cities of the state belongs more properly to a state-wide organization, and one which has no opinions to express regarding any pending legislation. The Union makes the main contribution toward the expense of the Voters Legislative Association and is the heaviest user of its information service. This service is in charge of men who received their training in the Citizens Union Bureau. PROVISIONS OF BILLS SUMURIZED In order to provide this the bureau some years ago began to prepare summaries of all bills introduced. These summaries tell clearly, and as briefly as clearness permits, what changes each bill would make in existing law. The summary of each ‘bill is prepared on the day of its introduc- tion from the manuscript copy presented to the legislature and is mani- folded in mimeograph copies so as to be available for use by all the news- paper correspondents as soon after the introduction of the bill as possible. Many of the summaries are ready for such use within an hour after the bills are introduced. These summaries contain no expressions of opinion regarding the merits of the bills, but merely a plain statement of their provisions. Great care is exercised to employ accurate language. The legislative correspondents have gained such confidence in the accuracy of the summaries that they rely upon them and use them freely in reports to their newspapers. The summaries are also delivered to some public 363 WATCHIXG LEGISLATION FOR THE PUBLIC 19141 officials, including the governor’s counsel, who find them helpful; and the state library places them in substantial bindings for preservation on its shelves, because no other reports-official or unofficial-contain the same information in an equally convenient form. q y The bureau, under direction of the committee, issues public statements and keeps up a running fire of constructive criticism of the work of the legislature. It prepares and publishes analyses of the bills affecting the city of New York. It contends for home rule, sound policies of government, and the protection of the city’s interests at every point. Memoranda are filed with legislative committees, with the mayor in case of special city bills, and with the governor, and representatives of the Union appear at hearings upon bills and public questions.2 WASTE O F ENERGY AVOIDED In addition to making as effective as possible the action of the com- mittee on legislation regarding pending bills, the legislative bureau makes up the calendar of bills to be considered by the committee, and facilitates its work in other ways. The most pressing business before the committee is placed upon a “submitted calendar,” a copy of which is sent to each member before each meeting. If the calendar of the committee on legislation becomes congested, the secretary put,s at the head of the “submitted calendar” of each meeting those bills whose passage by the legislature is most imminent: It always happens that at the close of the legislative session there are bills on the calendar of the committee upon which neither the committee nor the legislature has taken any action. The close watch kept upon the progress of events a t the capital enables the committee to center its criticism upon those bills that have a chance of passage, devoting time to the critical examination of other bills only when it has disposed of those that are most imminent. The bureau keeps files of sub-committee reports, briefs and memoranda, prepared each year, and makes this storehouse of information available for the committee in subsequent years. The same bill is frequently introduced in a number of successive sessions of the legislature, and every time such a bill comes before the committee on legislation, it can refer to the files of the bureau for what information regarding it was gathered in previous years. The work of the sub-committees, also, is facilitated by the bureau. Each member of each sub-committee receives from the bureau a copy of every bill referred to that sub-committee. This makes it possible for much of the work of the sub-committees to be performed without actual meetings. The chairman receives from the secretary in charge of the bureau at Albany a mail envelope containing a number of bills that have been placed on the calendar of the committee on legislation and referred to his sub-committee for examination and report. He divides the work among members of his sub-committee, and telephones to them their respective assignments. The work of detailed examination of a legislative bill is usually better performed, in fact, by a single lawyer or legislative expert, than by a group working together. WASTE O F ENERGY AVOIDED If the bill is specially important, two or more members of the sub-committee are called upon to examine it separately. “TICKLERS” ON PROGRESS OF BILLS The asociation hss heretofore been enabled by private contributions and the use of facilities of the legislative bureau placed freely at its disposal, to keep a number of civic organizations in Merent cities informed regarding the progress of legislation, aa well aa to publish bulletins for the information of its members, and annual rcports chronicling the principal events of the legislative sessions and the votes of each senator and assemblyman in important issues. J J. 0. H. NATIONAL MUNICIPAL REVIEW 3 64 [ApriI ler” system which enables the Citizens Union representative to see that objection is made to a bad bill at precisely the right moment, is apparent. REPORTS ON WORK OF LEGISLATORS After the close of the legislative session, the committee on legislation This report makes a final report which is printed for distribution. 19141 WATCHING LEGISLATION FOR THE PUBLIC 365 contains (1) the most complete chronicle of and commentary upon legislation affecting the city of New York that is anywhere obtainable, and (2) a thorough-going non-partisan analysis of the work of each legislator representing a New York City constituency, with a statement of the committee’s conclusions regarding the merits of his service. The comments on individual legislators are brief though specific and are usually published in full in a number of metropolitan daily newspapers. Here are some examples: Voted to sustain Stilwell charges. Otherwise made poor record of votes. Introduced reactionary primary legislation for machine. Fathered bad railroad consolidation bill. Active and influential member of majority. Voted to whitewash Stilwell and lauded him in speech as a “faithful legislator. ” Voted against bad police bill and for thorough-going direct primary bill; in other respects made bad record. p y p Voted to sustain Stilwell charges and made, on the whole, good record of votes except that he followed machine on direct primaries. Record greatly improved over previous years. On other issues, also, made bad record of votes. Introduced Massachusetts Ballot Bill but made no appreciable effort to secure its passage, and followed machine on direct primaries. Spoke and voted to whitewash Stilwell. Voted to sustaixi Stilwell charges, and rendered valuable service in advocating condemnation reform. Made, on the whole, a fair record, although he followed machine on direct primaries. As in previous years, made himself a nuisance by verbosity in debate, while seldom contributing anything to consideration of bills. Intro- duced attacks upon merit system in civil service. Thoroughly bad record of votes. Frequently unrecorded on important roll calls and voted with machine on most other occasions, although on two votes on electoral bills he showed independence. TWO of his four bills violated home rule. New member. Searly all his proposed legislation thoroughly objec- tionable. One of the two Kew York City members to vote against desirable bill to protect city water supply from pollution. Frequently unrecorded on important roll calls, and on other occasions followed ma- chine in his votes. New member. Introduced bad legislation affecting uniformed force of Fire Department. Unrecorded on five important roll calls on primary and election bills and on other roll calls followed machine. REPORTS ON WORK OF LEGISLATORS In seventh term showed no promise of usefulness. Frequently unrecorded, including six important roll calls on primary and election bills. His view of his legislative functions Sew member. NATIONAL MUNICIPAL REVIEW 366 [April indicated by his attempt to regulate by mandatory state law the color of uniforms in street cleaning department. g p Tables of the votes of New York City representatives on important issues, and other information upon which the conclusions of the com- mittee are based, appear in the printed report. pp p p Before the report is published, each legislator is invited to call at the Citizens Union office, examine the comments upon his record, and offer any objections, criticisms or additional information that he thinks should be considered. The committee then holds a special meeting to consider these criticisms upon its criticisms and makes any changes in the tentative draft of its report that seem to it to be justified. The committee’s comments on the work of legislators are given considerable weight by thoughtful voters and have an influence upon elections. p JOSEPH 0. HAMMIT IS UNEMPLOYNENT A MUNICIPAL PROBLEM? A AT LEAST nine American cities have answered this question in the affirmative by establishing municipal employment agen- cies. Nineteen states have established state agencies, but in every successful one, the state law empowers the city to establish and operate such agencies. The rise of the city as a self-governing unit, the growth of the home rule idea, the establishment of public welfare departments in place of the antiquated (( charities” still maintained by states, the fact that the city is so largely the source of progressive activities in government and civic organizations, these and many other facts bring to mind certain broad questions which it is pertinent to raise as municipal issues. There are two broad classifications of unemployed-the employable and the unemployable. At the present time these are greatly confused be- cause we insist upon treating the former as a relief matter, which can never be solved by relief measures, and the latter as an industrial problem which lowers the whole standard of business efficiency. The first need of American government is, therefore, some agency for classification of these groups, a task now handled very inadequately by volunteer organ- izations. I am here primarily concerned with the normal amount of unemploy- ment of employable people, due to seasonal occupations, casual labor, changing from one occupation to another, entrance of fresh workers into industry and the distribution of immigrants to industries, Unquestion- ably the centers of this exchange of labor are the great cities. These cities are of two kinds-those which attract numbers of workers because of large industries, and in which trade schools and special schools flourish,
https://openalex.org/W3125737196	http://repository.bilkent.edu.tr/bitstream/11693/53064/1/Measuring_euro_area_monetary_policy.pdf	English	null	Measuring Euro Area Monetary Policy	Social Science Research Network	2,019	cc-by	17,567	a r t i c l e i n f o Article history: Received 21 August 2019 Accepted 21 August 2019 Available online 30 August 2019 JEL classiﬁcation: E43 E44 E52 E58 G12 Keywords: G14 ECB Policy surprise Event-study Intraday Persistence Asymmetry We map ECB policy communication into yield curve changes and study the information ﬂow on policy dates. A byproduct is the publicly available Euro Area Monetary Policy Event-Study Database (EA-MPD), containing intraday asset price changes. We ﬁnd that Pol- icy Target, Forward Guidance and Quantitative Easing factors capture about all the varia- tion in the yield curve, with different factors appearing in the windows covering the policy decision announcement and the press conference, and having time-varying variance shares. We study sovereign yields, exchange rates, stock prices, persistence of effects and response asymmetry. Our methodology can be implemented for any policy-related event. © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) Carlo Altavilla a , ∗, Luca Brugnolini b , Refet S. Gürkaynak c , d , Roberto Motto e Giuseppe Ragusa f a European Central Bank, 60314 Frankfurt am Main, Germany b Unaﬃliated, London, UK c Department of Economics, Bilkent University, Ankara 06800, Turkey d Bilkent University, CEPR, CESIfo, & CFS, Turkey e European Central Bank, 60314 Frankfurt am Main, Germany f Department of Economics and Management, University of Pisa, Pisa 56124, Italy a European Central Bank, 60314 Frankfurt am Main, Germany p g E-mail addresses: carlo.altavilla@ecb.europa.eu , carlo.altavilla@ecb.int (C. Altavilla), lucabrugnolini@gmail.com (L. Brugnolini), refet@bilkent.edu.tr (R.S. Gürkaynak), roberto.motto@ecb.europa.eu (R. Motto), giuseppe.ragusa@unipi.it (G. Ragusa). We thank Burcin Kısacıko ˘glu, Sang Seok Lee, Kadir Özen, Giulio Nicoletti, Michael McMahon, Giovanni Ricco, and Jonathan Wright for comments. Gürkaynak’s research was supported by funding from the European Research Council ( ERC ) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 726400 ). The database introduced in this paper is available at https://www.ecb.europa.eu/pub/pdf/annex/ Dataset _ EA-MPD.xlsx and will be periodically updated by the authors. The code that implements the analysis is also available. The views expressed in this paper are those of the authors and do not necessarily reﬂect those of the European Central Bank or the Eurosystem. d h Journal of Monetary Economics 108 (2019) 162–179 Journal of Monetary Economics 108 (2019) 162–179 https://doi.org/10.1016/j.jmoneco.2019.08.016 0304-3932/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) https://doi.org/10.1016/j.jmoneco.2019.08.016 0304-3932/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. (http://creativecommons org/licenses/by/4 0/) 1. Introduction Monetary policy, for better or worse, has been on the forefront of cyclical policymaking in the past two decades, espe- cially during the Great Recession and the sovereign debt crisis. While there is an extensive literature on the US monetary policy, a comprehensive understanding of the ECB policy and its ﬁnancial market effects are yet to materialize. This is partly due to the lack of a systematic database of high frequency, intraday data for a broad class of asset prices in the euro area of the kind that has been employed in the US for more than a decade. We thank Burcin Kısacıko ˘glu, Sang Seok Lee, Kadir Özen, Giulio Nicoletti, Michael McMahon, Giovanni Ricco, and Jonathan Wright for comments. Gürkaynak’s research was supported by funding from the European Research Council ( ERC ) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 726400 ). The database introduced in this paper is available at https://www.ecb.europa.eu/pub/pdf/annex/ Dataset _ EA-MPD.xlsx and will be periodically updated by the authors. The code that implements the analysis is also available. The views expressed in this paper are those of the authors and do not necessarily reﬂect those of the European Central Bank or the Eurosystem. C di h https://doi.org/10.1016/j.jmoneco.2019.08.016 0304-3932/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. http://creativecommons.org/licenses/by/4.0/) 163 C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 This paper helps remedy both deﬁciencies by constructing a long (by euro area standards) time series of policy-date event studies for the euro area, which will be kept up to date, and by using this data to measure and assess the ECB monetary policy. While our substantive analysis focuses on the ﬁnancial market effects of monetary policy in the euro area, the dataset we construct to carry out the analysis is of independent interest. Our euro area monetary policy event-study database (EA-MPD) features price changes for a broad class of assets and various maturities, including Overnight Index Swaps (OIS), sovereign yields, stock prices, and exchange rates. We have exercised considerable care in studying each event window to clean outliers and misquotes, of which there were plenty early in the sample, to make sure that what we report accurately reﬂects the market reaction. 1. Introduction We provide a description of the data, and timing of events on ECB policy dates in Section 2 and offer details and event-by-event analysis in an accompanying online appendix. Monetary policy surprises in the euro area are not only multi-dimensional – we show the existence of perceived policy Target, Timing, Forward Guidance, and Quantitative Easing (QE) surprises – they are also revealed in a multi-step structure. A press release provides information on the policy decision with no rationale and discussion, later followed by a press conference. The information markets extract from these two events are distinct, and intraday data allows us to measure these different types of information separately. Our event-study database utilizes the intraday data to form event windows bracketing about 10 min before the release of monetary policy information, to about 10 min after. Employing tick data allows us to separately compute asset price changes around the release of the monetary policy decision at 13.45, and the reading of the statement and the following Q&A in the press conference beginning at 14.30. The separate releases of the policy decision and the narrative information differentiates euro area monetary policy information revelation from that of the US, where the two happen simultaneously. After brieﬂy presenting the event-study database, we use it to study monetary policy surprises in the euro area and their effects on various ﬁnancial market segments. To do so, we employ the methods developed by Gürkaynak et al. (2005) and Swanson (2017) for the US and use them in the two intraday windows of the euro area policy communication. The dif- ference in the nature of information released in these two windows provides a new understanding of market participants’ interpretation of monetary policy communication. Our results show that, naturally, the preponderance of surprises in the press release window are about the current setting of the policy rates, “Target” surprises, with no other statistically signiﬁcant policy surprise factors. In the press conference window, as expected, there are no Target surprises and the Path and QE surprises dominate. Interestingly, while our analy- sis suggests that a Target factor does not even appear as a statistically signiﬁcant factor in the press conference window, a different factor, “Timing” emerges. 1. Introduction This type of surprise captures the revision of policy expectations by shifting the expected policy action between the current meeting and the next or the one following, in a way that leaves longer-term policy expec- tations about unchanged. In essence, market participants extract two distinct types of guidance from the press conference. One that is informative about the medium run, peaking at about two years–Forward Guidance–and one for the near future, peaking at about six months maturity – what we call Timing. The methodology we employ to extract QE surprises yields continuous measures of the market surprise. Hence, in study- ing the asset price responses to QE, we are able to condition on the size of the surprises rather than only on a binary variable that shows when a QE announcement took place. This allows for a substantially more precise understanding of QE effects and helps distinguish QE from Forward Guidance surprises, which were also frequent during the Zero Lower Bound (ZLB) period. 1 We ﬁnd that both QE (after 2014) and Forward Guidance surprises are active in the press conference window and that while Forward Guidance affected the middle of the yield curve most heavily, with a peak effect at about two years, QE effects get larger as maturity increases, peaking at the 10-year maturity. Surprises about the current setting of monetary policy, which were present in the pre-ZLB period, never had noticeable effects on the long-end of the yield curve. Having the quantiﬁed QE surprises also allows us to study the persistence of their effects better. Unlike in the US, where QE effects were short lived, with a half-life of three months as estimated by Wright (2012) , we estimate a half-life of about one year in the euro area. Thus, ECB QE not only had substantial immediate effects on yields, it also had long lasting effects. We also study the effects of ECB policy surprises on different sovereign yields, exchange rates, and stock prices. Some of these were studied previously in the literature using the combined press release and press conference windows (such as Andrade and Ferroni, 2016 ) and in the separate windows but not including the QE surprises in the analysis (such as Brand et al., 2010 and Leombroni et al., 2017 ). 1 Although the effective lower bound turned out to be below zero, we maintain the convention of calling the bound ZLB. 2 Details of the database construction are presented in Appendices B, C, and D. The database is updated periodically and is available at https://www.ecb. europa.eu/pub/pdf/annex/Dataset _ EA-MPD.xlsx . 3 The Appendix is available online and is structured as follows: Appendix A provides details on the Governing Council meeting frequencies, the infor- mation release structure on policy dates and other relevant information since the inception of the ECB. It also contains a table that shows the policy rate decision and any other policy relevant announcements on each policy date. Appendix B describes our high-frequency dataset showing the Reuters Identiﬁcation Code (RIC) and data availability, which varies by instrument under consideration. Appendix C explains the procedure we follow in cleaning the tick data. Appendix D describes the structure of the database and the main features of the instruments it covers. Appendix E shows the consistency checks we have performed on the computed asset price/yield changes. Appendix F describes the econometric procedure used in the paper to identify the market-based surprises. Appendix G compares the market-based surprise time series we built with market commentary for the largest surprises of each type. Appendix H contains the tables for the responses of Italian and Spanish sovereign yields, and the euro-dollar exchange rate to ECB policy surprises. Appendix I presents the details of our analysis of persistence of effects, and lastly Appendix J does the same for our analysis of possible non-linearity of asset price responses. 1. Introduction This paper is the ﬁrst to look at intraday data to separately study the press release and press conference windows and extract both conventional and unconventional monetary policy communication surprises from both. It is also the ﬁrst not to assume only Target surprises take place in the press release and broadly deﬁned “communication” surprises in the press conference windows but to ask statistically how many factors are present in each and to estimate factors that can be attributed to speciﬁc types of communication. Importantly, this paper is also the ﬁrst in presenting a market-based identiﬁcation of QE surprises in the euro area that shows QE has narrowed spreads, rather than identifying QE surprises by assuming that these have narrowed spreads. In the limited cases where both the event window coverage and the monetary policy surprise deﬁnitions overlap with the existing literature, our ﬁndings are in line with what is already known–such as effects of Target surprises that are signiﬁcant for the short end of the yield curve–and instills conﬁdence for the new results we report on the difference between Timing and Forward Guidance, on the effects of QE, on persistence of these effects, on information and stock market reactions, 164 C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 on nonlinearity, and ﬁnally on using our methodology for the analysis of policy news that do not come out on Governing Council policy dates. nally on using our methodology for the analysis of policy news that do not come out on Governin On stock prices, we ﬁnd that the reaction of broad and banking indices can only be understood when the genuine policy surprises (perceived deviations of interest rates from the policy rule) are separated from information effects (perceived information signaled by the central bank on the current and future state). When studied using this lens, monetary policy has had signiﬁcant effects on stock prices but there was signiﬁcant time variance in the perceived variances of genuine policy and information surprises. We further ﬁnd that the policy surprise effects on stocks were persistent as were the effects of these surprises on longer-term interest rates, especially for Forward Guidance and QE surprises. Our ﬁndings on nonlinearity are noteworthy. We study whether the market responses to positive and negative surprises are different. 1. Introduction A nascent literature is suggesting that in the US, monetary policy has asymmetric real effects ( Barnichon and Matthes, 2017; Tenreyro and Thwaites, 2016 ). We ﬁnd that in the euro area ﬁnancial market participants do not perceive monetary policy effects to be asymmetric with respect to positive surprises and negative in providing asset price responses. Our ﬁndings on nonlinearity are noteworthy. We study whether the market responses to positive and negative surprises are different. A nascent literature is suggesting that in the US, monetary policy has asymmetric real effects ( Barnichon and Matthes, 2017; Tenreyro and Thwaites, 2016 ). We ﬁnd that in the euro area ﬁnancial market participants do not perceive monetary policy effects to be asymmetric with respect to positive surprises and negative in providing asset price responses. Lastly, we use our estimated factors of monetary policy surprises and show how to use them to decompose any policy- related news effects into these four factors. In two illustrative examples we show that policymaker speeches and newswire Lastly, we use our estimated factors of monetary policy surprises and show how to use them to decompose any policy- related news effects into these four factors. In two illustrative examples we show that policymaker speeches and newswire reports can have substantial effects on yields and that in the recent past there have been cases where the ﬁnancial market participants have extracted information on QE-related policy from such news. The paper is organized as follows. In Section 2 below, we discuss the ECB policy communication and our event-study database. Then, in Section 3 we construct our surprises in terms of Target, Timing, Forward Guidance, and QE factors of monetary policy in the two policy communication windows, and discuss their features and plausibility. Section 4 presents asset price responses to the monetary policy surprises using a linear model. In Section 5 we turn to stock price reactions and information effects. In Section 6 , we show how to adapt our methodology to speeches and other monetary policy events and in Section 7 present our main ﬁndings for persistence and non-linear effects, with the full analysis of those topics relegated to the Appendix. Lastly, in Section 8 we offer concluding thoughts. 2. Euro area monetary policy event-study database One of the contributions of this paper is to develop a Euro Area Monetary Policy Event-Study Database (EA-MPD). 2 This section is an introductory manual for the dataset, which will be periodically updated and made available. The underlying tick data come from the Thomson Reuters Tick History database, off of which we measure and report changes in asset prices over the relevant policy windows. Here, we ﬁrst describe the monetary policy communication process in the euro area and then concentrate on the features of the dataset that we develop to measure the monetary policy surprises, delegating more technical details on the dataset construction to online appendices. uction are presented in Appendices B, C, and D. The database is updated periodically and is available at https://www.ecb. _ EA-MPD.xlsx . l d d f ll d d d l h l f h f 2.1. A primer on euro area monetary policy communication As of March 2016, the content of the decisions on non-standard policy measures has been included in the press release. In constructing the database, we ﬁrst cleanse the data of misquotes, 4 then discretize the data within each window by taking the last quote of each minute within the window, and then use the median price in the 13:25–13:35 interval as the pre-press-release quote, and the median price in the 14:00–14:10 as the post-press-release quote. Similarly, we take the median price in the 14:15–14:25 interval as the pre-conference quote and the median price in the 15:40–15:50 interval as the post-conference quote. We make use of an interval rather than selecting a particular minute to measure the pre- and post-event quotes in order to minimize the risk of selecting a quote that is not representative. The changes reported in the database are changes from the pre-event quote to the post-event quote for each communication window. We deﬁne the “monetary policy event” as the union of the press release and conference, and measure changes in asset prices due to this event as the change from the pre-press release quote to the post-conference quote. Fig. 1 shows this timeline in stylized form. The EA-MPD reports the asset price/yield changes we construct for the three event windows in separate worksheets. In each worksheet a policy date is in the ﬁrst column on each row, and the following columns show changes in selected asset prices/yields. The assets covered are: OIS rates with 1, 3, 6 month, 1 to 10, 15, and 20 year maturities; German bund yields with 3 and 6 month, 1 to 10, 15, 20, and 30 year maturities, French, Italian, and Spanish sovereign yields with 2, 5, and 10 year maturities, the STOXX50E and the stock price index comprising banks (SX7E), and the exchange rate of the euro. The EA-MPD is made available as a supplement to this paper and will be regularly updated. In our substantive analysis we start the sample in 2002 because from 1999 (the beginning of the single currency in the Euro Area) to the end of 2001 our intraday OIS data are very noisy, with large spikes and sparse quotes. To provide an illustration of the (cleansed) intraday data, Fig. 2.1. A primer on euro area monetary policy communication At its inception in 1999, the ECB Governing Council took policy decisions twice a month, whereas a press conference took place only once a month, on the ﬁrst meeting of the month. After November 2001 only one meeting per month was a policy meeting, taking place on the ﬁrst Thursday of the month, regularly accompanied by the press conferences, with some exceptions ( Ehrmann and Fratzscher 2009 discuss this policy communication structure). As of January 2015, the frequency of monetary policy meetings has moved to a six-week cycle. The online appendix (Appendix A) shows this information in summary form and in meeting-by-meeting detail. 3 Part of our job in creating the EA-MPD was to compile this information in detail so that the intraday windows have the right coverage. On the day of a policy meeting, different from the US FOMC press release (and very helpfully for researchers), the ECB policy decision is announced in two separate steps. First, at 13.45 Central European Time (CET) a brief press release provides the policy decision without any explanation and rationale. Then, at 14.30 CET the ECB President reads a prepared text, the Introductory Statement (IS), on the rationale behind the decision, which market participants often perceive to be also informative about the future path of monetary policy. It takes about 15 min for the President to read out the statement and there is a follow up question-and-answer session with journalists that lasts for about 45 min. Up to December 2014 the press release referred to the decision on policy rates only, while announcements of non-standard measures were made in the C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 165 Fig. 1. ECB policy communication timeline. Note: The ﬁgure shows the typical policy communication structure during a day of the Governing Council policy meeting of the ECB. Fig. 1. ECB policy communication timeline. B policy communication timeline. ﬁgure shows the typical policy communication structure during a day of the Governing Council policy meeting of the ECB. Fig. 1. ECB policy communication timeline. Note: The ﬁgure shows the typical policy communication structure during a day of the Governing Council policy mee Introductory Statement during the press conference. Between January 2015 and January 2016 the press release mentioned whether “further measures” (without stating what measures were decided) will be announced during the press conference. 4 Such misquotes, which did not reﬂect actual market pricing, were prevalent especially early in the sample. 2.1. A primer on euro area monetary policy communication 2 shows the changes in the 2-year Overnight Index Swap (OIS) rate around the publication of the press release and around the press conference on four different policy meeting dates: 4 July 2013, 4 September 2014, 3 December 2015, and 7 September 2017. We select the 2-year rate as it is of suﬃciently long maturity to display movements in response to announcements of non-standard as well as standard measures. p y p These four panels are illustrative of the different cases in which the monetary policy surprises may arise within the policy meeting day. Panel (a) displays no reaction of the 2-year OIS rate in the press release window and a reaction in the conference window. This episode corresponds to the ECB announcement in the press conference, ﬁrst time ever, of formal Forward Guidance on the future path of its policy rates, by stating that policy rates are expected to remain at present or lower levels for an extended period of time. Panel (b) shows a reaction in the press release window, with no further news affecting the OIS rate in the conference window. This episode corresponds to the announcement of a cut in the ECB deposit rate announced in the press release. Panel (c) depicts a policy date in which there are sizable movements in both windows. This episode captures the ﬁnancial markets’ disappointment following the ECB decision to increase the size of its QE program: markets evidently were expecting a larger increase of QE, as well as a cut in the policy rate, as suggested also by survey expectations among ﬁnancial analysts gathered ahead of the policy meeting. Lastly, panel (d) shows a day in which there is no surprise, either in the press release or the conference windows. Policy dates like these are surprisingly rare; there is usually some news for the ﬁnancial markets, especially in the press conference window. While this discussion of the policy surprises on the basis of intraday changes in the 2-year OIS rate is suggestive of a Target/Path/QE decomposition, formally carrying out this analysis requires the simultaneous study of interest rates at different maturities, which are covered by our event-study database and analyzed in Section 3 below. C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 166 Fig. 2. Intraday 2-year OIS rate around the press release and the conference windows. 2.1. A primer on euro area monetary policy communication Note: The ﬁgure shows the intraday movement (percentage points on the y-axis, trading hours on the x-axis) in the 2-year OIS rate during four selected episodes. The vertical solid line marks the publication of the press release; the vertical dashed line marks the beginning of the press conference. d Fig. 2. Intraday 2-year OIS rate around the press release and the conference windows. Note: The ﬁgure shows the intraday movement (percentage points on the y-axis, trading hours on the x-axis) in the 2-year OIS rate during four selected episodes. The vertical solid line marks the publication of the press release; the vertical dashed line marks the beginning of the press conference. Before turning to the measurement of surprises it is useful to brieﬂy note some of the internal consistency and robustness checks we have carried out on the data (Appendix E contains details and further checks). The raw changes and surprises we measure, and the results of the analysis we do using these are mostly insensitive to changes in the measurement windows. Our estimates would have been more or less the same if we had taken the last quote instead of the median within the window or used wider or narrower windows. We have also veriﬁed that the changes and surprises are independent across the two windows, showing that we are indeed measuring reactions to unanticipated news in the two windows and not momentum effects that carry over from one window to the other. Another important issue has to do with the US data release calendar, which makes the initial unemployment claims release often overlap with the event windows in the Euro Area. In the window that contains both an ECB policy commu- nication and the US initial claims release, 5 although the two will be uncorrelated, if the short-term euro OIS rates respond to the initial claims surprise the monetary policy surprise measures will be subject to measurement error. We studied the impact of US initial claims surprises on short-term euro OIS rates and found that even in the very rare cases where there are statistically signiﬁcant coeﬃcients, the R 2 coeﬃcients are in single digits, implying that the simultaneous release of US initial claims does not introduce noticeable measurement error into the Euro Area monetary policy surprises that we de- velop. 2.1. A primer on euro area monetary policy communication For completeness we include these as control variables in the statistical work we present in Section 4 . It is reassuring that whether we include this control or not has no bearing on the results we report. 5 In the window that does not overlap with the initial claims release there is no correlation between the US surprise and the changes and surprises we measure for the Euro Area, again verifying that we are measuring proper surprises arising from monetary policy communication. 3. Measuring policy surprises in the euro area Understanding the effects of monetary policy requires identifying orthogonal changes in the policy stance. These changes may be orthogonal to the state of the economy, as in VAR analysis–in which case they are usually called policy shocks–or they may be orthogonal to the information set of ﬁnancial market participants–in which case they are called (market-based) policy surprises. Using monetary policy surprises measured in daily or higher frequency one can study asset price responses to mon- etary policy in a meaningful way. The very reasonable identifying assumption here is that monetary policy does not re- spond to asset price changes within the day, hence causality goes from monetary policy to asset prices and ﬁnancial mar- kets’ reaction to monetary policy can be studied. Work on related questions for the euro area have been done by Brand et al. (2010) , Jardet and Monks (2014) , Andrade and Ferroni (2016) , Leombroni et al. (2017) , Cieslak and Schrimpf (2018) , Kane et al. (2018) , Rogers et al. (2014) , and Jaroci ´nski and Karadi (2018) among others. These papers collectively study the C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 167 ﬁnancial and real effects of ECB policy measured using high frequency surprises, but do not do the decompositions of sur- prises as we do. ects of ECB policy measured using high frequency surprises, but do not do the decompositions of All of these papers had to construct their own event-study databases to carry out similar analyses. The EA-MPD we provide, by virtue of being regularly updated, will eliminate this sizable ﬁxed cost of doing research on Euro Area monetary policy. In terms of the exercises we carry out, our value added will be in estimating rather than assuming the number of different types of policy surprises market participants perceive and in naming these. The results of this exercise are interesting and different from what has been assumed so far. Our further value added will be in covering the crisis period and measuring the effects of ECB non-standard monetary policy measures over a range of ﬁnancial assets and comparing their transmission with that of standard policy measures, as well as measuring the persistence of responses and possible asymmetry of these responses to positive and negative surprises. 3.1. Identifying the surprises 3.1. Identifying the surprises We ﬁrst measure monetary policy as a potentially two dimensional process with possible Target/Timing and Path (For- ward Guidance) components, and then allow for a third dimension after the onset of the ﬁnancial crisis so as to capture the information about non-standard measures and especially QE. As noted above, the ECB policy communication extends over two separate windows where the policy action is ﬁrst announced in a press release with no motivation, and then a statement is read by the President, followed by a question-and-answer session. Market participants may update their beliefs about the current stance and future path of monetary policy in response to the press release as well as the press conference; hence on each policy date, using intraday data, we measure two sets of surprises. We construct the Target and Forward Guidance surprises following the methodology employed in Gürkaynak et al. (2005) and Gürkaynak (2005) , who in turn build on the work of Kuttner (2001) , using Federal Funds futures quotes to measure policy surprises in the US. The methodology of constructing the QE factor follows that of Swanson (2017) . In particular, we extract factors from changes in yields of risk-free rates at different maturities, spanning one-month to ten-years, in each of the two windows (press release and conference). Ideally, the risk-free rate curve in the euro area would be proxied by the term structure of the OIS. Unfortunately, however, at maturities longer than 2 years high-frequency data on the OIS rates is only available after August 2011. Therefore, prior to that date we use yields on the German sovereign yields as proxy for the risk-free rates. Using the German yields throughout the period makes no signiﬁcant difference. To extract monetary policy surprises that admit economic interpretation from these asset price changes we estimate latent factors from changes in yields and rotate these factors. The matrix X j , j = { press release, press conference } , has changes in 1, 3, and 6-month and 1, 2, 5, and 10-year yields in its seven columns, with each row corresponding to a policy date. This matrix is taken directly from the EA-MPD. The factor structure is X j = F j j + ϵ j , (1) where F are the common latent factors, are the factor loadings, and ϵ are the idiosyncratic variation of yields at different maturities. 3. Measuring policy surprises in the euro area In particular, we will be estimating the persistence of QE effects in the euro area using a precise, continuous measure of QE surprises for the ﬁrst time. 3.1. Identifying the surprises We analyze the press release and press conference windows separately and estimate the factors–common drivers of yield changes–by principal components. where F are the common latent factors, are the factor loadings, and ϵ are the idiosyncratic variation of yields at different maturities. We analyze the press release and press conference windows separately and estimate the factors–common drivers of yield changes–by principal components. We test the number of statistically signiﬁcant factors in the two windows over the full sample and the pre-QE samples. As shown in Table 1 , we consistently ﬁnd a single signiﬁcant factor in the press release window in both periods, but ﬁnd Table 1 Test of number of factors characterizing monetary policy announcements. Press release window Conference window Pre-QE Full sample Pre-QE Full sample H 0 : k = 0 46.20 49.12 105.50 108.49 (0.001) (0.000) (0.000) (0.000) H 0 : k = 1 18.77 22.55 33.73 39.42 (0.174) (0.068) (0.002) (0.000) H 0 : k = 2 14.87 17.44 (0.062) (0.026) H 0 : k = 3 4.07 (0.254) Note: The table reports the Wald statistics and associated p-values in paren- thesis of Cragg and Donald (1997) test for testing the null hypothesis of k = k 0 factors against the alternative that k > k 0 . The same test is performed on the two policy windows (press release and press conference) and for two different samples. The Pre-QE sample consists of all surprises due to monetary policy events taking places between January 2002 and December 2013. The full sam- ple contains all the monetary surprises (January 2002–September 2018). Table 1 Test of number of factors characterizing monetary policy announcements. Press release window Conference window Pre-QE Full sample Pre-QE Full sample H 0 : k = 0 46.20 49.12 105.50 108.49 (0.001) (0.000) (0.000) (0.000) H 0 : k = 1 18.77 22.55 33.73 39.42 (0.174) (0.068) (0.002) (0.000) H 0 : k = 2 14.87 17.44 (0.062) (0.026) H 0 : k = 3 4.07 (0.254) C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 168 two factors in the press conference window before QE and three in the full sample, suggesting the presence of a new factor in this window in the QE period. 3.1. Identifying the surprises The existence of guidance by central banks predates the global ﬁnancial crisis as well as the explicit designation of “Forward Guidance” and even the adoption of statements accompanying policy decisions. The Forward Guidance factor captures the revision in market expectations about the future path of policy rates that are orthogonal to the current policy surprise. The other factor that is always present in the press conference window does not have factor loadings that lead to a Target interpretation. That factor does not load on the 1-month OIS much. This is mechanically possible because the rotation forces one factor to be orthogonal to 1-month OIS but does not force the other factor to closely follow it. The other factor in the press conference window turns out to be a “Timing” factor that captures the shifts in market expectations over the next few meetings that leaves longer-term interest rates essentially unchanged. It is therefore important to note that estimating a Target factor in the press release window and a Timing factor in the press conference window are ﬁndings, not assumptions. Similarly, the QE factor that loads only on longer-term yields is a ﬁnding interpretable this way, not ex-ante assumed. y y g y g p y The interpretable surprises we measure using the factor rotation are suitable for our research questions. Other decom- positions of surprises are also possible and the EA-MPD will facilitate asking a variety of euro area monetary policy related questions. Some of the recent work in this vein are Jaroci ´nski and Karadi (2018) , who use stock-bond correlations to iden- tify central bank information signaling as opposed to classical monetary policy surprises; Andrade and Ferroni (2016) , using index-linked swaps for the same purpose; and Cieslak and Schrimpf (2018) , who use stocks and interest rates of short and long maturities to do a three-way decomposition between classical monetary policy surprises, information (on growth) surprises, and risk premium surprises. We have also experimented with alternative identifying assumptions. Since we found a single factor in the press release window, identifying the Target factor by orthogonalizing with respect to a second factor may have been misleading so we used the change in the one-month OIS directly as the Target factor, which unsurprisingly made no difference. 3.1. Identifying the surprises 6 The latent factors F j do not have clean interpretations as monetary policy surprises, so we rotate the factors to make them interpretable. This method has been used frequently since Gürkaynak et al. (2005) and was ﬁrst applied to Euro Area data by Brand et al. (2010) using intraday data. For our purposes, including the analysis of QE, the orthogonal factors are identiﬁed by imposing the following restrictions on the rotation matrix: (1) the second and third (when the third factor is present) factors do not load on the one-month OIS; (2) the rotation is such that the third factor has the smallest variance in the pre-crisis period (2 January 2002 - 7 Aug 2008). Essentially, we are forcing two factors not to be correlated with the one-month OIS (the standard measure of the immediate policy setting surprise) and allowing for one of them to affect the yield curve such that this factor was not important in the pre-crisis period. 7 This factor will turn out to contribute only to the movements in the long-end of the yield curve, and only be active post-2014, naturally leading to the QE factor label. Extraction of this last factor is an application of the Swanson (2017) methodology to euro area monetary policy data. It is important to recognize that what matters for these surprises is how market participants interpret the policy news and how their expectations change following the policy news; our ﬁndings are not about the type of signal the central bank aims to provide. The sequential nature of these orthogonalizations is important to keep in mind to understand the rotated factors. One factor is deﬁned by orthogonality to the 1-month OIS change. Another is deﬁned by orthogonality to this, such that the two explain most of the variance of the yields. The third factor is deﬁned such that it is orthogonal to the ﬁrst two and the 1-month OIS, and explains the minimal share of the pre-crisis variance. 8 With these rotations, the ﬁrst and only statistically signiﬁcant factor in the press release window turns out to be the “Target” factor, which loads only on the short rates. As expected, we ﬁnd a Forward Guidance (Path) factor that is always present in the press conference window. 7 Note that this would be a somewhat imperfect measure prior to November 2001, as over that period the Governing Council took policy decisions at a fortnightly frequency. (Note also that, as discussed above, although we make the event study entries for all dates available in the dataset, the analysis in this section and beyond uses data beginning in 2002.) In that case, the one-month OIS covers the next meeting as well, hence may capture changes in expectations about the next meeting in addition to the immediate policy surprise. There are very few, if any, quotes for the one-week OIS on many event dates. Otherwise utilizing that measure would have solved the problem of frequency of meetings being higher than the maturity of contract used for the period prior to November 2001. 8 A more elaborate and formal presentation of the factor rotations is presented in Appendix F. 3.1. Identifying the surprises Similarly, not using the one-month OIS in the conference window (where it has negligible variance) and using orthogonality to three- month OIS to do the factor rotation gave qualitatively the same results. Many other ways of identifying surprises are possible and will surely be used by other researchers. 6 The full sample press conference window having three factors might also have been due to the two sub-samples having two factors each, but one of these being different across the pre-QE and QE periods. But the ﬁrst two factors of the pre-QE and full samples are exactly the same, ruling out this interpretation. Further, although utilizing the test for only the QE sample is undesirable due to the very low degrees of freedom one would have, doing that nonetheless ﬁnds three factors in the press conference window in the QE period. 3.2. Policy surprises in the euro area 6 The full sample press conference window having three factors might also have been due to the two sub-samples having two factors each, but one of these being different across the pre-QE and QE periods. But the ﬁrst two factors of the pre-QE and full samples are exactly the same, ruling out this interpretation. Further, although utilizing the test for only the QE sample is undesirable due to the very low degrees of freedom one would have, doing that nonetheless ﬁnds three factors in the press conference window in the QE period. 7 Note that this would be a somewhat imperfect measure prior to November 2001, as over that period the Governing Council took policy decisions at a fortnightly frequency. (Note also that, as discussed above, although we make the event study entries for all dates available in the dataset, the analysis in this section and beyond uses data beginning in 2002.) In that case, the one-month OIS covers the next meeting as well, hence may capture changes in expectations about the next meeting in addition to the immediate policy surprise. There are very few, if any, quotes for the one-week OIS on many event dates. Otherwise utilizing that measure would have solved the problem of frequency of meetings being higher than the maturity of contract used for the period prior to November 2001. 8 A more elaborate and formal presentation of the factor rotations is presented in Appendix F. 3.2. Policy surprises in the euro area Guidance has unit effect on the two-year and QE has unit effect on the ten-year yields. This normalization has no effect on the variance shares and statistical signiﬁcance of the results we report. Note again that these factors are deﬁned by the factor loadings, the shapes shown in Fig. 3 are not assumed but es- timated. The assumptions we made are the orthogonality conditions discussed above. Our statistical tests tell us to look for a single factor in the press release window and three factors in the press conference window. (The third factor is only statistically present in the QE period but the factor loadings of all rotated factors are the same throughout the period. The QE factor has the same deﬁnition in the pre-QE period but is not contributing much to the overall variance, consistent with its label.) Those factors are rotated according to the orthogonality conditions assumed so that they may be interpretable. In the event, they turn out to have factor loadings shown in the ﬁgure and admit labels that have economic meaning. Notice in particular that the effect of QE was consistent with its design: the effect is greater the longer the maturity–recall that only the effect on the current-month OIS are set (to zero) by construction, whereas the effects on all other maturities are estimated. 9 Table 2 shows the relative contribution (variance shares) of our identiﬁed factors in explaining changes across the risk- free rate curve in the press release and conference windows. This is a striking table showing that in the press release window yield curve variance is greatest in the shortest maturity to begin with, and the Target factor captures about all of the short-end volatility. Since there is no other statistically signiﬁcant factor in this window and the Target factor does not load on the long-end at all, the volatility of the long-end is entirely in the idiosyncratic residual, but this volatility is small. Conversely, in the press conference window, the overall volatility is much higher and is concentrated on the longer-end of the yield curve, with a peak in volatility at two to ﬁve years, the maturities that are affected by both Forward Guidance and QE. 9 ECB used a variety of maturities for its QE program, with an average maturity of about eight years. Hence, ﬁnding largest effects of QE on the long-end of the yield curve is as reasonable in the euro area as in the US. 3.2. Policy surprises in the euro area Fig. 3 shows the loadings of the rotated factors over the seven maturities in the analysis. As the factors are identiﬁed up to scale, we scale them such that Target has unit effect on one-month OIS, Timing has unit effect on six-month OIS, Forward C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 169 Fig. 3. Factor loadings. Note: The ﬁgure shows the factor loadings for the press release (ﬁrst row) and the conference windows (second row), in basis points. In each window, for each maturity the loadings are obtained by regressing the surprises onto the factors also controlling for the standardized surprise associated with the release of the US initial jobless claims. The Target and the Timing factors are normalized to have unit effect on the 1-month and 6-month OIS, respectively. The Forward Guidance and QE factors are normalized to have unit effect on the 2-year and on the 10-year yields, respectively. The shaded areas indicate the 90% conﬁdence intervals. Fig. 3. Factor loadings. Note: The ﬁgure shows the factor loadings for the press release (ﬁrst row) and the conference windows (second row), in basis points. In each window, for each maturity the loadings are obtained by regressing the surprises onto the factors also controlling for the standardized surprise associated with the release of the US initial jobless claims. The Target and the Timing factors are normalized to have unit effect on the 1-month and 6-month OIS, respectively. The Forward Guidance and QE factors are normalized to have unit effect on the 2-year and on the 10-year yields, respectively. The shaded areas indicate the 90% conﬁdence intervals. Fig. 3. Factor loadings. Fig. 3. Factor loadings. g g Note: The ﬁgure shows the factor loadings for the press release (ﬁrst row) and the conference windows (second row), in basis points. In each window, for each maturity the loadings are obtained by regressing the surprises onto the factors also controlling for the standardized surprise associated with the release of the US initial jobless claims. The Target and the Timing factors are normalized to have unit effect on the 1-month and 6-month OIS, respectively. The Forward Guidance and QE factors are normalized to have unit effect on the 2-year and on the 10-year yields, respectively. The shaded areas indicate the 90% conﬁdence intervals. 3.2. Policy surprises in the euro area This ﬁnding is interesting and speaks to both the methodology of extracting the factors and the structure of monetary policy communication in the Euro Area. The methodology forces two of the factors, which we call Forward Guidance and QE factors, to be uncorrelated with the current interest rate surprise, in this case the change in the one-month OIS rate. The other factor is not forced to display the features of a Target factor (that is, not forced to be highly correlated with the change in the one month OIS rate) but in the press release window it turns out endogenously to be so, suggesting that Target is a dominant feature in the data. Recall that in the US policy window Target is always a dominant policy surprise as the policy decision and statement are simultaneously released; therefore this identiﬁcation strategy in the US leads to ﬁnding the ﬁrst factor to be Target. In the euro area, in the conference window we ﬁnd instead that the ﬁrst factor does not look like a Target factor; it is not correlated with the one-month OIS at all. This has to be expected because once the current setting of policy is announced in the press release, updates to beliefs about Target should no longer be a dominant source of variation in the conference window. Indeed, principal components and the factor rotations that deﬁne Forward Guidance (Path) and QE lead to ﬁnding a remaining factor that does not display the features of a Target factor, but the features of a factor that resembles the Timing factor discussed by Gürkaynak et al. (2007) . This is a factor that loads on the very near term (within six months) OIS rate changes and captures shifts in the perceived Timing of policy actions, such as beliefs that although a policy change did not happen today it is now more likely that it will happen in the next meeting or two. That is, the press conference makes the relative probabilities shift across very near-term meetings, as well as farther out. Forward Guidance captures changes in policy expectations affecting interest rates about two years out and Timing captures changes in expectations at a shorter horizon, affecting only to a small extent interest rates of longer maturities. 3.2. Policy surprises in the euro area Thus, in the cases where much of the variance of a particular maturity is attributed to the residual, such as 10-years in the press release window or one-month in the press conference window, these have the lowest variances across the yield curve and the maturities with largest variances, one-month in the press release and two and ﬁve-years in the press conference windows are fully explained by the measured policy surprises. C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 170 Table 2 Relative contribution of factors in explaining policy surprises, full sample. 1-month 3-month 6-month 1-year 2-year 5-year 10-year SD Factor Press release Target 97.8 91.3 82.7 60.4 32.9 11.9 1.5 2.2 Residual 2.2 8.7 17.3 39.6 67.1 88.1 98.5 NA SD OIS rel. 2.2 1.7 1.5 1.4 1.4 1.5 1.2 NA Conference Timing 54.8 86.7 70.3 50.0 29.6 14.7 9.5 2.3 Forward Guidance 0.0 9.1 28.1 49.0 68.9 67.3 37.2 3.6 QE 0.0 0.1 0.0 0.0 0.8 15.7 50.0 1.9 Residual 45.2 4.2 1.6 0.9 0.8 2.3 3.3 NA SD OIS conf. 1.1 2.1 2.8 3.9 4.4 4.1 2.7 NA Note: The table reports, for the maturity of the OIS rates indicated in the ﬁrst row, the fraction of the variance (in percentage points) explained by Target factor in the press release window and by each of the three factors (Timing, Forward Guidance, and QE) in the conference window. The row labeled “Residual” reports the variance not explained by the factors. The last row in each panel shows the variance of the OIS rate at the relevant maturity measured in the release and conference windows respectively. The last column reports the standard deviations of the four factors. As discussed above, we ﬁnd that (a) changes of interest rates at short maturities in the press release window are mainly explained by the Target surprise, while in the conference window the Target surprise is absent and a “Timing” surprise emerges; (b) the Path (Forward Guidance) factor is present in the conference window and affects interest rates across all maturities above one month, with a typical hump-shaped pattern, and (c) the QE factor, present in the conference window, is differentiated from Forward Guidance by having an impact that is stronger the longer the maturity. 3.2. Policy surprises in the euro area Although we name these factors Timing and Forward Guidance, it is important to recognize that in essence these are two separate Forward Guidance factors, one with a peak effect at shorter maturities and the other at relatively longer maturities. One can do the analysis with these factors, as we will, and keep in mind that the interpretations of factors across win- dows will be different: the ﬁrst factor is Target in the press release window, and Timing in the conference window. Of course, it is possible to force the ﬁrst factor to consistently be Target in both windows by imposing a large positive corre- lation of this factor with the change in the one-month OIS rate. In that case, the factor interpretations will be symmetric across windows, but this will come at the cost that less of the variance of the conference window will be explained: Timing, which we have shown is important, would have to turn into a residual. We continue with allowing for the Timing factor in the conference window. As discussed in detail in Appendix G, the largest readings of the surprises correspond to well known dates and accord well with the market commentary. While we present the surprises and the statements that led to these in the appendix, a formal mapping between quantiﬁed words, as in Hansen and McMahon (2016) , and the market perceptions we measure here has to be a separate paper. 4. Asset price response to policy Coeﬃcients are expressed in percentage per annum per standard deviation change in the factors. Robust standard errors in parentheses; ∗∗∗, ∗∗, and ∗denote statistical signiﬁcance at the 1%, 5% and 10% levels, respectively. In the conference window we continue to use the surprise in the US initial jobless claims as an additional control (labeled IJC) whenever we run regressions. Including or excluding the initial claims makes essentially no difference to our coeﬃcients of interest. In the conference window we continue to use the surprise in the US initial jobless claims as an additional control (labeled IJC) whenever we run regressions. Including or excluding the initial claims makes essentially no difference to our coeﬃcients of interest. Table 3 shows the intraday reaction of euro area risk-free rates to the Target surprise we have identiﬁed in the press release window (panel A) and the three surprises in the conference window (panel B). This table presents the information in Fig. 3 with numbers, the normalized factor loadings using an OLS regression framework and controlling for the US Ini- tial Jobless Claims surprises in the press conference window. The loadings, which are essentially unchanged compared to Fig. 3 (the ECB policy surprises and US IJC surprises are not correlated) show the estimated weights over maturities in the full sample. An interesting question is whether these factors have behaved the same way throughout the sample or whether the policy surprises elicited different responses before the crisis, during it before QE became a policy tool, and when QE was actively used. To study this, we run these regressions with samples up to 20 08, 20 08–2014, and 2014 to September 2018, corresponding to pre-crisis, crisis before QE, and QE periods. The QE factor is of course only active in the last period. Tables 4–6 show the results. Here, remember that these factors are deﬁned as before (loadings do not change across tables) and are normalized such that Target, Timing, Forward Guidance, and QE factors have unit effects on one-month, six-month, two-year and 10-year yields in the full sample. Hence, the coeﬃcients are comparable across tables and strongly show that these surprises functioned almost exactly the same way throughout our sample. Once the ZLB was binding there were fewer Target surprises but to the extent they took place, their effects were conﬁned to the short end, with a rapid decay. 10 Effects on German rates are indistinguishable from effects on OIS rates when both are available and effects on French rates very closely follow German ones. We do not separately report or discuss these. 4. Asset price response to policy An important contribution of this paper is deﬁning the interpretable policy surprise factors for the euro area. Once the policy surprises are measured as described in Section 3 , causality is established, and the response of other asset prices can be studied via OLS. We estimate the impact of monetary policy on sovereign yields, the exchange rate, and the stock market. C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 171 C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162 179 Table 3 Estimated effects of monetary policy surprises on OIS yields. Panel (A): Press release window (1) (2) (3) (4) (5) (6) (7) VARIABLES OIS 1M OIS 3M OIS 6M OIS 1Y OIS 2Y OIS 5Y OIS 10Y Target 1.00 ∗∗∗ 0.74 ∗∗∗ 0.63 ∗∗∗ 0.50 ∗∗∗ 0.37 ∗∗∗ 0.24 ∗∗∗ 0.07 (0.02) (0.03) (0.04) (0.06) (0.08) (0.08) (0.06) Observations 185 185 185 185 185 185 185 R -squared 0.98 0.91 0.83 0.60 0.33 0.12 0.02 Panel (B): Conference window (1) (2) (3) (4) (5) (6) (7) VARIABLES OIS 1M OIS 3M OIS 6M OIS 1Y OIS 2Y OIS 5Y OIS 10Y Timing 0.33 ∗∗∗ 0.84 ∗∗∗ 1.00 ∗∗∗ 1.17 ∗∗∗ 1.01 ∗∗∗ 0.68 ∗∗∗ 0.36 ∗∗∗ (0.07) (0.01) (0.02) (0.02) (0.02) (0.03) (0.02) FG 0.00 0.17 ∗∗∗ 0.41 ∗∗∗ 0.75 ∗∗∗ 1.00 ∗∗∗ 0.94 ∗∗∗ 0.46 ∗∗∗ (0.03) (0.01) (0.01) (0.01) (0.02) (0.03) (0.02) QE 0.00 0.03 −0.02 ∗∗ 0.00 0.20 ∗∗∗ 0.85 ∗∗∗ 1.00 ∗∗∗ (0.03) (0.02) (0.01) (0.02) (0.02) (0.04) (0.03) IJC 0.04 −0.04 0.02 0.00 −0.01 0.02 −0.01 (0.05) (0.03) (0.03) (0.03) (0.03) (0.04) (0.03) Observations 180 180 180 180 180 180 180 R -squared 0.55 0.96 0.98 0.99 0.99 0.98 0.97 Note: The table reports the reaction of OIS and German sovereign yields at different maturities to surprises in monetary policy using intraday data. Coeﬃcients are expressed in percentage per annum per standard deviation change in the factors. Robust standard errors in parentheses; ∗∗∗, ∗∗, and ∗denote statistical signiﬁcance at the 1%, 5% and 10% levels, respectively. Table 3 Estimated effects of monetary policy surprises on OIS yields. Note: The table reports the reaction of OIS and German sovereign yields at different maturities to surprises in monetary policy using intraday data. 10 Effects on German rates are indistinguishable from effects on OIS rates when both are available and effects on French ones. We do not separately report or discuss these. 4. Asset price response to policy Timing always has an early hump with little effect on the shortest and longest maturities. Forward guidance always affects intermediate maturities the most and QE, when present, affects the long-end only. Importantly, these statements are true quantitatively as well, the coeﬃcients differ little across subsamples, and ﬁts, as measured by R 2 are also similar, showing that the relevance of the factors have not changed in terms of aggregate variance explained. Notice that this analysis is only possible with our factor estimation methodology. While assuming that a “communica- tion” surprise is present in the press conference window may be appropriate for some purposes, that approach would ﬁnd a changing response to the communication surprise over time because OIS rate changes in this window would be captur- ing different types of information; dominantly Forward Guidance before 2014 and dominantly QE afterwards. That is, the surprise, not the response would be changing. Our methodology tracks different types of surprises, keeping their deﬁnitions the same across sub-samples, and shows that reactions to these have not changed markedly over time. Having veriﬁed the consistency of our surprise measures across subsamples for the euro area risk-free rates, we now turn to their their effects on the sovereign yields of Italy and Spain. 10 The results, shown in detail in Appendix H, document that these factors affected the sovereign yields the same way they affected the safe rates. Importantly, QE, when active, has 172 C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 Table 4 Estimated effects of monetary policy surprises on OIS, 01/20 02-12/20 07. Table 5 Table 5 Estimated effects of monetary policy surprises on OIS, 01/2008-12/2013. Panel (A): Press release window (1) (2) (3) (4) (5) (6) (7) VARIABLES OIS 1M OIS 3M OIS 6M OIS 1Y OIS 2Y OIS 5Y OIS 10Y Target 1.00 ∗∗∗ 0.77 ∗∗∗ 0.67 ∗∗∗ 0.58 ∗∗∗ 0.47 ∗∗∗ 0.31 ∗∗∗ 0.10 (0.03) (0.03) (0.02) (0.04) (0.06) (0.09) (0.08) Observations 71 71 71 71 71 71 71 R -squared 0.98 0.97 0.95 0.79 0.53 0.20 0.04 Panel (B): Conference window (1) (2) (3) (4) (5) (6) (7) VARIABLES OIS 1M OIS 3M OIS 6M OIS 1Y OIS 2Y OIS 5Y OIS 10Y Timing 0.34 ∗∗∗ 0.81 ∗∗∗ 1.02 ∗∗∗ 1.18 ∗∗∗ 0.98 ∗∗∗ 0.62 ∗∗∗ 0.27 ∗∗∗ (0.08) (0.02) (0.02) (0.03) (0.03) (0.04) (0.06) FG 0.02 0.18 ∗∗∗ 0.39 ∗∗∗ 0.73 ∗∗∗ 1.01 ∗∗∗ 0.87 ∗∗∗ 0.38 ∗∗∗ (0.03) (0.01) (0.01) (0.01) (0.01) (0.04) (0.06) IJC 0.21 ∗∗ −0.05 −0.07 ∗∗ −0.00 0.01 −0.27 −0.28 (0.09) (0.05) (0.04) (0.05) (0.05) (0.27) (0.27) Observations 71 71 71 71 71 71 71 R -squared 0.64 0.98 0.99 0.99 0.99 0.86 0.51 Note: The table reports the reaction of OIS and German sovereign yields at different maturities to surprises in monetary policy using intraday data. Coeﬃcients are expressed in percentage per annum per standard deviation change in the factors. Robust standard errors in parentheses; ∗∗∗, ∗∗, and ∗denote statistical signiﬁcance at the 1%, 5% and 10% levels, respectively. Table 5 Estimated effects of monetary policy surprises on OIS, 01/2008-12/2013. Note: The table reports the reaction of OIS and German sovereign yields at different maturities to surprises in monetary policy using intraday data. Coeﬃcients are expressed in percentage per annum per standard deviation change in the factors. Robust standard errors in parentheses; ∗∗∗, ∗∗, and ∗denote statistical signiﬁcance at the 1%, 5% and 10% levels, respectively. affected Spanish and especially Italian long-term yields more than the safe rate, narrowing spreads on impact, consistent with the evidence in Altavilla et al. (2015) . 2 affected Spanish and especially Italian long-term yields more than the safe rate, narrowing spreads on impact, consistent with the evidence in Altavilla et al. (2015) . Table 4 Table 4 Estimated effects of monetary policy surprises on OIS, 01/20 02-12/20 07. Note: The table reports the reaction of OIS and German sovereign yields at different maturities to surprises in monetary policy using intraday data. Coeﬃcients are expressed in percentage per annum per standard deviation change in the factors. Robust standard errors in parentheses; ∗∗∗, ∗∗, and ∗denote statistical signiﬁcance at the 1%, 5% and 10% levels, respectively. 4. Asset price response to policy Panel (A): Press release window (1) (2) (3) (4) (5) (6) (7) VARIABLES OIS 1M OIS 3M OIS 6M OIS 1Y OIS 2Y OIS 5Y OIS 10Y Target 1.03 ∗∗∗ 0.62 ∗∗∗ 0.42 ∗∗∗ 0.22 ∗∗∗ 0.01 -0.04 -0.07 (0.02) (0.03) (0.03) (0.06) (0.09) (0.11) (0.10) Observations 72 72 72 72 72 72 72 R -squared 0.98 0.85 0.69 0.22 0.00 0.01 0.03 Panel (B): Conference window (1) (2) (3) (4) (5) (6) (7) VARIABLES OIS 1M OIS 3M OIS 6M OIS 1Y OIS 2Y OIS 5Y OIS 10Y Timing 0.23 ∗∗∗ 0.87 ∗∗∗ 0.98 ∗∗∗ 1.18 ∗∗∗ 1.07 ∗∗∗ 0.57 ∗∗∗ 0.44 ∗∗∗ (0.07) (0.05) (0.04) (0.04) (0.04) (0.14) (0.07) FG 0.02 0.15 ∗∗∗ 0.42 ∗∗∗ 0.78 ∗∗∗ 0.99 ∗∗∗ 1.14 ∗∗∗ 0.63 ∗∗∗ (0.03) (0.03) (0.01) (0.02) (0.05) (0.13) (0.06) IJC −0.03 −0.05 0.10 ∗∗ 0.00 −0.11 ∗∗∗ −0.02 −0.17 ∗ (0.06) (0.04) (0.05) (0.03) (0.04) (0.12) (0.09) Observations 67 67 67 67 67 67 67 R -squared 0.34 0.92 0.97 0.99 0.98 0.88 0.78 Note: The table reports the reaction of OIS and German sovereign yields at different maturities to surprises in monetary policy using intraday data. Coeﬃcients are expressed in percentage per annum per standard deviation change in the factors. Robust standard errors in parentheses; ∗∗∗, ∗∗, and ∗denote statistical signiﬁcance at the 1%, 5% and 10% levels, respectively. Table 5 2 As judged by the high R 2 s of the regressions, the monetary policy surprises we extracted capture large fractions of yield changes for risk-free and sovereign rates, especially in the conference window for the last subsample for long rates, as the QE factor helps explain the 10-year yield changes. In the earlier samples the R 2 for long rates are lower but the variance of these rates are also lower without QE surprises that move them. This is in contrast to Leombroni et al. (2017) , who ﬁnd that spreads were widened due to Forward Guidance surprises in a study that did not separately assess QE effects. We therefore ﬁnd that the ﬁrst stage of monetary policy transmission is not different across the largest euro area countries. The real effects may be different due to non-ﬁnancial differences across them, as argued by Corsetti et al. (2018) but that is a separate topic of study. C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 173 Table 6 Estimated effects of monetary policy surprises on OIS, 01/2014-09/2018. Panel (A): Press release window (1) (2) (3) (4) (5) (6) (7) VARIABLES OIS 1M OIS 3M OIS 6M OIS 1Y OIS 2Y OIS 5Y OIS 10Y Target 0.88 ∗∗∗ 1.05 ∗∗∗ 1.11 ∗∗∗ 1.04 ∗∗∗ 1.09 ∗∗∗ 0.92 ∗∗∗ 0.45 ∗ (0.08) (0.12) (0.18) (0.14) (0.17) (0.18) (0.25) Observations 42 42 42 42 42 42 42 R -squared 0.91 0.85 0.80 0.79 0.65 0.43 0.11 Panel (B): Conference window (1) (2) (3) (4) (5) (6) (7) VARIABLES OIS 1M OIS 3M OIS 6M OIS 1Y OIS 2Y OIS 5Y OIS 10Y Timing 0.58 ∗∗∗ 0.91 ∗∗∗ 0.92 ∗∗∗ 1.03 ∗∗∗ 1.06 ∗∗∗ 0.83 ∗∗∗ 0.21 ∗∗∗ (0.13) (0.07) (0.04) (0.05) (0.04) (0.10) (0.08) FG −0.05 0.14 ∗∗ 0.48 ∗∗∗ 0.74 ∗∗∗ 1.00 ∗∗∗ 0.91 ∗∗∗ 0.49 ∗∗∗ (0.08) (0.05) (0.02) (0.03) (0.02) (0.05) (0.04) QE −0.06 ∗ −0.04 −0.00 0.09 ∗∗∗ 0.21 ∗∗∗ 0.73 ∗∗∗ 1.10 ∗∗∗ (0.03) (0.03) (0.02) (0.03) (0.02) (0.06) (0.05) IJC −0.04 −0.02 0.04 0.03 −0.06 ∗ 0.04 −0.02 (0.10) (0.11) (0.07) (0.04) (0.03) (0.06) (0.05) Observations 42 42 42 42 42 42 42 R -squared 0.71 0.87 0.95 0.96 0.99 0.97 0.99 Note: The table reports the reaction of OIS rates at different maturities to surprises in mone- tary policy using intraday data. 4.1. Exchange rate In exchange rates, where we again relegate the reporting of the detailed results to Appendix H, we ﬁnd that the surprises exert a statistically signiﬁcant effect on the exchange value of the euro. The R -squared of the regression for the conference window is about 0.6, which is very large for work involving exchange rates on the left-hand-side. It is also noteworthy that in our samples the Rogers et al. (2014) “preserving the euro effect” where easing surprises make the euro appreciate, does not manifest itself. This effect, while certainly present on some days, is dominated by the standard uncovered interest parity channel in the periods of our analysis. These ﬁndings are broadly consistent with the work of Faust et al. (2007) , who show that “good news” in data releases in the US lead to an appreciation of the dollar and relate this to uncovered interest parity. We ﬁnd that higher rates than anticipated in the euro area similarly lead to an appreciation of the euro, as expected. There is not much of a literature on whether current rates or expected future interest rates, or short versus long-term interest rates theoretically should be and empirically are the main drivers of exchange rates. Our empirical ﬁndings suggest that changing interest rate expectations at all maturities as a result of monetary policy communication affect the exchange value of the euro. Table 6 Table 6 Estimated effects of monetary policy surprises on OIS, 01/2014-09/2018. Note: The table reports the reaction of OIS rates at different maturities to surprises in mone- tary policy using intraday data. Coeﬃcients are expressed in percentage per annum per standard deviation change in the factors. Robust standard errors in parentheses; ∗∗∗, ∗∗, and ∗denote sta- tistical signiﬁcance at the 1%, 5% and 10% levels, respectively. 4.1. Exchange rate Table 5 Coeﬃcients are expressed in percentage per annum per standard deviation change in the factors. Robust standard errors in parentheses; ∗∗∗, ∗∗, and ∗denote sta- tistical signiﬁcance at the 1%, 5% and 10% levels, respectively. 5. Stock prices and the information surprise Table 7 shows the intraday reaction of the euro area stock market to the Target surprise (panel A) and the three surprises in the conference window (panel B). The response is statistically signiﬁcant and negative (surprises that increase yields lead to stock index declines) for the Target factor in the full sample and the pre-crisis sample, and for the Timing and Forward Guidance factors in the post-2014 sample. Results for the stock market index comprising only euro area banks are similar. p p p g y Lack of signiﬁcance for some of the factors in some sub-samples may be expected in light of the recent literature ar- guing that any monetary policy decision (or no decision) may be interpreted by markets in two different ways: a textbook monetary policy shock or a perceived revelation of information about the state of the economy. This latter type of policy is called Delphic in Campbell et al. (2012) and information shock in Miranda-Agrippino and Ricco (2018) and Jaroci ´nski and Karadi (2018) . For our analysis, it is important to note that the responses of nominal rates to the two types of surprises are qualitatively similar. A positive policy surprise increases yields if it is a genuine shock, but perceived information that the state of the business cycle is stronger than thought also similarly increases yields by increasing future expected short rates. Therefore, possible information effects do not interfere with our identiﬁcation strategy of the factors. However, the two types of surprises have opposite effects on stocks. Lower rates are good news (lower discount rates and higher demand) but learning that the cyclical state is worse than previously thought is bad news (lower dividends). One can see similar effects on inﬂation expectations; a positive policy surprise should decrease inﬂation compensation implied by indexed securities unless the surprise is perceived to be signalling information about high inﬂationary pressures, in which case inﬂation compensation will increase. The macroeconomic impacts of these policy surprises are also found to C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 174 Table 7 Estimated effects of monetary policy surprises on stock prices. Table 7 E i able 7 stimated effects of monetary policy surprises on stock prices. Note: The table reports the reaction of the general euro area stock market index over different samples (columns 1 to 4), and the reaction of the euro area bank stock market sub-index over the same samples (columns 5 to 8) to surprises in monetary policy using intraday data. Coeﬃcients are expressed in percentage points per standard deviation change in the factors. Robust standard errors in parentheses; ∗∗∗, ∗∗, and ∗denote statistical signiﬁcance at the 1%, 5% and 10% levels, respectively. differ depending on whether the policy surprise triggers a positive or negative response of the stock market ( Jaroci ´nski and Karadi, 2018 ) or, similarly, a positive or negative response of inﬂation-linked swaps ( Andrade and Ferroni, 2016 ), as measured at high-frequency around policy events. 11 Therefore, the presence of these two types of policy can make the response of the stock market, on average, insigniﬁcant and can produce the results reported in Table 7 . differ depending on whether the policy surprise triggers a positive or negative response of the stock market ( Jaroci ´nski and Karadi, 2018 ) or, similarly, a positive or negative response of inﬂation-linked swaps ( Andrade and Ferroni, 2016 ), as measured at high-frequency around policy events. 11 Therefore, the presence of these two types of policy can make the response of the stock market, on average, insigniﬁcant and can produce the results reported in Table 7 . To assess whether there is evidence of these two types of surprises in our dataset, for each policy event we compare the sign of the response of the stock market and inﬂation-linked swaps. If nominal rates, stock prices and inﬂation-linked swaps all move in the same direction, this would suggest that information shocks may be prevailing. To overcome the problem that data on inﬂation compensation are not reliably available at intraday frequency, we adapt our analysis to daily frequency and measure the interest rate reaction as the ﬁtted value of the one-day change (around the policy events) of the 2-year OIS regressed on the intraday factors. We do the same for the one-day change of inﬂation-linked swaps and the one-day log-difference of stock prices. Fig. 11 Jaroci ´nski and Karadi (2018) use intraday data employing stocks whereas Andrade and Ferroni (2016) use daily data due to lack of reliable intraday data for inﬂation-linked swaps. 12 In about 80% of the policy dates stock prices and inﬂation-linked swaps move in the same direction. In the other 20%, in almost all of the cases the stock price reaction is about zero and the inﬂation-linked swap reaction is very small. Hence, these two measures almost never meaningfully disagree. Therefore, incidentally, our results verify Andrade and Ferroni (2016) and Jaroci ´nski and Karadi (2018) methods against each other and ﬁnd that they are in agreement. 5. Stock prices and the information surprise Panel (A): Press release window (1) (2) (3) (4) (5) (6) (7) (8) VARIABLES STOXX50E STOXX50E STOXX50E STOXX50E SX7E SX7E SX7E SX7E Target −0.04 ∗∗∗ −0.08 ∗∗∗ −0.03 −0.07 −0.03 −0.07 ∗∗∗ −0.03 0.14 (0.02) (0.02) (0.02) (0.14) (0.02) (0.01) (0.02) (0.24) Observations 185 72 71 42 185 72 71 42 R -squared 0.08 0.36 0.06 0.03 0.01 0.46 0.03 0.03 Panel (B): Conference window (1) (2) (3) (4) (5) (6) (7) (8) VARIABLES STOXX50E STOXX50E STOXX50E STOXX50E SX7E SX7E SX7E SX7E Timing −0.00 −0.01 0.02 −0.24 ∗ 0.00 −0.00 0.02 −0.30 ∗∗ (0.02) (0.03) (0.02) (0.12) (0.03) (0.03) (0.03) (0.11) FG −0.02 0.01 −0.03 −0.23 ∗∗ −0.04 ∗ −0.00 −0.05 −0.25 ∗∗∗ (0.02) (0.02) (0.02) (0.09) (0.02) (0.02) (0.03) (0.08) QE −0.01 −0.07 0.07 0.02 (0.04) (0.04) (0.08) (0.07) IJC −0.07 ∗ −0.11 ∗∗ −0.04 −0.05 −0.06 −0.12 ∗∗∗ −0.06 −0.03 (0.04) (0.05) (0.06) (0.10) (0.06) (0.04) (0.10) (0.14) Observations 180 67 71 42 180 67 71 42 R -squared 0.03 0.12 0.05 0.35 0.04 0.10 0.03 0.17 Note: The table reports the reaction of the general euro area stock market index over different samples (columns 1 to 4), and the reaction of the euro area bank stock market sub-index over the same samples (columns 5 to 8) to surprises in monetary policy using intraday data. Coeﬃcients are expressed in percentage points per standard deviation change in the factors. Robust standard errors in parentheses; ∗∗∗, ∗∗, and ∗denote statistical signiﬁcance at the 1%, 5% and 10% levels, respectively. Table 7 Estimated effects of monetary policy surprises on stock prices. Table 7 E i (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 4. Information and monetary policy surprises around GC meetings. g y p y p g Note: The ﬁgure shows the changes in 2-year OIS on ECB Governing Council Policy Meeting Dates in the pre- and post-QE periods in the ﬁrst and second panel respectively. Days in which the 2-year OIS, log STOXX50E, and 2-year Inﬂation-linked swap variables comove (red bars) have predominantly informa- tion surprises (Delphic), while days in which bond yields move in the opposite direction of the other two (blue bars) have predominantly pure monetary policy surprises (Odyssean). Grey bars represent the other cases (Unclassiﬁed). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) g y p y p g Note: The ﬁgure shows the changes in 2-year OIS on ECB Governing Council Policy Meeting Dates in the pre- and post-QE periods in the ﬁrst and second panel respectively. Days in which the 2-year OIS, log STOXX50E, and 2-year Inﬂation-linked swap variables comove (red bars) have predominantly informa- tion surprises (Delphic), while days in which bond yields move in the opposite direction of the other two (blue bars) have predominantly pure monetary policy surprises (Odyssean). Grey bars represent the other cases (Unclassiﬁed). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) where Y t is a vector consisting of the 2-year OIS, the log EUR-USD exchange rate, the log of the stock market index (Euro Stoxx 50), and the 2-year inﬂation linked swap (ILS2Y). As we are only interested in the effects of monetary policy shocks, our objective is to identify the column of the matrix A 0 corresponding to the contemporaneous effect of the monetary policy shock. ust satisfy the relevance and exogeneity assumptions: The instrument must satisfy the relevance and exogeneity assumptions: (3) E(Z t u m t ) = α ̸ = 0 , E(Z t u o t ) = 0 , where Z t is the instrument, and u m t and u o t denote the monetary and the non-monetary shocks. Our instruments will be the policy surprises we have identiﬁed. Table 7 E i 4 shows the dates for which stock prices and inﬂation swaps move in the same direction so that the market per- ception of policy can be clearly interpreted as information (Delphic) or pure policy (Odyssean) surprises. 12 The ﬁgure shows that there is a marked difference across sub-samples in terms of the information market participants extract from policy surprises. Whereas in the post-2014 sub-sample (bottom panel) information shocks (deﬁned as nominal interest rates, stock prices, and inﬂation-linked swaps moving in the same direction) are rare, these are frequent during the crisis sub-sample (top panel). That is, in the crisis period market participants attributed more of the surprises they perceived to information they thought the ECB has, consistent with a similar ﬁnding for the US by Lunsford (2018) . To delve deeper into this issue we build a daily VAR. The identiﬁcation strategy is based on the idea of using high- frequency monetary policy surprises to isolate the variation in the reduced-form residuals in the VAR due to monetary policy shocks. The use of external instruments for identiﬁcation in macroeconometric models goes back to Stock and Wat- son (2012) and Mertens and Ravn (2013) . We start the analysis by estimating the reduced form VAR as described in Eq. (2) . Y t = c + p j=1 B j Y t−j + A 0 u t , (2) (2) C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 175 C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162 179 175 Fig. 4. Information and monetary policy surprises around GC meetings. Note: The ﬁgure shows the changes in 2-year OIS on ECB Governing Council Policy Meeting Dates in the pre- and post-QE periods in the ﬁrst and second Fig. 4. Information and monetary policy surprises around GC meetings. Note: The ﬁgure shows the changes in 2-year OIS on ECB Governing Council Policy Meeting Dates in the pre- and post-QE periods in the ﬁrst and second panel respectively. Days in which the 2-year OIS, log STOXX50E, and 2-year Inﬂation-linked swap variables comove (red bars) have predominantly informa- tion surprises (Delphic), while days in which bond yields move in the opposite direction of the other two (blue bars) have predominantly pure monetary policy surprises (Odyssean). Grey bars represent the other cases (Unclassiﬁed). Table 7 E i / Journal of Monetary Economics 108 (2019) 162–179 176 , g y / J f y ( ) Fig. 5. Financial VAR. Note: The ﬁgures show the IRFs of a ﬁnancial 4-variable VAR, including 2Y OIS, log STOXX50E, ILS2Y and the log EUR/USD exchange rate. Blue-dotted lines show the IRFs for the pre-crisis period (20 05–20 08), green-dash-dotted lines for the pre-QE period (2008–2014), and the red-dashed lines, for the QE period (2014–2018). The x-axis units are days. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 5. Financial VAR. Fig. 5. Financial VAR. Note: The ﬁgures show the IRFs of a ﬁnancial 4-variable VAR, including 2Y OIS, log STOXX50E, ILS2Y and the log EUR/USD exchange rate. Blue-dotted lines show the IRFs for the pre-crisis period (20 05–20 08), green-dash-dotted lines for the pre-QE period (2008–2014), and the red-dashed lines, for the QE period (2014–2018). The x-axis units are days. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) sample of 20 02–20 07 we ﬁnd that stock prices go down in response to positive Target surprise while there is no clear pattern for Timing and Forward Guidance surprises. Over that sub-sample we cannot use inﬂation-linked swaps as they become available only in 2005. Overall, these results suggest that there is evidence of information shocks and that stock prices and inﬂation-linked swaps may both help in telling apart these shocks from the more traditional policy shocks. We ﬁnd that these results are fairly persistent, they do not only manifest themselves on policy dates then disappear. The results also suggest that it is possible to extend and generalise the macroeconomic analysis of Andrade and Ferroni (2016) and Jaroci ´nski and Karadi (2018) . They use the overall policy surprise as measured by the high-frequency change of the 2-year OIS around policy events. Our analysis shows that it is possible to compute the macroeconomic response to each of the factors we have identiﬁed including QE. The important ﬁnding of this section is that ECB policy surprises do have effects on stock prices but to properly study these one needs to separate traditional surprises from perceived information revelation, as we have brieﬂy shown here. Table 7 E i We do not go into a discussion of whether perceived “information” is about the current or future state, or about the reaction function of the central bank, although this is an important topic for future research. Table 7 E i The external instruments are Target (press release), the Timing, the Forward Guidance, and the QE factor (press conference) factors. As the VAR residual is a linear combination of structural shocks, we instrument the residuals of the VAR with one instrument at a time, as we aim to extrapolate the component correlated with the Target, Timing, Forward Guidance and Quantitative Easing surprises respectively. As a different exercise, one can also include all the instruments simultaneously, however, this will only help to identify a single monetary policy shock which reﬂects a linear combination of the four. To be consistent with the previous analysis, we use the intraday factors as external instruments for the VAR estimated on all sub-samples. When analyzing Quantitative Easing, the instrument is used on the period 2014–2018. where Z t is the instrument, and u m t and u o t denote the monetary and the non-monetary shocks. Our instruments will be the policy surprises we have identiﬁed. The external instruments are Target (press release), the Timing, the Forward Guidance, and the QE factor (press conference) factors. As the VAR residual is a linear combination of structural shocks, we instrument the residuals of the VAR with one instrument at a time, as we aim to extrapolate the component correlated with the Target, Timing, Forward Guidance and Quantitative Easing surprises respectively. As a different exercise, one can also include all the instruments simultaneously, however, this will only help to identify a single monetary policy shock which reﬂects a linear combination of the four. To be consistent with the previous analysis, we use the intraday factors as external instruments for the VAR estimated on all sub-samples. When analyzing Quantitative Easing, the instrument is used on the period 2014–2018. Fig. 5 shows that in the post-2014 sub-sample each factor exerts a signiﬁcant impact, with a decline in interest rates triggering a rise in stock prices and inﬂation-linked swaps, and a depreciation of the euro. In the 2008–2013 sub-sample the impact of each factor is signiﬁcant, but we ﬁnd that this time a decline in interest rates triggers a decline in stock prices and inﬂation linked-swaps, consistent with a perceived revelation of negative news. The exchange rate is found to depreciate, suggesting that it does not react differently, at least qualitatively, to an information shock. In the pre-crisis sub- C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. 13 Since we are interested in the market perception, we do not go into the details of the events but provide Bloomberg headlines for each event to give context. On the 2017 speech: “Draghi Sees Room for Paring Stimulus Without Tightening Policy”, “ECB president says forces damping inﬂation are temporary”. On the 2016 news report: “Informal consensus among ECB policymakers is that QE will need to be wound down gradually when decision is taken to end the program, say people familiar with the matter”, “One scenario is to taper QE in steps of EU10b/month, people say”. 6. Decomposing various policy news Policy events that are not covered by our data set–policy communications that are not Governing Council policy decisions–can also be analyzed using our methodology. Policymaker speeches, releases of minutes and the like are also policy communication and have ﬁnancial market effects. We can treat these events as if they are Governing Council policy C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 177 Fig. 6. Estimated factors for the two events. Note: The ﬁgures show the estimated factor decomposition into Target, Timing, FG and QE factors of the two monetary policy events not included in our sample. In each subplot the ﬁrst bar is for the June 2017 Draghi speech and the second one is for the October 2016 Bloomberg news article. Fig. 6. Estimated factors for the two events. Fig. 6. Estimated factors for the two events. Note: The ﬁgures show the estimated factor decomposition into Target, Timing, FG and QE factors of the two monetary sample. In each subplot the ﬁrst bar is for the June 2017 Draghi speech and the second one is for the October 2016 Bloo Fig. 6. Estimated factors for the two events. Note: The ﬁgures show the estimated factor decomposition into Target, Timing, FG and QE factors of the two monetary policy events not included in our sample. In each subplot the ﬁrst bar is for the June 2017 Draghi speech and the second one is for the October 2016 Bloomberg news article. e two events. imated factor decomposition into Target, Timing, FG and QE factors of the two monetary policy events not included in ou st bar is for the June 2017 Draghi speech and the second one is for the October 2016 Bloomberg news article. dates and, given the factor loadings we estimated for our factors, use the changes in the OIS yields in those event windows to decompose the market perception into Target, Timing, Forward Guidance, and QE surprises. This is an exercise in ﬁnding the combination of monetary policy surprise factors that best ﬁt the change in yields around the relevant window. The methodology is straightforward; for a particular event i –e.g., a speech–we take a window long enough to bracket the beginning and the end of the event, and compute the change in the 1M, 3M, 6M, 1Y, 2Y, 5Y, and 10Y OIS. 6. Decomposing various policy news We collect these yield changes in a vector OIS i , and, given the (rotated) factor loadings ˆ we estimated from the EA-MPD, we ﬁnd the factors ˆ F i which minimize the sum of squared residuals of OIS i −F i ˆ . That is ˆ F i = arg min F i OIS i −F i ˆ ′ OIS i −F i ˆ (4) The solution of this minimization problem can be recognized as the OLS estimator of F i , in a regression of OIS i onto the space spanned by ˆ . The solution of this minimization problem can be recognized as the OLS estimator of F i , in a regression of OIS i onto the space spanned by ˆ . We apply our methodology to two illustrative events that elicited noticeable market reactions. The ﬁrst is a speech given by Mario Draghi on 27 June 2017 at the ECB Forum on Central Banking in Sintra “Accompanying the economic recovery”. The second is a Bloomberg news article by Jana Randow, Alessandro Speciale, and Jeff Black which was released on October 4, 2016 and hinted at a decision on tapering by the ECB. 13 Fig. 6 shows our estimated factors for these two events, in terms of the Target, Timing, FG, and QE decomposition. To make the factors comparable to what were shown earlier in this paper, we rescale them as in Section 3 , and we report them 178 C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 as a fraction of the average absolute value of the in-sample surprise of each type. Note, as before, that the different scalings imply that magnitudes are comparable across different dates for the same type of surprise, but not across different types of surprises. as a fraction of the average absolute value of the in-sample surprise of each type. Note, as before, that the different scalings imply that magnitudes are comparable across different dates for the same type of surprise, but not across different types of surprises. As expected, our exercise ﬁnds a large QE factor in both events. For the ﬁrst event, we found a relatively large Forward Guidance component as well. “Large” here is with respect to the in-sample average size of these surprises. 14 The “Whatever it takes” speech by Mario Draghi is perhaps the most famous of euro area policy communications. We do not use it as an example here because we do not have data on the intraday ﬁve-year OIS yield for that event. However, the minor increase in the 10-year OIS yield around that speech suggests that our methodology would have interpreted this event, if anything, as a small QE tightening surprise. Identifying QE surprises by utilizing spreads would not lead to this ﬁnding for this speech but in that case QE would narrow spreads by assumption, and we would not be able to discuss effects of QE on spreads. 7. Extensions: persistence and non-linearity Two other issues that we study in depth but relegate to the appendix to keep the paper of reasonable length are the persistence and possible non-linearity of responses. Here we brieﬂy note the main ﬁndings for these topics. On persistence (Appendix I), we ﬁnd that the effects are notably persistent, especially for Forward Guidance and QE. Using a daily VAR with many combinations of variables we show that, very robustly, QE affected safe rates and sovereign yields at the 10-year maturity for a long duration, with a half-life of about one year. This is much longer than the about three month half-life Wright (2012) and Swanson (2017) ﬁnd for the US, although we caution that methodological differences do not allow direct comparisons. On non-linearity (Appendix J), our results are notable for the lack of asymmetry they imply. Market reactions have been remarkably symmetric with respect to positive and negative surprises. In particular, we ﬁnd no evidence that easing sur- prises elicit a smaller market reaction than tightening ones in contrast to the nascent literature (for the US) that monetary policy easings may have smaller effects on the real economy ( Barnichon and Matthes, 2017; Tenreyro and Thwaites, 2016 ). 6. Decomposing various policy news The normalization we used imply that a reading of any factor above unity means that factor was larger than its average absolute reading in- sample. A few notes on this exercise are in order. First of all, we show that it is possible to map our understanding of identiﬁed policy surprises based on the analysis of Governing Council policy announcements into any other kind of policy news. This is of independent interest. Secondly, the Target factor is also estimated here. One can of course treat these news as analogous to press conferences and limit the possible factors to Timing, Forward Guidance, and QE but to the extent that market participants update their beliefs about outcomes of policy meetings within a month, one may measure a reaction interpretable as Target. Lastly, this is a good place to discuss methodological choices. Our methodology is based only on the changes in safe rates of different maturities: the factors, including QE, do not load on individual country yields or spreads. Thus, the QE surprise we identify is one that lowers the long-term euro area safe rate and we can show, as a ﬁnding, that this QE surprise also lowers spreads. One should think of this as a “macroeconomic easing QE.” An alternative is QE that is perceived to particularly affect the Italian and Spanish yields, as in Rogers et al. (2014) . Measuring this requires having spreads in the matrix from which one extracts the factors; we chose not to follow this path. For example, as spreads narrowed sharply around the “whatever it takes” speech, this second type of QE factor would have signaled a very large QE easing surprise by deﬁnition. 14 Using our methodology, we are able to show that the macroeconomic easing QE factor that we identify also narrows spreads but we are silent about QE that may be perceived to differentially affect sovereign yields rather than providing overall stimulus. Clearly it is possible to measure a second type of QE and indeed to measure the two simultaneously, by allowing for two different types of QE surprises. Our research questions are best answered by the rotated factors we identiﬁed, but work on speeches of the “whatever it takes” type, or on the particular experiences of periphery countries may require alternative surprise identiﬁcations and associated factor rotations. 8. Conclusions Our aims in writing this paper were twofold. First, we wanted to explain and make available what we expect to be the standard euro area monetary policy event-study database, the EA-MPD. The paper discusses the construction of this data set. This is data we carefully analyzed and cleaned so that misquotes that were prevalent in the early period of ECB policymaking do not lead to spurious ﬁndings. The data will be continuously updated and made available online. Our second aim was to show the use of this data in constructing euro area monetary policy surprises, to decompose these into surprises in policy action, policy path, and QE news. The two-stage nature of ECB policy news dissemination turns out to be very helpful in both increasing the number of data points and making statistical analysis more precise, and also in providing a validity check for our surprise measures. Comfortingly, we ﬁnd that the Target surprises were dominant in the announcement window but that in the conference window this factor does not even exist, with news about future path of policy the main driver of yield changes before QE, and that QE news were present in the latter part of our sample, only in the conference window. These all dovetail with C. Altavilla, L. Brugnolini and R.S. Gürkaynak et al. / Journal of Monetary Economics 108 (2019) 162–179 179 the general understanding of how the ECB policy communication is designed to operate. We show that in practice policy communication has indeed worked as designed. We studied the response of various asset prices to these surprises and learned that surprises in the immediate setting of monetary policy, Target surprises, have effects only on the short end of the yield curve while Timing, Forward Guidance, and QE surprises all affect longer-term yields but in different ways. the general understanding of how the ECB policy communication is designed to operate. We show that in practice policy communication has indeed worked as designed. We studied the response of various asset prices to these surprises and learned that surprises in the immediate setting of monetary policy, Target surprises, have effects only on the short end of the yield curve while Timing, Forward Guidance, and QE surprises all affect longer-term yields but in different ways. Supplementary material Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jmoneco.2019. 08.016 . 8. Conclusions It is important to emphasize again that rather than assuming the presence of predeﬁned surprises in different windows, we estimated and identiﬁed these, ﬁnding a multifaceted information structure in the press conference window. We further learned that sovereign yields and exchange rates respond to ECB policy communication in interpretable ways, based on the source of the surprise. Importantly, QE turns out to have lowered all yields and narrowed spreads. Also importantly, we show that this effect was long-lived, with a half life of about a year. This is much longer than what was found earlier in the literature, when QE surprises were not quantiﬁed, and effects were based only on the dates of QE announcements. Among other extensions, we showed that measuring the information surprise in ECB policy employing stock prices or inﬂation compensation yield similar results and this decomposition is needed to understand stock price reactions to mone- tary policy. We also showed how to use the identiﬁed policy surprise factors to analyze any policy communication, not only Governing Council policy releases and statements on meeting dates. We hope that the event-study dataset that we have compiled and will regularly update will foster more research on monetary policy and its effects in the euro area. Questions about the effects of monetary policy on markets in different countries, how these differ by the fundamentals of those countries, using the surprises to help identify VAR-based real effects in the Gertler and Karadi (2015) fashion, and the transmission of ECB policies to non-euro area countries are some of the questions that continue to be important for academics and policymakers alike. While we shed more light than before on measuring and assessing monetary policy in the euro area, there certainly is much to be done. References , Mann, S. , 2018. 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W4254604622.txt	https://zenodo.org/record/2507199/files/article.pdf	fr		Relations officielles entre la Croix-Rouge et l'Armée	Bulletin international des sociétés de la Croix-Rouge	1,912	public-domain	3,276	— 142 — y aura a la Havane une assemblee supreme et un comite executif. La premiere sera composee d'un president effectif, qui sera en meme temps le delegue officiel, nomme par le gouvernement, de deux vice-presidents, d'un secretaire-general, d'un vice-secretaire, d'un tresorier, d'un vice-tresorier, d'un comptable, d'un vice-comptable, d'un directeur en chef, de huit membres et d'un inspecteur general. Le comite executif se compose du president, des vice-presidents, du secretaire-general, du tresorier, du comptable et du directeur en chef. Le comite executif repartit lui-meme les charges entre ses membres. Le reglement general organique determinera les devoirs et les droits correspondant a chacune de ces charges. Le droit de faire des paiements appartient au president de l'assemblee supreme et du comite executif, lequel pourra deleguer ce pouvoir au premier vice-president Le decret gouvernemental a officielle du 14 octobre 1911. ete publie dans la Gazette Relations officielles entre la Croix-Rouge et I'Armee Ces rapports sont deflnis par le decret IF 85 du 5 fevrier 1910, sur les relations de la Croix-Rouge ayec le corps de sante militaire de 1'armee cubaine. En voici la teneur : ARTICLE PREMIER. — La Societe national© cubaine de la CroixRouge, etablie dans la Republique de Cuba sous les auspices des conventions internationales de Geneve de 1864 et de 1906, est un auxiliaire du corps sanitaire militaire et de la fiotte, en temps de guerre, conformement a ce qui est prevu dans son reglement general organique, approuve par le decret n° 406 du 10 mai 1909 et par le Comit6 international de la Croix-Rouge le 7 septembre de meme annee ; elle est, par suite, un corps volontaire de la reserve des forces armees pour tout ce qui touche au but humanitaire qu'elle poursuit. ART. 2. — Parmi les principaux elements de secours que la CroixRouge pourra preter a 1'armee en campagne, il y a lieu de citer : 1° La creation d'hopitaux auxiliaires on complementaires de ceux etablis par le corps de sante militaire. 2° Le transport de malades ou de blesses par trains-hopitaux, par ambulances hippomobiles ou automobiles ou tirees par quelque moyen que ce soit, approprie aux necessites ou aux circonstances, chaque fois que le corps de sante militaire lui fera connaitre qu'il ne dispose pas des elements necessaires a ces services. — 143 — 3° L'etablissement de postes de seoours ou d'ambulances a postes fixes. ART. 3. — Tout auxiliaire volontaire, en provenance de n'importe quelle corporation, institution ou association, doit etre presente et associe aux forces armees par Fintermediaire de la seule Societe nationale cubaine de la Croix-Rouge. ART. 4. — Le president representera la Croix-Rouge cubaine dans ses rapports avec le Secretaire du gouvernement ; les autorites militaires pourront inspecter le service des chefs d'ambulanoes, d'hopitaux, etc., installes par la Croix-Rouge et le corps sanitaire militaire pourra veiller a ce que le combattant soit secouru de la meilleure maniere. ART. 5. — Le delegue du secretariat du gouvernement aupres du Comite de la Croix-Rouge cubaine sera un membre du corps de sante militaire. ART. 6. — Aupres de chaque caserne, ou quartier general, il y aura un delegue de la Croix-Rouge cubaine representant cette Societe. ART. 7. — Ce delegue, apres autorisation du general en chef des forces armees ou du general en chef des troupes en campagne, s'entendra avec le chef du corps sanitaire dependant de ce dernier, pour l'etablissement d'hopitaux et de postes de secours. ART. 8. -— Les autorites militaires, en leur qualite d'inspecteurs naturels de toutes les sections de la Croix-Rouge, etablies sur leur terrain, veilleront au bon fonctionnement de Finstitution, soit ellesmemes, soit en deleguant a cette inspection un chef quelconque des forces armees et du corps de sante militaire. ART. 9. — Tout service ou installation de la Croix-Rouge cubaine devra porter son embleme. ART. 10. — Le personnel de la Croix-Rouge cubaine, une fois accepte par Fautorite militaire, est soumis aux lois et reglements militaires. En temps de guerre ou en manoeuvres, si les services du personnel ou du materiel de la Societe deviennent necessaires, Fautorite militaire en fait la demande au president de l'Association. ART. 11. — Pour porter secours en qualite de medecin pratiquant, pour faire une operation aux soldats appartenant aux forces armees, il est necessaire de posseder le diplome de docteur ou de licencie en medecine, delivre par, l'universite nationale ou par une universite etrangere, a la condition que ce dernier ait ete rendu valable legalement. ART. 12. -— La Croix-Rouge cubaine est responsable du personnel qui figure sur les listes qu'elle soumet aux autorites militaires. ART. 13. — Elle veillera sous son entiere responsabilit^, a ce que les elements sanitaires, qu'elle s'est engagee a fournir a des endroits — 144 — determines, ne manquent jamais a seS'delegues ou representants, a moins de cas de force majeure, qui devront etre immediatement signales au general en chef. A cet effet, toute installation nouvelle devra faire Fobjet d'un rapport detaille, mentionnant les ressources dont elle dispose et signalant a l'autorite militaire superieure les elements qui lui manquent, afin qu'en aucun cas, les malades ou les blesses qui y seraient envoyes ne manquent des ressources sur lesquelles croyaient pouyoir compter les autorites militaires ou le chef du corps de sante de l'armee. Le rapport devra faire mention du personnel, du materiel de logement et de transport, aussi bien que des medicaments, des instruments de chirurgie, des bandages et des vivres de chaque installation. Les augmentations ou diminutions seront signalees tous les dix jours, ou plus souvent si possible. La Societe sera responsable du manque de services offerts par elle et acceptes par les forces armees. ABT. 14. — Les generaux des forces armees et les chefs commandant des colonnes etabliront des certiflcats relatifs aux merites du personnel de la Croix-Rouge ; de meme, le Comite executif de Vassemblee supreme de cette Societe octroiera aux membres de 1'armee, selon la hierarchie, les distinctions honoriflques qu'elle possede, lorsqu'ils s'en seront rendus dignes. ABT. 15. — Les chefs de colonnes pourront accorder des rations de vivres et de fourrage au personnel et au betail affecte au service de la Croix-Rouge cubaine, a la charge de l'institution quand son etat de prosperity le permettra. ART. 16. — Les autorites militaires resoudront toutes les questions relatives a l'installation d'infirmeries, de postes sanitaires ou d'hopitaux pour l'assistance aux blesses ou malades a la charge de la Societe et toujours derriere les lignes de combattants. ART. 17. — L'action de la Societe ne pourra etre etendue au service d'avant-garde ni aux hopitaux d'evacuation, a moins de circonstances exceptionnelles qu'appreciera le general en chef ou ceki* a qui incombe la direction du combat. ABT. 18. — Pour maintenir les etablissements precites dans la zone susmentidnnee, leur transfert d'un lieu a un autre devra se faire conformement aux mouvements des troupes. ART. 19. — Dans des circonstances extraordinaires, on pourra utiliser les services de la Croix-Rouge cubaine, surtout lorsqu'il s'agit d'evenements prolonges, ou de marches rapides, qui forcent les chefs a separer le gros des troupes des hopitaux ou des ambulances ; la Croix-Rouge remplacera alors les ambulances et on lui confiera les malades qui ne peuvent suivre leur corps. ART. 20. — Pour ce qui concerhe lai maniere d'effectuer les changements de residence, les haltes a faire et les endroits designes — 145 — pour le repos du personnel et du materiel de la Croix-Rouge oubaine, ainsi que pour ce qui a trait a la presence de eelle-ci sur le champ de bataille, apres le combat, pour seconder le corps de sante militaire, on appliquera les instructions du general en chef de l'armee en campagne et les prescriptions du reglement general organique deja cite. ART. 21. — La Croix-Rouge cubaine fournira un materiel de logement, de transport et un materiel chirurgical identique a celui de l'armee ; s'il en differait, il devrait, avant tout usage, etre declare utilisable par les chefs du corps sanitaire des divers quartiers generaux de l'armee. AKT. 22. — Les depots de materiel sanitaire pour les besoins de la Croix-Rouge cubaine sur le territoire ou se deroulent les operations de guerre seront autorises, moyennant une demande prealable de la Societe. ART. 23. — La Societe adressera chaque semestre, en temps de paix, aux chefs des quartiers generaux, un memoire detaille des ressources dont elle dispose en personnel et en materiel. ART. 24. — Au moment d'entrer en campagne, quel que soit le temps ecoule depuis le moment ou elle a fourni le precedent memoire, elle adressera un rapport aussi detaille que possible sur sa situation au double point de vue, personnel et materiel. ART. 25. — Avec l'autorisation du general en chef ou de celui qui dirige le combat, les membres de la Croix-Rouge cubaine pourront identifier les morts. ART. 26. — Avant de repartir des dons ou des secours aux blesses et aux malades de l'armec, le delegue de la Croix-Rouge cubaine. sollicitera l'autorisation de l'autorite militaire ; sans cette permission, il ne pourra commencer la distribution des dons ou secours. ART. 27. — Aucun membre de la Societe nationale cubaine de la Croix-Rouge ne pourra prendre Pinitiative d'accorder un conge, une permission a des soldats blesses ou malades ou de reformer ces derniers ; 1'intervention des societaires est limitee aux soins medicaux a donner a ces soldats. ART. 28. — En cas de desertion d'un soldat recueilli dans un hopital ou une ambulance, le medecin directeur en avisera immediatement l'autorite militaire la plus rapprochee. Hopitaux auxiliaires ART. 29. — On n'autorisera pas l'ouverture d'un hopital de la Croix-Rouge cubaine sans s'etre assure qu'il possede tous les elements necessaires a l'assistance, au traitement et a Palimentation des blesses ou malades militaires. ART. 30. — On veillera a ce que l'installation de ces hopitaux — 146 — se fasse dans des edifices reunissant les meilleurs conditions d'hygiene, dans des localites pourvues du plus grand nombre d'elements d'assistance, sous tous leurs aspects, et desservies par les voies de communications les plus appropriees et les plus rapides par rapport a i'armee et au theatre des operations. ART. 31. — Les hopitaux de campagne a la charge de la OroixBouge, aux conditions anterieurement indiquees, ne devront pas avoir, en moyenne, plus de 100 lits, repartis au nombre de 25 par salle de malades ou de blesses ; le corps de sante militaire devra pouvoir ainsi veiller a l'assistance donn^e a ces derniers avec les facilites que lui concedent les articles 4 et 8. ABT. 32. — Le personnel de la Societe sera nomine suivant la forme reglementaire. ART. 33. — La 8oci6te veillera a ce qu'il y ait dans les hopitaux au moins un medecin par salle ou section, avec le personnel subalterne n^cessaire ; il devra y avoir egalement un medecin de garde pour faire face aux exigences du service. ART. 34. — Les menus seront, a moins de force majeure, conformes a ceux des hopitaux militaires. Les incidents qui pourraient surgir a ce propos et au sujet des prescriptions medicaies seront consignes dans un rapport quotidien, signe pnr le chef de clinique et qui pourra etre examine par le corps de sante militaire. ART. 35. — Le prix de sejour des individus hospitalises dans ces etahlissements ne pourra exceder, en cas de paiemeiit par l'Etat, le prix fixe pour des sejours de ce genre dans les >hopitaux militaires. ART. 36. — S'il surgissait une maladie contagieuso ou infectieuse, ou une epidemie, on devra en rondre compte immediatement a l'autorite militaire et au chef du corps de sante de I'armee. ART. 37. — Le rapport quotidien contenant le chiffre des deces, etc... sera egalement identique a celui des hopitaux de I'armee ; il devra faire preuve de la plus grande exactitude dans la partie relative aux morts. ART. 38. — Dans des cas urgents, resultant des multiples incidents d'une campagne ou les blesses ou malades entreraient a 1'ambulance avec leur equipement et leurs armes, cet ^quipement serait place dans des magasins convenables, de facon a ne pas se deteriorer et a ne pas etre vole ; il en sera donne une description detaillee a l'autorite militaire de la place ou du camp le plus rapproche, et au delegue de la Croix-Rouge cubaine aupres du general en chef, afin que celui-ci en ait connaissance. On agira de meme pour les valeurs trouvees en la possession des malades ou blesses ; ces valeurs entreront dans la caisse de l'etablissement ; on en donnera un recu aux interesses et on les leur rendra le jour de leur sortie. En cas de d^ces, les valeurs seront — 147 — remises au corps auquel appartenait le soldat decode. S'il existe dans la locality un pare d'artillerie ou s'il s'y trouve des troupes, les munitions et les armes du soldat mort seront imm^diatement remises au chef de ces forces. ART. 39. — Outre qu'elle devra s'en tenir, ainsi que cela a deja 6te dit, pour ses rapports journaliers, aux modeles en vigueur dans les hopitaux militaires, la Croix-Rouge cubaine devra posseder dans ses bureaux les reglements du service sanitaire en campagne, ceux des hopitaux militaires et tous ceux concernant le service du corps de sante de l'armee, pour pouvoir approprier ses reglements a ceux precites, dans la mesure du possible, et pour pouvoir trancher toutes les questions qui provoqueraient une hesitation de sa part. Service de la Croix-Rouge cubaine, sur les voies ferrees, fluviales et maritimes ART. 40. — Le transport des blesses ou malades, derriere les lignes de troupes, peut etre confie a la Croix-Rouge cubaine, quand le corps sanitaire militaire le juge opportun. AET. 41. — Le general en chef determine la composition des equipes accompagnant les trains-hopitaux et le reste des services incombant a la Croix-Rouge cubaine. ART. 42. — Ces trains ne porteront pas de signe special (a part ceux prevus par les reglements de chemins de fer), autre que l'embleme de la Croix-Rouge cubaine. ART. 43. — Chaque fois que ce sera possible, un chef ou un ofneier du corps de sante militaire, ou tout au moins un officier de l'armee, accompagnera ces trains ; il sera charge de veiller au transport des blesses ou malades et de leur temoigner la sollicitude a laquelle ils ont droit. ART. 44. — Lorsqu'on organisera des trains-hopitaux a la charge de la Croix-Rouge cubaine, il y aura lieu d'etablir un rapport complet et d'une exactitude scrupuleuse. Ce rapport contiendra une liste detaillee des soldats transported et fera mention de tous les incidents survenus au cours du voyage. II sera etabli en triple expedition ; l'un des exemplaires sera conserve par le medecin, chef du convoi, qui en remettra un second au medecin du corps de sante militaire de la localite de depart; celui-ci signera Pexemplaire qui reste entre les mains du medecin chef de convoi, qui, a son tour, signera la prise en charge des blesses ou malades sur l'exemplaire qu'il laisse au chef du corps sanitaire ou a l'autorite militaire, et a defaut de ceux-ci, au maire ou a l'autorite civile de la localite ou a lieu le depart. ART. 45. — Le m^decin chef de convoi refusera tout individu — 148 — expose par la gravite de son etat a mourir en oours de route, ou a etre dangereux pour les autres. Si, dans des circonstances extraordinaires, il etait oblige de l'admettre, il ferait constater le cas dans le rapport (journal de train) pour sauver sa responsabilite professionnelle. ART. 46. — La Croix-Rouge nationale cubaine pourra etablir des postes de seeours, convenablement dotes, dans les gares de chemins de fer situees pres du theatre des operations, dans les ports de debarquement habituel des blesses ou malades arrives par les voies maritimes, fluviales ou ferrees et, en general, dans les localites ou le passage des troupes en temps de guerre est probable. Ces postes de seeours devront etre pourvus des elements sanitaires necessaires. Us devront en outre : 1° Envoyer a l'hopital le plus rapproche les malades ou blesses qui ne peuvent continuer le voyage. 2° Secourir ceux qui sont gravement atteints et les conduire ensuite a l'hopital. 3° Donner des medicarnents et des soins a ceux qui souffrent de blessures legeres pour leur permettre de poursuivre leur route. 4° Provoquer la repartition des malades ou blesses entre les hopitaux locaux et faciliter leur transport a ces hopitaux. 5° Porter seeours immediatement au blesse qui le reclame. Le materiel affecte a ces postes de seeours sera celui fixe par le reglement et sera soumis a l'inspection de l'autorite militaire et au chef du corps de sante de l'armee. II devra y avoir dans chaque poste de seeours un medecin aide du personnel subalterne ad hoc. ART. 47. — La Croix-Rouge pourra etre autorisee a utiliser, pour le transport fluvial et cotier des blesses et malades, des bateauxhopitaux soumis aux memes reglements que ceux edictes pour les trains. Dans le second cas (e'est-a-dire pour ce qui concerne les transports le long des cotes), il y aura lieu de faire etat des dispositions prises par la marine nationale. Services de la Croix-Rouge dans les villes en etat de siege, pendant les emeutes, etc. ART. 48. Quand 1'ordre public est trouble, en temps de paix, la Societe pourra installer des postes de seeours ou des hopitaux et porter seeours aux blesses, sans s'interposer entre les combattants. ART. 49. — Ces postes de seoours et hopitaux seront indiques a l'autorite militaire qui saura ainsi ou ils se trouvent et pourra les approuver ; elle devra en avoir connaissance, soit que les postes aient ete installes depuis que l'autorite militaire a pris le commandement de la ville, soit qu'ils aient existe anterieurement. ART. 50. — Dans les villes en etat de siege, la Societe, quand elle portera seeours a la troupe, se conformera au present reglement — 149 — sur les postes de secours et hopitaux. Elle pourra, dans ces etablissements, seconder Ie service de sante militaire, chaque fois que cela lui paraitra necessaire. Dispositions generates ART. 51. — Les postes de secours, hopitaux et autres services de la Croix-Rouge cesseront de fonctionner quand le corps de sante militaire lui aura fait connaitre qu'il dispose de tout le materiel et personnel necessaires pour l'assistance aux blesses et malades militaires. ART. 52. — La Societe ne pourra, en aueun cas, retirer son personnel ou son materiel des endroits ou elle a ete autoris^e a les mettre par le chef des forces armees, sans le consentement de ce dernier. ART. 53. — Les prisonniers faits a l'ennemi seront l'objet des memes soins que les nationaux, mais toujours avec l'autorisation de l'autorite militaire, qui prendra les mesures utiles pour leur garde et leur s^cu- rite. ART. 54. — Les societaires de la Croix-Rouge pourront recevoir a leur domicile particulier, des blesses ou malades, moyennant que l'autorite militaire les y autorise et qu'il s'agisse de convalescents ou de soldats grievement blesses ou assez malades pour etre empeches pendant longtemps de retourner sur le theatre des operations. ART. 55. — Le present reglement entrera en vigueur a la date de sa publication dans le Journal officiel et aura son effet a partir du moment ou la Societe nationale cubaine de la Croix-Rouge entrera en fonctions conjointement avec les forces armees de la nation. ART. 56. — Afin qu'il regne toujours la plus parfaite harmonie entre les membres de la Croix-Rouge cubaine et ceux des forces armees, et la plus complete discipline, il est etabli qu'ils respecteront mutuellement leur hierarchie. Le secretaire du Gouvernement est charge de l'execution du present d^cret qui entrera en application a la date de sa publication dans le Journal officiel 1. Le Secretaire, F. LOPEZ-LEIVA 1 Le president, JOSE GOMEZ Ce decret a ete publie dans la Gazeta oficial du 7 fevrier 1910. 11
https://openalex.org/W4366549261	https://zenodo.org/records/7854701/files/Herpetozoa_article_99985.pdf	English	null	First record of a male-male aggressive interaction in the golden Alpine salamander Salamandra atra aurorae (Caudata, Salamandridae)	Herpetozoa	2,023	cc-by	1,812	Herpetozoa 36: 91–93 (2023) DOI 10.3897/herpetozoa.36.e99985 First record of a male-male aggressive interaction in the golden Alpine salamander Salamandra atra aurorae (Caudata, Salamandridae) Milos Di Gregorio1, Raoul Manenti2, Danilo Borgatti2 Milos Di Gregorio1, Raoul Manenti2, Danilo Borgatti2 1 Dipartimento di Biologia, Università degli Studi di Pisa, Via Volta 4, 56127 Pisa, Italy 2 Dipartimento di Scienze e Politiche Ambientali, Università degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy 1 Dipartimento di Biologia, Università degli Studi di Pisa, Via Volta 4, 56127 Pisa, Italy 2 Dipartimento di Scienze e Politiche Ambientali, Università degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy 1 Dipartimento di Biologia, Università degli Studi di Pisa, Via Volta 4, 56127 Pisa, Italy 2 Dipartimento di Scienze e Politiche Ambientali, Università degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy Corresponding author: Milos Di Gregorio (milosdigre@gmail.com) Academic editor: Günter Gollmann ♦ Received 6 January 2023 ♦ Accepted 5 April 2023 ♦ Published 20 April 2023 Abstract The golden Alpine salamander Salamandra atra aurorae Trevisan, 1982 is an endemic subspecies found in Sette Comuni and Vezzena plateau in Veneto, Italy. We describe an aggressive interaction between two males which fought for four minutes, trying to go on top of each other and rubbing their chin on the antagonist’s head. This is the first documented case of aggressive behaviour in Salamandra atra aurorae. Copyright Milos Di Gregorio et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Key Words The survey started at 6.45 am, the temperature was 14 °C and the relative humidity was 76%, measured with a Walfront HT-86 Hu- midity meter (resolution of 0.01% rh, 0.01 °C). At 7.00 am we found two individuals fighting near a white fir. The two salamanders had a highly visible swollen cloaca, a distinctive male characteristic, and unique dorsal patterns that made them recognizable throughout the duration of the interaction. The first salamander, hereafter ‘sal1’, had a heavy golden colouration on the back, head and limbs; almost all of its dorsal pattern was gold. In contrast, the second one, hereafter ‘sal2’, had a golden colouration only in the central part of the back, the first part of the limbs and on the head.i without change of focus, adjustment of the camera and movements of the operator. At the beginning of the video (Fig. 1A) sal1 was on top of sal2 trying to rub its chin on the rival’s head. After a brief stop sal1 went on trying to grab the competitor’s fore- limbs and to rub its chin on the opponent’s head. During this time sal2 was moving, trying to escape from the rival. After a quick interruption, during which sal1 seemed to leave the opponent, sal1 turned around and resumed the attack towards sal2 by climbing on top of him and rubbing its chin on the head of sal2. Also this time sal2 was moving, and this was effective in making the oppo- nent fall, even if sal1 was able to quickly recover and get back on top. A few moments later sal2 managed to free itself from the hold of sal1, and sal2 was able to grasp the opponent’s forelimbs after getting on top of sal1 using both anterior and posterior legs (Fig. 1B).l The whole interaction was filmed using a Canon EOS 5D Mark III, mounting a 24–70 mm f/2.8 L USM lens. Immediately after spotting the two males, we started re- cording, capturing four videos of 47, 116, 66 and 5 sec- onds each for a total of little less than 4 minutes. The videos were cut and merged in order to create a single movie (accessible at https://youtu.be/sVpBYo-E_y0) After sal1 was able to escape briefly, sal2 continued its attack which was made easier by sal1 being completely still. Key Words Amphibians, behaviour, ecology, male-male interaction mandra (Di Nicola et al. 2022). Although also male-male interactions for S. atra atra are known (Di Nicola et al. 2022), no records for the highly threatened golden Alpine salamander Salamandra atra aurorae Trevisan, 1982 ex- ist so far. It is important to understand the behavioural patterns that allow amphibians to exploit their terrestrial habitats so we can plan proper management actions and enhance con- servation policies. Intraspecific interactions recorded on members of the genus Salamandra in terrestrial environ- ments show a relatively wide range of sexual and territo- rial behaviours, which, apart from mating, include hom- ing, site fidelity, displaying postures to protect territories and detect conspecifics (Werner et al. 2014; Manenti et al. 2017; Di Nicola et al. 2022). Salamandra atra aurorae is endemic to a narrow area ranging from Sette Comuni and Vezzena plateau (Ro- manazzi and Bonato 2014) in Veneto region (north-east Italy). It can be found mostly in mixed woods with prev- alence of beech and white fir, but there are populations in woods with prevalence of spruce and shrubbed meadow (Bonato and Fracasso 2003). If the environment is suitable the golden Alpine salamander can reach high abundances. For example, Bonato and Fracasso (2003) reported a den- sity up to 475 individuals/ha with a sex-ratio of 1:1 and home range of only a few square meters. In the present note we report the first documented case of a male-male aggressive interaction in the golden Alpine salamander. Several male-male interactions have been document- ed, but mostly in the grey literature. Although they are difficult to interpret because they could be linked to ter- ritoriality, mating competition, mistaken mating attempts or sex recognition (Guex and Grossenbacher 2004; Di Nicola et al. 2022), such observations are worth report- ing on as they increase the understanding of salamanders’ behavioural and ecological requirements. A recent review showed that male-male interactions have been reported for S. algira, S. lanzai and several subspecies of S. sala- The observation was made on 4 June 2022, in Bosco del Dosso, near Asiago (Province of Vicenza), the type Milos Di Gregorio et al.: Male aggressive behaviour in the golden Alpine Salamander 92 locality of the golden Alpine salamander (Bonato and Grossenbacher 2000), while making a survey focused on the colouration of S. a. aurorae. herpetozoa.pensoft.net Key Words Even if sal2 was not able to completely get on top of sal1, the head rubbing was very intense since sal2 was in a good position to firmly use its legs to sustain the weight. After some time sal1 escaped the grasp of sal2, which 1. Stages in the encounter of two male salamanders. A. Start of the interaction: sal1 climbing on top of sal2; B. Roles are : sal2 is now on top of sal1; C. The interaction has ended. re 1. Stages in the encounter of two male salamanders. A. Start of the interaction: sal1 climbing on top of sal2; B. Roles are Figure 1. Stages in the encounter of two male salamanders. A. Start of the interaction: sal1 climbing on top of sal2; B. Roles are inverted: sal2 is now on top of sal1; C. The interaction has ended. Herpetozoa 36: 91–93 (2023) 93 immediately stopped its chase: the two salamanders stayed near each other (30 cm apart) for about one minute (Fig. 1C), after which the two salamanders began to move in opposite directions. This led us to assume that the in- teraction was over. Only at this point sal2 was collected: it had a total length of 12.14 cm and weighed 11 g. The other individual was not collected, but had a similar size. updated review (Urodela: Salamandridae). Herpetozoa 13: 171–180. https://www.zobodat.at/pdf/HER_13_3_4_0171-0180.pdf Camp CD, Lee TP (1996) Intraspecific spacing and interaction within a population of Desmognatus quadramaculatus. Copeia 1996: 78–84. https://doi.org/10.2307/1446943 Davis TM (2002) An ethogram of intraspecific agonistic and display behavior for the wandering salamander, Aneides vegrans. Herpeto- logica 58: 371–382. https://doi.org/10.1655/0018-0831(2002)058[0 371 AEOIAA]2 0 CO 2 The sequence of behaviours displayed by the two males is similar to that reported in the other species of the genus Salamandra (Di Nicola et al. 2022) and in sev- eral North American salamanders, which, in addition to mounting attempts, forelimb grasping and snout-rubbing behaviours, also display more aggressive behaviours such as bite, bite-hold, and cannibalism as the ultimate aggressive response (Staub 1993; Camp and Lee 1996; Davis 2002; Deitloff et al. 2014). The biting behaviour was associated with Salamandra salamandra by Verrell (1989) but without mention of any occurrence. As of to- day, more research is needed to confirm this behaviour in the genus Salamandra. It can now be confirmed that aggressive male-male interactions are present in the golden Alpine salamander. Key Words Considering the rarity of in- formation about this behaviour in this species, and more generally in the genus Salamandra, it is important to address the factors that enhance the probability of male- male aggressive interactions, to assess the frequency of these interactions and their biological meaning in terms of advantages for mating and trophic resources access. g p g ( ) [ 371:AEOIAA]2.0.CO;2 Deitloff J, Alcorn MA, Graham SP (2014) Variation in mating systems of salamanders: Mate guarding or territoriality? Behavioural Pro- cesses 106: 111–117. https://doi.org/10.1016/j.beproc.2014.04.006 Di Nicola MR, Zabbia T, Mezzadri S, Cerullo A, Bruni G (2022) Male- male interactions in Alpine Salamanders, Salamandra atra atra (Laurenti, 1768), with an overview of the main cases reported for the genus Salamandra (Amphibia: Salamandridae). Herpetology Notes 15: 601–604. https://www.biotaxa.org/hn/article/view/72519 otes 15: 601–604. https://www.biotaxa.org/hn/article/view/72519 Guex GD, Grossenbacher K (2004) Salamandra atra Laurenti, 1768. Alpensalamander. In: Thiesmeier B, Grossenbacher K (Eds) Hand- buch der Reptilien und Amphibien Europas 4 (IIB): Schwanzlurche (Urodela). Wiesbaden, Germany, Aula-Verlag, 975–1028. (Urodela). Wiesbaden, Germany, Aula-Verlag, 975–1028. Manenti R, Conti A, Pennati R (2017) Fire salamander (Salamandra salamandra) males’ activity during breeding season: effects of mi- crohabitat features and body size. Acta Herpetologica 12: 29–36. https://doi.org/10.13128/Acta_Herpetol-18115 Romanazzi E, Bonato L (2014) Updating the range of the narrowly dis- tributed endemites Salamandra atra aurorae and S. atra pasubiensis. Amphibia-Reptilia 35: 123–128. https://doi.org/10.1163/15685381- 00002923 Staub NL (1993) Intraspecific agonistic behavior of the salamander Aneides flavipunctatus (Amphibia: Plethodontidae) with compar- isons to other plethodontid species. Herpetologica 49: 271–282. https://www.jstor.org/stable/3892804 herpetozoa.pensoft.net References Bonato L, Fracasso G (2003) Movements, distribution pattern and density in a population of Salamandra atra aurorae (Cauda- ta: Salamandridae). Amphibia-Reptilia 24: 251–260. https://doi. org/10.1163/156853803322440736 Verrell PA (1989) The sexual strategies of natural populations of newts and salamanders. Herpetologica 45: 265–282. https://www.jstor.org/ stable/3892882 Bonato L, Grossenbacher K (2000) On the distribution and chromatic differentiation of the Alpine Salamander Salamandra atra Laurenti, 1768, between Val Lagarina and Val Sugana (Venetian Prealps): an Werner P, Lötters S, Schmidt BR (2014) Analysis of habitat determi- nants in contact zones of parapatric European salamanders. Journal of Zoology 292: 31–38. https://doi.org/10.1111/jzo.12079 Werner P, Lötters S, Schmidt BR (2014) Analysis of habitat determi- nants in contact zones of parapatric European salamanders. Journal of Zoology 292: 31–38. https://doi.org/10.1111/jzo.12079 herpetozoa.pensoft.net
https://openalex.org/W4388205309	https://journal-center.litpam.com/index.php/linov/article/download/1084/778	Indonesian	null	Pelatihan Pengajaran Bahasa Inggris dengan Tehnik Storytelling dalam Kelompok Kecil bagi Siswa SMPN 2 Alalak	Lumbung Inovasi	2,023	cc-by	4,651	Abstrak Kegiatan pengabdian kepada masyarakat yang berjudul Pelatihan Pembelajaran Bahasa Inggris dengan Tehnik Storytelling dalam Kelompok Kecil di SMPN 2 Alalak Kabupaten Barito Kuala ini dilaksanakan sebagai salah satu bentuk implementasi dari Tridharma Perguruan Tinggi yang sesuai dengan Visi dan Misi Universitas Lambung Mangkurat yang relevan dengan perkembangan ipteks dan berfokus pada program unggulan lingkungan lahan basah. Pengenalan Bahasa Inggris dengan baik dan benar kepada anak-anak pada usia sekolah tentunya akan memberikan kesan baik dan manfaat untuk mereka kedepannya untuk lebih termotivasi belajar Bahasa Inggris. Pengabdian ini bertujuan untuk memberikan contoh secara faktual dan akurat tentang mengajarkan Bahasa Inggris untuk meningkatkan kemampuan berbicara anak-anak dengan menggunakan cerita anak-anak berbahasa Inggris, yang meliputi proses persiapan dan pelaksanaan. Selain itu, pengabdian ini akan menjabarkan tentang bagaimana mengajarkan bahasa Inggris yang menyenangkan untuk menghindari kejenuhan dan rasa bosan atau bahkan trauma terhadap bahasa Inggris. Hasil pengabdian ini memfasilitasi para guru untuk memberikan pengajaran yang efektif dan bermakna melalui cerita anak-anak berbahasa Inggris yang dilakukan dalam kelompok-kelompok kecil. hasil kegiatan menunjukkan 80% dari siswa menunjukkan respon positif dengan memperlihatkan keaktifan dalam pembelajaran, serta 70% dari mereka juga mampu mengingat dan mengucapkan vocabulary dari cerita yang telah diajarkan. Sehingga dapat disimpulkan bahwa tehnik mengajar dengan menggunakan storytelling dan dilakukan dalam kelompok kecil membawa manfaat dan hasil yang lebih baik. Kata kunci: Storytelling, Bahasa Inggris, Cerita Anak-Anak, Kelompok Kecil, Siswa SMP How to Cite: Febriyanti, E. R., Listia, R., & Chandra, N. E. (2023). Pelatihan Pengajaran Bahasa Inggris dengan Tehnik Storytelling dalam Kelompok Kecil bagi Siswa SMPN 2 Alalak. Lumbung Inovasi: Jurnal Pengabdian Kepada Masyarakat, 8(1), 70–78. https://doi.org/10.36312/linov.v8i1.1084 Pelatihan Pengajaran Bahasa Inggris dengan Tehnik Storytelling dalam Kelompok Kecil bagi Siswa SMPN 2 Alalak Emma Rosana Febriyanti, Rina Listia, Noor Eka Chandra English Department, Faculty of Teacher Training and Education, Universitas Lambung Mangkurat. Jl. Brigjen H. Hasan Basri, Pangeran, Kec. Banjarmasin Utara, Indonesia. Postal code: 70123 Corresponding Author e-mail: emma.rosana@ulm.ac.id Postal code: 70123 Corresponding Author e-mail: emma.rosana@ulm.ac.id Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat https://journal-center.litpam.com/index.php/linov Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat https://journal-center.litpam.com/index.php/linov Maret 2023 Vol. 8, No. 1 e-ISSN: 2541-626X pp.70-78 Pelatihan Pengajaran Bahasa Inggris dengan Tehnik Storytelling dalam Kelompok Kecil bagi Siswa SMPN 2 Alalak Emma Rosana Febriyanti, Rina Listia, Noor Eka Chandra English Department, Faculty of Teacher Training and Education, Universitas Lambung Mangkurat. Jl. Brigjen H. Hasan Basri, Pangeran, Kec. Banjarmasin Utara, Indonesia. Postal code: 70123 *Corresponding Author e-mail: emma.rosana@ulm.ac.id ata kunci: Storytelling, Bahasa Inggris, Cerita Anak-Anak, Kelompok Kecil, Siswa SMP PENDAHULUAN Bahasa, baik secara lisan maupun tertulis, adalah alat komunikasi yang memegang peranan yang sangat penting bagi umat manusia untuk bersosialisasi, berkomunikasi, dan saling berhubungan dengan manusia yang lainnya dan dengan lingkungannya. Hal ini adalah karena manusia adalah makhluk sosial yang tidak dapat terlepas dengan manusia yang lainnya. Dengan saling menyampaikan pikiran, saling mengemukakan tentang pengalaman, ataupun saling memberikan atau menerima informasi dari orang lain yang hanya dapat dilakukan dengan memakai bahasa; terlepas dari bahasa apapun yang dikuasai oleh orang tersebut. Selain itu, hal lain yang dapat dilakukan oleh manusia adalah saling bertukar cerita, yaitu dengan tujuan memberikan informasi kepada orang lain dengan suasana yang santai dan lebih ringan. g Bercerita adalah penyampaian pesan atau cerita secara naratif, berdasarkan urutan pada kejadian tertentu ataupun kisah hidup seseorang yang dilakukan secara lisan. Dengan bercerita seseorang akan dapat mengutarakan berbagai pengalaman atau pengetahuan yang pernah dijumpainya, dilihat, dialami, serta informasi yang dimiliki dan kehidupan pernah dijalaninya kepada orang lain (Tarigan, 2013 dalam Nurharyadi, 2018). Akan tetapi, kemampuan bercerita ini belum tentu dapat dimiliki oleh setiap orang, karena ini menyangkut tentang berbagi perasaan, kemauan serta keinginan untuk berbagi tentang pengalaman yang diperolehnya kepada orang lain. Sehingga kemampuan bercerita ini perlu dibangun dan dikembangkan sejak dini untuk menghasilkan individu yang percaya diri dan dapat berfungsi sosial dimasyarakat nantinya. Pada jenjang pendidikan, bahasa tidak hanya dapat dimanfaatkan untuk penyampaian informasi dari guru ke muridnya, akan tetapi juga untuk pemerolehan ilmu pengetahuan dan interaksi dari guru ke murid ataupun sebaliknya. Mengembangkan keterampilan berbahasa Inggris adalah salah satu keterampilan yang perlu dikuasai anak-anak. (Sari, 2012) menunjukkan bahwa peran guru sangat penting dalam memberikan kesempatan kepada peserta didiknya untuk menggunakan bahasa terutama bahasa Inggris baik secara individu maupun di dalam kelompok. Guru juga harus kreatif dalam menyediakan sumber belajar dan perlu memperhatikan perkembangan peserta didiknya ketika menggunakan bahasa Inggris sebagai alat komunikasi verbal. Untuk alasan ini, guru perlu memilih dari berbagai kegiatan yang akan dinikmati anak-anak untuk memaksimalkan kegiatan berbicara mereka dalam Bahasa Inggris. gg Pembelajaran Bahasa Inggris selalu dikaitkan dengan kemampuan membaca, mendengarkan, berbicara, dan menulis. Dalam hal ini, kemampuan bercerita terutama dalam Bahasa Inggris berkaitan dengan kemampuan berbicara (speaking skill). Kemampuan berbicara dalam Bahasa Inggris termasuk kemampuan yang dianggap sulit oleh siswa karena selain Bahasa Inggris adalah bukan Bahasa ibu mereka, hal ini juga terkait rendahnya rasa percaya diri mereka dan khawatir akan ditertawakan oleh teman-temannya. Training on Teaching English with Storytelling Technique in Small Groups for Students at SMPN 2 Alalak Abstract This community service program entitled Learning English through Storytelling Technique in Small Groups at SMPN 2 Alalak, Barito Kuala Regency was carried out as a form of implementation of the Tridharma of Higher Education in accordance with the Vision and Mission of Lambung Mangkurat University. It was relevant to the development of science and technology that focused on the flagship program of the wetland environment. The proper and correct introduction of English to children at school will certainly give a good impression and benefit them in the future so they are more motivated to learn English. This program aims to provide factual and accurate examples of teaching English to improve children's speaking skills by using English children's stories, which include preparation and implementation processes. In addition, the program explains how to teach English in a fun way to avoid boredom or even trauma to English. It is hoped that the results of this program can facilitate teachers to provide effective and meaningful teaching through children's stories in English. The results of showed that 80% of students gave positive responses by showing activeness in learning, and 70% of them were also able to pronounce and remember vocabulary from the stories that had been taught. Therefore, it can be concluded that teaching technique using storytelling and carried out in small groups bring better benefits and results. Keywords: storytelling, English, children’s stories, small groups, Junior High School Students How to Cite: Febriyanti, E. R., Listia, R., & Chandra, N. E. (2023). Pelatihan Pengajaran Bahasa Inggris dengan Tehnik Storytelling dalam Kelompok Kecil bagi Siswa SMPN 2 Alalak. Lumbung Inovasi: Jurnal Pengabdian Kepada Masyarakat, 8(1), 70–78. https://doi.org/10.36312/linov.v8i1.1084 \| \|70 Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 Pelatihan Pengajaran Bahasa Inggris… Febriyanti et al https://doi.org/10.36312/linov.v8i1.1084 Copyright© 2023, Febriyanti et al This is an open-access article under the CC-BY-SA License. Copyright© 2023, Febriyanti et al article under the CC-BY-SA License. https://doi.org/10.36312/linov.v8i1.1084 PENDAHULUAN Hal ini menjadi hal yang sangat umum terjadi di kelas-kelas Bahasa Inggris di seluruh Indonesia, yaitu dimana siswa hanya menjadi pendengar dan menuruti apa yang diperintahkan oleh gurunya. Selain faktor-faktor yang telah disebutkan sebelumnya, kemampuan berbicara siswa dalam Bahasa Inggris juga dikarenakan kurangnya penguasaan kosakata (vocabulary), takut salah \| \|71 Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 Pelatihan Pengajaran Bahasa Inggris… Febriyanti et al dalam mengucapkan kata (pronunciation), serta tidak memiliki ide tentang topik apa yang akan dibicarakan (Rohyeni, 2021)Sehingga, ketika mereka diminta untuk berbicara dalam Bahasa Inggris, mereka takut dan lebih memilih diam yang membuat kemampuan berbicara mereka menjadi semakin tidak terlatih. Lebih khusus, ada beberapa permasalahan yang dihadapi oleh sekolah mitra dalam melatih kemampuan berbicara siswa dan membangun kepercayaan diri mereka, antara lain adalah pembelajaran Bahasa Inggris dilaksanakan dengan satu orang guru yang menangani 30 orang siswa ataupun lebih dalam satu kelas sehingga tidak memungkinkan setiap individu siswa mendapat perhatian penuh dari guru tersebut. Kemudian, hal itu ditambah lagi dengan kurangnya rasa percaya diri siswa dalam menggunakan Bahasa Inggris di kelas, sehingga mereka lebih sering menggunakan Bahasa Indonesia dalam interaksi pembelajaran Bahasa Inggris. Sebagai tambahan, guru juga tidak menggunakan tehnik pembelajaran Bahasa Inggris yang bervariasi sehingga tidak memungkinkan terjadinya interaksi yang diharapkan ataupun munculnya aksi yang diminta akan terjadi. Salah satu kegiatan yang dapat dilakukan oleh guru untuk mengatasi rendahnya rasa percaya diri anak-anak didiknya dan melatih keberanian dalam berbicara adalah dengan melakukan berbagai kegiatan pembelajaran yang dapat meningkatkan rasa percaya diri mereka, yaitu salah satunya dengan menggunakan tehnik bercerita atau storytelling (Abasi & Soori, 2014). Selain itu, Abasi & Soori (2014) juga berhasil membuktikan bahwa dengan kegiatan bercerita, kosakata Bahasa Inggris anak-anak meningkat. Selain dengan tehnik tersebut, model pembelajaran yang berpusat pada siswa juga perlu dilaksanakan untuk membangun suasana yang santai dan memberikan kesempatan agar dapat terjadi interaksi antar siswa, yaitu dengan model berkelompok. Dengan model ini, diharapkan siswa akan lebih memiliki kepercayaan diri dan secara aktif membangun keberanian yang dimilikinya dengan bertukar pikiran bersama dengan teman dalam kelompok kecilnya dan guru mengenai hal-hal yang tidak dimengertinya. g y Adapun tujuan pelaksanaan pengabdian ini adalah untuk mengajarkan Bahasa Inggris kepada siswa SMPN 2 Alalak dengan tehnik storytelling melalui cerita berbahasa Inggris agar motivasi belajar mereka akan Bahasa Inggris pada umumnya jadi meningkat, dan dapat menambah kepercayaan diri mereka untuk berbicara dalam Bahasa Inggris pada khususnya yang dilakukan dalam kelompok kecil. PENDAHULUAN Selain itu, para guru diharapkan dapat menggunakan tehnik dan metode ini secara berkelanjutan untuk mendapatkan tidak hanya manfaat jangka pendek, akan tetapi juga manfaat jangka panjang untuk siswa mereka terutama dalam bidang Bahasa Inggris. Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 TEHNIK PELAKSANAAN Pelaksanaan kegiatan penerapan pengabdian ini dibagi dalam tiga tahap yaitu tahap persiapan, pelaksanaan dan evaluasi. Yang menjadi peserta dalam kegiatan pengabdian ini adalah 84 orang mahasiswa Program Studi Pendidikan Bahasa Inggris dan seluruh siswa SMPN 2 Alalak yang berjumlah 451 orang dari kelas 7 – 9. Pengabdian ini dilaksanakan dalam 6 pertemuan dari 7 Maret – 24 Maret 2022. Tahap yang pertama adalah persiapan yang dibagi dalam 2 tahap. Tahap persiapan 1 atau pertemuan pertama adalah pembagian kelompok mahasiswa yang akan menjadi instruktur dalam pelaksanaan pengabdian ini yaitu sebanyak 84 orang yang dibagi menjadi 14 kelompok yang masing-masing terdiri dari 6 orang. Kemudian, setiap kelompok memilih satu cerita yang akan digunakan dan harus memilih judul yang berbeda dengan kelompok lainnya. Pemilihan cerita ini harus disesuaikan dengan tema dan waktu yang disediakan oleh pihak sekolah. Cerita yang cukup \| \|72 Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 Pelatihan Pengajaran Bahasa Inggris… Febriyanti et al panjang akan disederhanakan sedemikian rupa sampai kira-kira 7-10 menit untuk penyampaian cerita. Cerita yang disampaikan juga harus dapat menambah perbendaharaan kata atau vocabulary siswa dan memiliki pesan moral yang tentunya dapat diambil hikmahnya. Adapun judul cerita yang dipilih oleh setiap kelompok adalah sebagai berikut: Group 1: Red Riding Hood Group 6: I Found a Frog Group 7: The Pied Piper of Hamelin Group 8: The Umbrella Group 9: Magical Moonlight Group 10: The Ramadan Lantern Story Group 11: Snow White and the Seven Dwarfs Group 12: Thumbelina Group 13: The Old, Rough Stone and the Gnarled Tree p g Group 14: An Adventure with the Water-Snake Group 14: An Adventure with the Water-Snake Pemilihan cerita ini didasarkan pada tingkat kesulitan kata atau kalimat yang terdapat didalamnya dan kemudahan untuk menceritakannya kembali. Selain itu, cerita yang dipilih tidak memiliki lebih dari 500 - 600 kata agar dapat mudah dipahami oleh siswa SMPN 2 Alalak. Untuk pemilihan cerita, para pelatih menyarankan mengambil dari https://learnenglishkids.britishcouncil.org/short-stories dan https://www.shortkidstories.com/ ataupun dari sumber lainnya. Hal ini disarankan karena selain tulisan dalam Bahasa Inggris, ceritanya juga memiliki gambar-gambar yang menarik dan berwarna sehingga akan memudahkan pemahaman siswa akan isi cerita. Tahap persiapan 2 adalah terkait dengan pelatihan storytelling kepada mahasiswa yang berperan sebagai instruktur. Dalam tahap ini juga, mahasiswa dibekali dengan lesson plan atau rencana pembelajaran dengan langkah-langkah yang lengkap. Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 TEHNIK PELAKSANAAN Kemudian, mereka berlatih mengajarkan cerita tersebut dengan tehnik storytelling dan berlatih mengucapkan kata-kata yang dianggap sulit dengan benar agar dapat mengajarkannya dengan benar juga. Kemudian mereka juga membuat media pembelajaran yang diperlukan dan sesuai dengan cerita yang mereka bawakan seperti word cards, gambar-gambar, caption dan lain sebagainya. Tahap kedua yaitu tahap pelaksanaan yang dilakukan 1 jam pelajaran (45 menit) pada 2 hari yang telah ditentukan oleh pihak sekolah yaitu 23 dan 24 Maret 2022. Dalam pelaksanaannya pada hari 1, 42 orang instruktur dan hari 2 sebanyak 42 orang yang disebar ke setiap kelas dari kelas 7-9 dan masing-masing instruktur membagi siswa dalam kelompok kecil sekitar 7-10 orang. Dalam memberikan materi, para instruktur diberikan keleluasaan tidak hanya bisa melakukannya dalam kelas, tapi juga bisa diluar kelas atau outdoor. Kebanyakan dari mereka memilih mengajar secara outdoor seperti di lapangan sekolah, jalan selasar depan ruangan kelas, ataupun dibawah pohon dengan beralaskan tikar. Setelah menemukan tempat yang sesuai, para instruktur memulai pembelajarannya. Setiap instruktur mengajar sesuai dengan lesson plan yang telah dibuat, menggunakan media yang telah disiapkan sebelumnya, dan melakukan improvisasi yang diperlukan untuk pemberian cerita. Proses pembelajaran dimulai dengan kegiatan apersepsi atau pre-activity sekitar 10 menit. Dalam kegiatan ini, instruktur memulai dengan mengucapkan salam, \| \|73 Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 Pelatihan Pengajaran Bahasa Inggris… Febriyanti et al lalu mengenalkan dirinya secara singkat dan meminta siswa melakukan hal yang sama. Inti kegiatan ini adalah untuk membangun hubungan pertemanan dengan siswa dan agar mereka tidak merasa takut dengan pembelajaran yang akan dilakukan. Setelah itu, instruktur menanyakan hal-hal yang berkaitan dengan isi cerita yang akan disampaikan untuk mengetahui sejauh mana mereka mengenal atau mengetahui isi cerita. Kemudian instruktur menunjukkan kata-kata kunci yang terdapat dalam cerita dalam bentuk cardboard. Setelah itu, instruktur mencontohkan pengucapan kata-kata tersebut agar siswa dapat mengulanginya dengan benar. p g p g p g g y g Kegiatan selanjutnya adalah while activities selama 25 menit dimana instruktur membaca cerita secara nyaring dengan pengucapan yang benar sambil siswa mendengarkan dengan tenang. Setelah itu, semua siswa secara bergiliran membaca nyaring cerita yang telah didengarkan dengan pengucapan yang benar. Kemudian, mendiskusikan secara bersama-sama kata-kata sulit yang muncul dalam cerita serta menyimpulkan inti cerita yang telah dibaca. Untuk selanjutnya, instruktur meminta siswa untuk berbicara mengemukakan pendapatnya ataupun mengajukan pertanyaan dalam Bahasa Inggris mengenai cerita yang telah dibacakan. TEHNIK PELAKSANAAN Setelah itu, secara berpasangan, mereka diminta untuk menceritakan kembali cerita yang telah didengar dengan bahasa mereka sendiri dan harus dengan pengucapan yang benar. g g g p g p y g Kegiatan terakhir adalah post-activity selama 10 menit dimana instruktur memberikan soal latihan mengenai cerita untuk dijawab oleh siswa dengan tujuan mencek pemahaman siswa akan isi cerita yang telah dibahas sebelumnya. Soal latihan yang diberikan bisa berupa pilihan ganda, benar atau salah, mencocokkan, ataupun berupa essai singkat. Setelah selesai, instruktur dan siswa secara bersama- sama mendiskusikan jawabannya. Untuk kegiatan terakhir, instruktur memberikan motivasi kepada siswa akan pentingnya belajar Bahasa Inggris dan agar mereka jangan merasa takut dan harus percaya diri untuk berbicara dalam Bahasa Inggris. Penutup, instruktur mengucapkan salam perpisahan kepada siswa yang diajarnya. Gambar 1. Tahapan kegiatan Pengabdian kepada Masyarakat Tahap Persiapan 1. Pembagian Kelompok dan Pemilihan cerita Tahap Persiapan 2. Pembuatan RPP dan media, serta Pelatihan pengajaran Tahap Pelaksanaan. Praktik Mengajar di Sekolah Tahap Persiapan 1. Pembagian Kelompok dan Pemilihan cerita Gambar 1. Tahapan kegiatan Pengabdian kepada Masyarakat HASIL DAN DISKUSI Hal ini juga memiliki tujuan untuk memudahkan mereka belajar sehingga akan lebih mudah juga bagi mereka untuk berbicara dalam Bahasa Inggris, mengekpresikan pendapat ataupun ide-ide, meningkatkan kepercayaan diri dan menambah penguasaan kosakata (vocabulary) Bahasa Inggris mereka. Selain itu, kelompok kecil juga berfungsi untuk mendorong, memotivasi dan merangsang siswa untuk berpikir kritis dan memudahkan guru untuk memberikan perhatian dan rangsangan yang diperlukan oleh setiap individu siswa (Argawati, 2014; La’biran, 2017; Fauzi 2017) Gambar 2. Dosen pembimbing (pelatih) memberikan pelatihan pengajaran kepada mahasiswa (instruktur) Selanjutnya, tahapan pelaksanaan atau kegiatan inti dari PkM ini yaitu pembelajaran Bahasa Inggris dengan menggunakan tehnik storytelling dalam kelompok kecil. Menurut Helmiati (2013), kelompok kecil terdiri dari 3 – 8 orang yang memungkinkan guru memberikan perhatian kepada setiap siswa sehingga terjadi hubungan yang lebih akrab antara guru dengan siswa dan siswa dengan siswa lainnya. Sedangkan Rocks (1981) dalam La’biran (2017) kelompok kecil adalah sekelompok orang yang beranggotakan sekitar 4 – 6 orang. Akan tetapi, dalam kegiatan pengabdian ini, siswa disetiap kelas di SMPN 2 Alalak dibagi kedalam kelompok kecil yang beranggotakan 5 – 10 orang karena menyesuaikan dengan jumlah instruktur. Hal ini juga memiliki tujuan untuk memudahkan mereka belajar sehingga akan lebih mudah juga bagi mereka untuk berbicara dalam Bahasa Inggris, mengekpresikan pendapat ataupun ide-ide, meningkatkan kepercayaan diri dan menambah penguasaan kosakata (vocabulary) Bahasa Inggris mereka. Selain itu, kelompok kecil juga berfungsi untuk mendorong, memotivasi dan merangsang siswa untuk berpikir kritis dan memudahkan guru untuk memberikan perhatian dan rangsangan yang diperlukan oleh setiap individu siswa (Argawati, 2014; La’biran, 2017; Fauzi 2017) p g (p ) p p g j p mahasiswa (instruktur) Selanjutnya, tahapan pelaksanaan atau kegiatan inti dari PkM ini yaitu pembelajaran Bahasa Inggris dengan menggunakan tehnik storytelling dalam kelompok kecil. Menurut Helmiati (2013), kelompok kecil terdiri dari 3 – 8 orang yang memungkinkan guru memberikan perhatian kepada setiap siswa sehingga terjadi hubungan yang lebih akrab antara guru dengan siswa dan siswa dengan siswa lainnya. Sedangkan Rocks (1981) dalam La’biran (2017) kelompok kecil adalah sekelompok orang yang beranggotakan sekitar 4 – 6 orang. Akan tetapi, dalam kegiatan pengabdian ini, siswa disetiap kelas di SMPN 2 Alalak dibagi kedalam kelompok kecil yang beranggotakan 5 – 10 orang karena menyesuaikan dengan jumlah instruktur. Hal ini juga memiliki tujuan untuk memudahkan mereka belajar sehingga akan lebih mudah juga bagi mereka untuk berbicara dalam Bahasa Inggris, mengekpresikan pendapat ataupun ide-ide, meningkatkan kepercayaan diri dan menambah penguasaan kosakata (vocabulary) Bahasa Inggris mereka. HASIL DAN DISKUSI Kegiatan pengabdian yang dilaksanakan yaitu Pelatihan Pengajaran Bahasa Inggris dengan tehnik storytelling dalam kelompok kecil bagi siswa SMPN 2 Alalak telah diselenggarakan dengan baik dan berjalan dengan lancar. Semua tahapan yang direncanakan mulai dari tahap persiapan sampai pelaksanaan sudah dilakukan tanpa ada kendala berarti baik dari tim PkM maupun dari peserta pengabdian yaitu para siswa SMPN 2 Alalak. Pada kegiatan pertama yaitu persiapan, tim PkM membagi para instruktur ke dalam 14 kelompok yang terdiri dari 6 orang. Masing-masing kelompok kemudian mencari cerita yang sesuai dengan karakteristik siswa SMP sebagai topik pengajaran. Pemilihan cerita yang bagus untuk diceritakan dan jenis cerita apa yang disukai oleh siswa sangat penting untuk memotivasi mereka untuk tetap mendengarkan cerita tersebut. Selain itu, mendengarkan cerita juga membantu menumbuhkan minat dan Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 \| \|74 Pelatihan Pengajaran Bahasa Inggris… Febriyanti et al kebiasaan membaca anak sehingga mereka cenderung menjadi pembaca yang handal dan tampil lebih baik di sekolah (Tampubolon, 1991). Juga, belajar bahasa Inggris melalui cerita sejak dini memungkinkan siswa untuk memperluas wawasan mereka dan merangsang semangat belajar mereka semenjak dini, serta menumbuhkan kesadaran akan pentingnya bahasa Inggris (Febriyanti & Hidayat, 2023) Pada tahapan persiapan kedua, masih dalam kelompoknya, para instruktur membuat lesson plan yang kemudian didiskusikan dengan tim PkM dan melakukan revisi apabila diperlukan. Setelah itu, mereka membuat media yang dibutuhkan agar cerita yang disampaikan dapat menarik perhatian siswa dan memotivasi mereka untuk belajar Bahasa Inggris. Masing-masing anggota kelompok memiliki media nya masing-masing karena mereka akan mengajarkannya kedalam kelompok siswa yang berbeda. Setelah itu, para instruktur berlatih mengajar dengan lesson plan dan media yang telah dibuat dengan bimbingan tim PkM (pelatih). Gambar 2. Dosen pembimbing (pelatih) memberikan pelatihan pengajaran kepada mahasiswa (instruktur) Selanjutnya, tahapan pelaksanaan atau kegiatan inti dari PkM ini yaitu pembelajaran Bahasa Inggris dengan menggunakan tehnik storytelling dalam kelompok kecil. Menurut Helmiati (2013), kelompok kecil terdiri dari 3 – 8 orang yang memungkinkan guru memberikan perhatian kepada setiap siswa sehingga terjadi hubungan yang lebih akrab antara guru dengan siswa dan siswa dengan siswa lainnya. Sedangkan Rocks (1981) dalam La’biran (2017) kelompok kecil adalah sekelompok orang yang beranggotakan sekitar 4 – 6 orang. Akan tetapi, dalam kegiatan pengabdian ini, siswa disetiap kelas di SMPN 2 Alalak dibagi kedalam kelompok kecil yang beranggotakan 5 – 10 orang karena menyesuaikan dengan jumlah instruktur. HASIL DAN DISKUSI Selain itu, kelompok kecil juga berfungsi untuk mendorong, memotivasi dan merangsang siswa untuk berpikir kritis dan memudahkan guru untuk memberikan perhatian dan rangsangan yang diperlukan oleh setiap individu siswa (Argawati, 2014; La’biran, 2017; Fauzi, 2017). \| \|75 Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 Pelatihan Pengajaran Bahasa Inggris… Febriyanti et al Membagi siswa dalam kelompok kecil merupakan tehnik pengajaran yang efektif dalam pengajaran bahasa terutama Bahasa Inggris. Ur (1996) menyatakan bahwa kegiatan belajar yang dilakukan dalam kelompok kecil membantu siswa berinteraksi dengan lingkungannya terutama teman-temannya, lebih banyak bertanya, dan bekerjasama dengan temannya. Hal tersebut memungkinkan lebih banyak latihan penggunaan Bahasa Inggris sebagai sarana berkomunikasi yang akhirnya dapat meningkatkan pemahaman dan pembelajaran. Berdasarkan hasil praktik mengajar yang dilakukan oleh para instruktur, 8 dari 10 siswa di dalam setiap kelompok (80% siswa) lebih aktif dalam tanya jawab dan berdiskusi dalam kelompok. kemudian, 7 dari 10 orang dapat mengingat kosakata yang telah diajarkan dan dapat mengucapkannya kembali dengan pronunciation yang benar pula. Hal ini dikarenakan mereka dapat berlatih dengan lebih intensif dalam kelompoknya dan juga karena kesempatan berbicara yang diberikan juga lebih banyak dibandingkan ketika pembelajaran yang dilakukan berpusat pada guru. Gambar 2. Kegiatan pengajaran Bahasa Inggris yang dilakukan di luar kelas Pembelajaran yang dilakukan selain di dalam kelas adalah sebagai bentuk variasi dalam tehnik mengajar. Hal ini bertujuan untuk menciptakan suasana yang baru dan menyenangkan bagi siswa agar pembelajaran tidak monoton dan membosankan. Diharapkan siswa akan menunjukkan ketekunan, antusiasme dan penuh partisipasi dalam pembelajaran yang dilakukan (Helmiati, 2013). Berdasarkan pengamatan yang dilakukan dilapangan, para siswa terlihat antusias dan fokus mendengarkan cerita yang disampaikan oleh instruktur di kelompok mereka. Para siswa terlihat menikmati pembelajaran yang dilakukan apalagi mereka bisa duduk leluasa menikmati udara dan lingkungannya yang dikarenakan sebagian besar dari mereka tidak duduk di dalam kelas. Menurut (Faraziah, 2015), pembelajaran yang dilakukan di luar kelas akan membuat kegiatan pembelajaran lebih menarik dan tidak membosankan, sehingga diharapkan motivasi belajar siswa akan lebih tinggi. Gambar 2. Kegiatan pengajaran Bahasa Inggris yang dilakukan di luar kelas Pembelajaran yang dilakukan selain di dalam kelas adalah sebagai bentuk variasi dalam tehnik mengajar. Hal ini bertujuan untuk menciptakan suasana yang baru dan menyenangkan bagi siswa agar pembelajaran tidak monoton dan membosankan. Diharapkan siswa akan menunjukkan ketekunan, antusiasme dan penuh partisipasi dalam pembelajaran yang dilakukan (Helmiati, 2013). Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 HASIL DAN DISKUSI Berdasarkan pengamatan yang dilakukan dilapangan, para siswa terlihat antusias dan fokus mendengarkan cerita yang disampaikan oleh instruktur di kelompok mereka. Para siswa terlihat menikmati pembelajaran yang dilakukan apalagi mereka bisa duduk leluasa menikmati udara dan lingkungannya yang dikarenakan sebagian besar dari mereka tidak duduk di dalam kelas. Menurut (Faraziah, 2015), pembelajaran yang dilakukan di luar kelas akan membuat kegiatan pembelajaran lebih menarik dan tidak membosankan, sehingga diharapkan motivasi belajar siswa akan lebih tinggi. Dalam wawancara yang dilakukan kepada Kepala Sekolah SMPN 2 Alalak, beliau menyatakan bahwa kegiatan ini sangat membawa manfaat bagi siswa dan menjadi inspirasi bagi para guru Bahasa Inggris untuk mengajarkan Bahasa Inggris kepada siswa didiknya dengan menggunakan tehnik-tehnik yang dapat membuat siswa termotivasi untuk belajar Bahasa Inggris. Kedepannya, sekolah juga akan melaksanakan pembelajaran outdoor karena dapat menciptakan suasana pembelajaran yang baru dan menyenangkan. Selanjutnya, pihak sekolah nantinya akan membuat jadwal bagi guru-guru, menyiapkan papan tulis besar, alas/tikar, dan media lainnya yang akan mendukung proses pembelajaran outdoor. Selain itu, pihak \| \|76 Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 Pelatihan Pengajaran Bahasa Inggris… Febriyanti et al sekolah juga terbuka untuk menjadi mitra lagi kedepannya untuk kegiatan-kegiatan baru yang berkaitan dengan pengajaran/pendidikan/pelatihan/penelitian/pengabdian dan lain sebagainya untuk meningkatkan mutu sekolah. sekolah juga terbuka untuk menjadi mitra lagi kedepannya untuk kegiatan-kegiatan baru yang berkaitan dengan pengajaran/pendidikan/pelatihan/penelitian/pengabdian dan lain sebagainya untuk meningkatkan mutu sekolah. Gambar 3. Foto Bersama dengan Dosen, Guru dan Mahasiswa setelah Refleksi Gambar 3. Foto Bersama dengan Dosen, Guru dan Mahasiswa setelah Refleksi KESIMPULAN Kegiatan pengabdian kepada masyarakat yang dilaksanakan oleh tim PkM dan diikuti oleh 84 orang mahasiswa yang berperan sebagai instruktur dan seluruh siswa SMPN 2 Alalak telah dilaksanakan dengan baik dan lancar tanpa kendala apapun. Para instruktur menguasai pembelajaran dengan baik sehingga dapat memberikan materi cerita yang santai dan menyenangkan namun tetap mendidik. Hal ini terlihat dengan antusias dan keaktifan siswa dalam mengikuti pembelajaran sejak dimulainya kegiatan pembelajaran. Pemberian cerita berbahasa Inggris dengan menggunakan tehnik storytelling yang dilakukan dalam kelompok kecil terbukti efektif dan berhasil dalam meningkatkan kepercayaan diri siswa untuk berbicara dalam Bahasa Inggris. Meskipun pada awalnya para siswa tersebut ragu, akan tetapi dengan penguatan yang diberikan oleh instruktur dapat mengembalikan kepercayaan diri dan menekan rasa takut tersebut. Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 REKOMENDSI Setelah menyelenggarakan kegiatan pengabdian kepada masyarakat mengenai Pelatihan Pengajaran Bahasa Inggris dengan Tehnik Storytelling dalam Kelompok Kecil bagi Siswa SMPN 2 Alalak, diharapkan akan membawa manfaat kepada para guru Bahasa Inggris di sekolah tersebut khususnya dan guru Bahasa Inggris di sekolah lain pada umumnya. membacakan cerita kepada anak dalam Bahasa Inggris dan meminta mereka mendengarkan dengan baik akan memberikan banyak keuntungan bagi mereka kedepannya. Diharapkan juga mereka akan mampu mengemukakan kembali cerita tersebut dengan keberanian dan percaya diri yang mereka miliki. Sehingga, nantinya mereka akan selalu dapat mengasah kemampuan berbicara dalam Bahasa Inggris mereka. Juga, para guru diminta untuk lebih memikirkan perkembangan dan motivasi belajar yang dirasakan oleh anak didiknya sehingga kedepannya menggunakan metode atau tehnik pengajaran Bahasa Inggris yang akan mengakomodasi hal-hal tersebut. Apabila dilaksanakan pengajaran Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 \| \|77 Pelatihan Pengajaran Bahasa Inggris… Febriyanti et al dengan menggunakan teknik storytelling dan juga diskusi kelompok kecil nantinya, diharapkan agar dapat membuat pelatihan dan pengabdian lebih mendalam mengenai respon dan pendapat siswa mengenai hal tersebut. dengan menggunakan teknik storytelling dan juga diskusi kelompok kecil nantinya, diharapkan agar dapat membuat pelatihan dan pengabdian lebih mendalam mengenai respon dan pendapat siswa mengenai hal tersebut. ACKNOWLEDGMENT Kami mengucapkan terima kasih yang sebesar-besarnya kepada pihak SMPN 2 Alalak yang telah mengijinkan kami untuk melaksanakan kegiatan pengabdian masyarakat. Ucapan terima kasih juga kami sampaikan kepada pihak-pihak yang telah membantu memberikan saran dan kontribusi yang mendalam sehingga terlaksananya kegiatan pengabdian tersebut dan sampai pada tahap penulisan artikel ini. DAFTAR PUSTAKA Abasi, M., & Soori, A. (2014). Is Storytelling Effective in Improving the English Vocabulary Learning among Iranian Children in Kindergartens? International Journal of Education and Literacy Studies, 2(3). https://doi.org/10.7575/aiac.ijels.v.2n.3p.7 Argawati, N. O. (2014). Improving Students’ Speaking Skill using Group Discussion. ELTIN Journal: Journal of English Language Teaching in Indonesia, 2(2), 74–81. Argawati, N. O. (2014). Improving Students’ Speaking Skill using Group Discussion. ELTIN Journal: Journal of English Language Teaching in Indonesia, 2(2), 74–81. Faraziah, R. (2015). Pengaruh Penggunaan Metode Pembelajaran Outdoor Learning Terhadap Motivasi Belajar Siswa Kelas III dalam Pembelajaran Ilmu Pengetahuan Sosial (IPS) di Madrasah Ibtidaiyah Nurul Huda Pondok Karya Tangerang Selatan [Thesis, Univesitas Islam Negeri Syarif Hidayatullah]. https://repository.uinjkt.ac.id/dspace/bitstream/123456789/29901/1/RIZA%20FA RAZIAH%20%281111018300018%29.pdf Febriyanti, E. R., & Hidayat, F. (2023). Developing Picture Storybook in English with Wetlands Theme for Young Learners. Acitya: Journal of Teaching and Education, 5(1), 171–187. https://doi.org/10.30650/ajte.v5i1.3515 ( ) p g j Helmiati. (2013). Micro Teaching Melatih Keterampilan Dasar Mengajar (1st ed.). CV. Aswaja Pressindo. La’biran, R. (2017). Improving Speaking Ability Through Small Groups Discussion for the Eight Year Students of SMPN 2 Saluputti in Tana Toraja. Elite : English and Literature Journal, 4(1), 51–62. https://doi.org/10.24252/elite.v4i1a5 Nurharyadi. (2018). Penerapan Metode Storytelling Pembelajaran Tematik untuk Meningkatkan Keterampilan Bercerita pada Siswa Kelas III Sekolah Dasar Negeri 187/X Desa Bangun Karya [Thesis, Universitas Islam Negeri Sulthan Thaha Saifuddin]. http://repository.uinjambi.ac.id/id/eprint/889 Rohyeni. (2021). Upaya Peningkatan Kemampuan Storytelling Bahasa Inggris Melalui Media Audio Visual Berbasis Experience. Jurnal Education of Batanghari, 4(3), 106–116. https://ojs.hr-institut.id/index.php/JEB/article/download/135/124/ Sari, M. (2012). Peningkatan Pengenalan Bahasa Inggris Anak dengan Total Physical Response di Taman Kanak-kanak Negeri Pembina Kabupaten Sijunjung. Jurnal Pesona PAUD, 1(1), 1–10. Ur, P. (1996). A course in language teaching: Practice and theory. Cambridge University Press. Lumbung Inovasi: Jurnal Pengabdian kepada Masyarakat, Maret 2023 Vol. 8, No. 1 \| \|78
https://openalex.org/W2749565118	http://www.fortunejournals.com/articles/generation-of-bioelectricity-from-sewage-sludge-using-single-chamber-microbial-fuel-cell.pdf	English	null	Generation of Bio-Electricity from Sewage Sludge Using Single Chamber Microbial Fuel Cell	Journal of environmental science and public health	2,017	cc-by	2,891	Generation of Bio-Electricity from Sewage Sludge Using Single Chamber Microbial Fuel Cell Kumar Sonu* Received: 03 July 2017; Accepted: 20 July 2017; Published: 02 August 2017 Received: 03 July 2017; Accepted: 20 July 2017; Published: 02 August 2017 Journal of Environmental Science and Public Health doi: 10.26502/JESPH.007 Journal of Environmental Science and Public Health doi: 10.26502/JESPH.007 Volume 1, Issue 2 Volume 1, Issue 2 Kumar Sonu* *Corresponding Author: Kumar Sonu, Department of Mechanical Engineering, Anand International College of Engineering, Near Kanota, Agra Road, Jaipur - 303012, Rajasthan, India, Tel: 01429 234 994; E-mail: kumar_sonuasg@yahoo.com Abstract A Single Chamber Microbial Fuel Cell (MFC) has been fabricated to generate electricity from the sludge of the sewage treatment plant at Anand International College of Engineering, Jaipur, at two different ambient temperature range of 25 ± 4°C and 32 ± 4°C under aerobic condition. The maximum voltage obtained was about 2890 mV after 80 (hrs.) at temperature range of25 ± 4°C, with the surface power density of 1108.29 mW/m2. When the ambient temperature was 32 ± 4°C, the maximum voltage obtained was 1652 mV after 40 (hrs.) surface power density reduced to 865.57 mW/m2.While changing the amount of substrate for certain area of the electrode at 25 ± 4°C range it showed that the electricity generation decreased with the available substrate and it also shortens the time to peak voltage. On the other way when the ambient temperature increased to 32 ± 4°C, the maximum potential energy generated is less than the previous temperature for the same substrate per unit area of the electrode and also the time to peak voltage decreases to 40 hrs. At the end of the 152 (hrs.), the maximum COD reduction for the sewage sludge was 30% for 32 ± 4°C. When comparing with other single chambers MFC, the present model is generating more electricity that any MFC using sewage sludge as substrate except platinum electrode, which is much costlier that electrode used in the present study. Keywords: Graphite electrode; Microbial fuel cell; Sewage sludge; Used dry cell J Environ Sci Public Health 2017; 1 (2): 68-74 1. Introduction The microbial fuel cell is a device which generates electricity by the metabolic activities of the microbes. Microorganisms transfer the electrons obtained from the metabolism of organic matters in the anode and thereby to the J Environ Sci Public Health 2017; 1 (2): 68-74 68 J Environ Sci Public Health 2017; 1 (2): 68-74 68 cathode through an external circuit, hence generate electricity [1, 2]. Though a century old technique, which was initially recognized for treatment of wastewater, MFC is gaining its interest for the generation of electricity, bio-hydrogen and also used as biosensor [3, 4]. Any biodegradable organic ranging from pure compounds such as glycerol, acetate, starch, glucose, cysteine, and ethanol [3, 5] to complex mixtures of organic matter such as wastewater [6], cow dung [2], and kitchen waste [6] are the ideal candidates of sustainable energy source for MFC. The performance of microbial fuel cell depends upon the types of electrode materials, microbe & substrate and its concentration, pH, temperature and ionic strength. The various types of the electrode that can be used in the construction of the microbial fuel cell are graphite; graphite felt, Platinum (Pt.), Pt. black, carbon paper, reticulated vitreous carbon (RVS). The design of MFC may be 1. Two compartment MFC systems consisting of an anodic chamber and catholic chamber separated by proton exchange membrane (PEM) or sometime salt bridge to allowing the proton to move across the blocking the diffusion of oxygen into the anode [3]. 2. Single –compartment MFC systems consisting of the single anodic chamber without the any aerated catholic chamber [3]. The anode chamber is coupled to a porous air-cathode exposing directly to the air. The Single–compartment MFC systems is cheaper than that of two compartments MFC. The traditional dual chamber which is bulky due to two separate anode and cathode chambers, the single chamber containing single anode chamber omitting the cathode chamber where the cathode is placed as membrane and exposed to air is much more efficient and cost effective than dual chamber MFC. Table 1 shows the performance of single chamber microbial fuel cell for the different substrate on the basis of maximum power density. The maximum surface power density of 6000 mW/m2 using platinum electrode modified polyanilineco and sewage sludge was found from the literature available [7]. When the sewage sludge used with graphite electrode 152 mW/m2 maximum surface power density could be achieved [7]. Sl. No. 1. Introduction Industry/Substrate System configuration Maximum surface power density Refs. 1 Sewage sludge Electrode: Single chamber (graphite electrode) 152 mW/m2 [7] Single chamber (Platinum and polyanilineco-modified) 6000 mW/m2 [7] 2 Glycerol -waste water Single-chamber Temperature (°C): 30°C pH: 7 600 mW/m2 [8] 3 Meat packing wastewater One-chamber, Carbon paper loaded with0. 35 mg platinum/cm2 Temperature (°C): NA pH: NA 80 ± 1 mW/m2 [9] J Environ Sci Public Health 2017; 1 (2): 68-74 J Environ Sci Public Health 2017; 1 (2): 68-74 69 4 Starch processing wastewater One-chamber air-cathode MFC with carbon paper anode (25 cm2) Temperature (°C):30 ± 1 °C pH: 7.1 ± 0.1 239.4 mW/m2 [10] 5 Swine wastewater Single-chambered air cathode Temperature (°C):30°C pH: NA. 261 mW/m2 [11] 6 Brewery wastewater One-chamber air-cathode MFC with non-wet proofed carbon cloth as anode (7 cm2) and wet proofed carbon cloth containing Pt as cathode Temperature (°C):30°C pH: 6.5 ± 0.2 205 mW/m2 [12] 7 Paper recycling wastewater One-chamber MFC with graphite fiber-brush anode (5418 m2/m3 brush volume) Temperature (°C): 22-26°C (Room Temperature) pH: 7 501 ± 20 mW/m2 [13] Table 1: Performance of single chamber MFC under different types of industrial waste. Table 1: Performance of single chamber MFC under different types of industrial waste. Table 1: Performance of single chamber MFC under different types of industrial waste. In conventional single chamber MFC the anode and cathode are separated by a membrane or sometime cathode is directly exposed to air. In both the cases, anode and cathode are completely different material in design and composition. In our present studies, we are using single chamber MFC where the carbon rod extracted from the used dry cell is used as anode and copper as a cathode. The substrate used here is domestic sewage sludge. J Environ Sci Public Health 2017; 1 (2): 68-74 Design 2.2 Six numbers of single chamber MFCs having carbon electrode as anode and copper as a cathode connected externally in series with copper wire have been used in this present study. Each electrode is submerged into the sludge and copper cathode is exposed to the atmosphere. Figure 1 shows the experimental setup in details. The various components in microbial fuel cell are as follow; 2.2.1 Electrode: The carbon electrodes were taken from the used dry cell to absorb electrons from the substrate, before using the electrodes were cleaned with 0.1 M HCl and stored in deionized water for 12 hr. The dimension of the electrode is 10 mm diameter and 57 mm, respectively having 40 mm (70% length) as the anode and 17 mm as cathode over which the copper wire was bounded acting as the cathode. Hence the surface area of an anode is 1256 mm2. 2.2.2 Copper wires: The copper wires of 0.7 ohms resistance were connected with the electrodes. The external circuit was in series connections. 2.2.2 Copper wires: The copper wires of 0.7 ohms resistance were connected with the electrodes. The external circuit was in series connections. 2.2.3 Calibrated multimeter: To measure the voltage output (Model No.MAS83OL). 2.2.4 pH meter: To measure the pH of the sewage sludge ( Make: Systronics). g p ( ) 2.2.4 pH meter: To measure the pH of the sewage sludge ( Make: Systronics). pH meter: To measure the pH of the sewage sludge ( Make: Systronics). The carbon electrodes were fixed in the containers with the help of the copper wires and are connected in series externally. The collected sewage sludge was mixed uniformly and was poured into the container and allowed the bacteria present in it to grow and start the metabolic activities in aerobic condition (Figure 1). Figure 1: Single chamber MFC (a) Circuit diagram of the single cell of single chamber single MFC. (b) Schematic representation of the circuit diagram of single chamber MFC in series (c) Photograph of the experimental setup of the combination of the single chamber. (d) Schematic representation of the three different setups of MFC studied. gle chamber MFC (a) Circuit diagram of the single cell of single chamber single MFC. (b) Schemat Figure 1: Single chamber MFC (a) Circuit diagram of the single cell of single chamber single MFC. 2.1 Collection and preservation of sample The sewage sludge is collected from the waste water treatment plant at Anand International College of Engineering, Jaipur. The sludge was preserved at 4°C after collection and before using in MFC [12]. The physiochemical characteristics of the sludge used in MFC are shown in the Table 2. Sl.No Parameters Values 1 COD 80000 mg/l 2 % of Total Solid 17.2 3 % of Volatile Solid 11.6 5 Colour Grey 6 pΗ 5.3 Table 2: Physiochemical properties of sewage sludge used in MFC Table 2: Physiochemical properties of sewage sludge used in MFC. Table 2: Physiochemical properties of sewage sludge used in MFC. J Environ Sci Public Health 2017; 1 (2): 68-74 70 J Environ Sci Public Health 2017; 1 (2): 68-74 Design (b) Schematic representation of the circuit diagram of single chamber MFC in series (c) Photograph of the experimental setup of the combination of the single chamber. (d) Schematic representation of the three different setups of MFC studied. J Environ Sci Public Health 2017; 1 (2): 68-74 71 5. Acknowledgement 5. Acknowledgement We are very grateful to Anand International College of Engineering, Jaipur for the financial support to carry out the above project work. Performance evaluation criteria For SET II the pH reduced from 5.3 to 4.6 But in SET III the pH increased from 5.3 to 6.1. At the end of the152 (hrs.), the COD of the sewage sludge for the SET I was 58000 mg/l (reduction of 27.5%), In case of SET II the COD was 72000 mg/l (reduction of 10%), finally for the SET III it is 56000 mg/l (reduction of 30%). The pH of the sewage sludge found to decrease from 5.3 to 4.5 in SET I (Table 3). For SET II the pH reduced from 5.3 to 4.6 But in SET III the pH increased from 5.3 to 6.1. At the end of the152 (hrs.), the COD of the sewage sludge for the SET I was 58000 mg/l (reduction of 27.5%), In case of SET II the COD was 72000 mg/l (reduction of 10%), finally for the SET III it is 56000 mg/l (reduction of 30%). Results obtained from the SET III clearly indicate that at elevated temperature the metabolic activities of the microbes reduce and even the pH of the sewage sludge is increased, thereby reducing the surface power density. But at the same time there is the slight improvement in the COD reduction. 4. Conclusion The present study is a novel and economical (cost of each cell Rs.250/-only) single chamber microbial fuel cell for the generation of electricity. Result found is very encouraging when compared to other single chamber MFC. The highest surface power density found to be 1108.29 mW/m2 which is more than any other single chamber MFC excluding platinum electrode (Table 1). Both SET I and SET II are experimented at the same ambient temperature and substrate, where SET II is giving less potential energy. This is because the amount of substrate (hence COD) available for anode at SET II is less than SET I. SET I and SET II are designed in such a way that the same amount of surface area of the electrode is exposed to the substrate in both cases. Thus, in SET II reducing the volume of the substrate in half will also decrease in substrate availability per unit area of the electrode to half. The reduced available substrate per unit area of the electrode not only reduced the peak potential energy, but it also shortens the time to peak voltage. SET III which is operated at the elevated ambient temperature (32 ± 4°C) gives less peak potential energy than SET I and SET II thus indicating that among the two different range of ambient temperature 25 ± 4°C and 32 ± 4°C, the previous one is more promising. As per the very preliminary study conducted on the presented single chamber, MFC is can be undoubtedly said that the current model is capable of generation of electricity from the domestic sewage sludge economically and effectively. J Environ Sci Public Health 2017; 1 (2): 68-74 Performance evaluation criteria Performance evaluation criteria 2.3 For all the three sets the voltage output was recorded by the multimeter across the resistor of 1000 ohm at the regular interval of 8 hrs and continuously operated until the energy generation is diminished to zero (least count of the multimeter is 0.01 mV). The first two sets were operated at the ambient temperature of 25 ± 4°Cwhile the third set is operated at the ambient temperature of 32 ± 4°C, which was within 10 days. The surface power density P (mW/m2) is calculated as 𝑃= 𝑉2/(𝑅× 𝐴× 1000), where V (mV) is the measured fuel cell voltage, R(ohm) external resistant and A ( m2) projected surface area of the anode, 1000 is needed for maintaining the units. 3. Result and Discussion Figure 2: Voltage (mV) vs. Time (hrs.) for all the 3 SETs. 0 500 1000 1500 2000 2500 3000 3500 0 16 32 48 64 80 96 112128 144 Voltage(mV) Time (hrs.) SET I SET II SET III Figure 2: Voltage (mV) vs. Time (hrs.) for all the 3 SETs. The maximum voltage obtained from the study was about 2890 mV after 80 (hrs.) for the SET Iat ambient temperature of 25 ± 4°C. At the same average ambient temperature in SET II , we could able to achieve 2554 mV after 40 (hrs.) where the quantity (hence the amount of available COD) of the sewage sludge is half that of SET I. In SET III though the quality of sewage sludge is same as that of SET I but we could able to achieve only 1652 mV after 40 (hrs.) at an average ambient temperature of 32 ± 4°C. SET I SET II SET III Initial Final Initial Final Initial Final pH 5.3 4.5 5.3 4.6 5.3 6.1 COD (mg/L) [% decrease] 80000 58000 [27.5%] 80000 72000 [10%] 80000 56000 [30%] Table 3: Change of pH and COD in three different sets of Microbial Fuel Cell. Table 3: Change of pH and COD in three different sets of Microbial Fuel Cell. Table 3: Change of pH and COD in three different sets of Microbial Fuel Cell. J Environ Sci Public Health 2017; 1 (2): 68-74 72 The pH of the sewage sludge found to decrease from 5.3 to 4.5 in SET I (Table 3). References 1. Potter MC. Electrical effects accompanying the decomposition of organic compounds. Proc R Soc London Ser B 84 (1911): 260-276. 2. Guang Zhao, Fang Ma, Li Wei, et al. Electricity generation from cattle dung using microbial fuel cell technology during anaerobic acidogenesis and the development of microbial populations. Waste Management 32 (2012): 1651-1658. J Environ Sci Public Health 2017; 1 (2): 68-74 73 3. Logan BE, Hamelers B, Rozendal R, et al. Microbial Fuel Cells: Methodology and Technology. Environ Sci Technol 40 (2006): 5181-5192. 4. Wang H, Park JD, Ren ZJ. Practical Energy Harvesting for Microbial Fuel Cells: A Review. Environ Sci Technol 49 (2015): 3267-3277. 5. Kim JR, Jung SH, Regan JM et al. Electricity generation and microbial community analysis of alcohol powered microbial fuel cells. Biores Technol 98 (2007): 2568-2577. 6. Das S, Mangwani N. Recent development in microbial fuel cell: a review. Journal of Scientific and Industrial Research. 69 (2010): 727-731. 7. Rahimnejad M, Adhami A, Darvari S, et al. Microbial fuel cell as new technology for bioelectricity generation: A review. Alexandria Engineering Journal 54 (2015): 745-756. 8. Nimji VR, Chen CJ, Chen CC, et al. Glycerol degradation in single-chamber microbial fuel cells. Bioresource Technolog 102 (2010): 2629-2634. 9. Heilmann J, Logan BE. Production of electricity from proteins using a microbial fuel cell. Water Environ Res 78 (2006): 531-537. 10. Liu H, Cheng S A, Logan BE. Production of electricity from acetate or butyrate using a single-chamber microbial fuel cell. Environ Sci Technol 39 (2005): 658-662. 11. Min B, Kim JR, Oh S, et al. Electricity generation from swine wastewater using microbial fuel cells. Water Res 39(2005): 4961-4968. 12. Feng Y, Wang X, Logan BE, et al. Brewery wastewater treatment using air-cathode microbial fuel cells. Appl Microbiol Biotechnol 78 (2008): 873-880. 13. Huang LP, Logan BE. Electricity generation and treatment of paper recycling wastewater using a microbial fuel cell. Appl Microbiol Biotechnol 80 (2008): 349-355. J Environ Sci Public Health 2017; 1 (2): 68-74 74 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license 4.0 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license 4.0 J Environ Sci Public Health 2017; 1 (2): 68-74 74
https://openalex.org/W2801881053	http://cinej.pitt.edu/ojs/index.php/cinej/article/download/166/474	English	null	Stereotype Representation of Women in Nigerian Films	CINEJ cinema journal	2,018	cc-by	7,242	Stereotype Representation of Women in Nigerian Films Andrew Ali Ibbi, alibbie@yahoo.co.uk Stereotype Representation of Women in Nigerian Films Andrew Ali Ibbi, alibbie@yahoo.co.uk Stereotype Representation of Women in Nigerian Films Andrew Ali Ibbi, alibbie@yahoo.co.uk Stereotype Representation of Women in Nigerian Films Andrew Ali Ibbi, alibbie@yahoo.co.uk Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu olume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu Abstract The stereotype representation of women in Nollywood films has attracted criticisms from the society with feminists clamoring for a review of the way women are projected. This study looks at the various issues associated with stereotype representation as a concept in film. The Feminist Media Theory was used as supporting theory for the paper. Part of the recommendations for the paper is the need for research to be properly conducted on the society before screenplays are written, to avoid misleading the public. Keywords: Representation, Gender, Stereotype, Femme Fatale, Sexploitation, Nollywood. New articles in this journal are licensed under a Creative Commons Attribution 4.0 United States License. This journal is published by the University Library System of the University of Pittsburgh as part This journal is published by the University Library System of the University of Pittsburgh as part of its D-Scribe Digital Publishing Program and is cosponsored by the University of Pittsburgh Press. Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films Introduction The issue of gender has been one that has attracted so many commentaries in the media. For time now, rights groups and feminists have decried the level of negative representation of women in the mass media. McDougall (2012: 52) defines representation as the sum of various micro parts, stating that it relates to the broader theories of collective identity, cultivation and ideology. Buckingham (2003: 57) explained that the notion of representation is one of the founding principles of media education. “The media do not offer us a transparent window on the world but a mediated version of the world. They don’t just present reality, they re-present it.” This implies that what the media projects to the audience stands the possibility of being accepted by the audience as a true reflection of reality. To this point, women groups have taken to various media platforms to protest the negative representation of women in the media especially film, television and advertisements. In many cultures around Africa, a clear line is often drawn to separate the men from the women. This has been defined and enshrined in the unwritten laws of the land and despite the advancement in civilization, this difference is still very much visible. Nigeria being an Africa country still has this practice going on. According to Dutt (2014: 3), since the women’s liberation movement in the 1960s, their roles in social, cultural, political and economic life has drastically changed and progressed for the better, seemingly giving women an equal footing to men in most aspects of life. But the male dominance of the film industry, like many other industries around the world, is still evident in the 21st century. Amobi (2013) brings this scenario to Nigeria by stating that even after four world conferences on women, Nigerian women continue to experience marginalization in every sphere of human endeavor. 50 The Nigerian film industry has grown to be the largest in Africa and one of the three largest in the world. Amobi (2013) further expressed concerns that in spite of the unprecedented growth and success of the industry, the content of these movies rather than reflect messages that correct societal ills appear to reinforce gender disparity in their portrayal of women. Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films Theoretical Framework Theoretical Framework Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu Introduction The nature of societies within Nigeria are structured along patriarchal lines where the girl child is seen as lesser than a man hence, there are certain societal roles that they are forbidden from participating in. This line of thought has reflected in the types of roles that women are given in movies in the country. Nigeria is a multi-ethnic country with more than 100 tribes each with a distinct language and culture from the other. Nigeria is the third most ethnically and linguistically diverse country in the world, after New Guinea and Indonesia (Blench, 2003). If there is anything that these different cultures have in common, it is the issue of marginalization of women. Cultures within the country share the common notion that the woman must be under the control of the man at all time. As the protest against the negative representation of women continue to dominate the agenda of feminists the world over, there is hope that this trend will take a different dimension especially with the coming of women as directors and producers in the Nigerian film industry. Downing and Husband (2005) quoted Winant (1994) as saying the concepts of race, ethnicity, feminism and their likes are essentially metaphors for institutionalized social relationships that combine processes of exploitation and domination, on the one hand, with processes of subjection and representation, that is, with struggles over meaning and identity, on the other. Having said this, it is worthy to note that in Nigeria, representation along the lines of these concepts will continue to assume a more productive dimension if Nigerian movie makers will inject funds into research on the various ethnic groups in Nigeria. Similarly, the idea that a woman should always play second fiddle or are symbols of sexploitation can be watered down in the face of more sensitive issues that will foster national unity and development. 51 Theoretical Framework Feminist Media Theory According to McDougall (2012), Feminist Media Theory is a complex range of approaches. Central to this range of approaches is the questioning of powerful gender norms that are reinforced to a greater or lesser extent in media and the relationship between media representation of gender and inequalities in broader social life. At the center of this theory is the concept of feminism. McDougall (2012: 178), stated that the word is often misunderstood as an extreme, militant politics. “Feminism is nothing more outrageous than the belief that we should oppose media texts that represent women as in-equal to men or as mere unthinking objects for male scrutiny.” Scholars like Humm (1992: 403) explained that historically, people and movements have been called feminists when they recognized the connections between social inequalities, deprivations and oppressions and gender differences. To McQuail (2010), the concept of feminism is a political as well as a cultural project. According to many feminist scholars, the mass media have the power to influence people’s thinking and for that reason, certain stereotype representation of women have the tendencies of encouraging some of the stereotype representations of women to thrive in the society. McQuail (2010: 123) outlined the following which form the basis for the argument in the feminist media theory:  Media have marginalized women in the public sphere.  Media purvey stereotypes of feminity and masculinity.  Production of content of media are gendered.  Female perspective offers alternative criteria of equal.  The personal is political.  Media offer positive and supportive as well as negative role models. CINEJ Cinema Journal: Andrew Ali Ibbi 52 Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.ed From the above, when taking a closer look at representation of gender in the media, the women have suffered from stereotype representation in Nigerian movies, ranging from prostitutes, to witches, to gold diggers, to husband killers and so on. Considering the fact that the media offer positive and supportive as well as negative role models, the society stands the risk of having its youth copying how the media are portraying the women. Conceptual Model nceptual Model Fig 1: Conceptual framework of female stereotype representation in Nigerian films Source: (Ibbi, 2016). Feminist Media Theory female stereotype representation in Nollywood femme fatale, girl next door, second choice gold diggers and trophy wives house wife, object of barter witch, mistress/ prostitute femme fatale, girl next door, second choice female stereotype representation in Nollywood witch, mistress/ prostitute gold diggers and trophy wives house wife, object of barter Fig 1: Conceptual framework of female stereotype representation in Nigerian films Source: (Ibbi, 2016). CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films 53 CINEJ Cinema Journal: Andrew Ali Ibbi According to Allanana (2013: 115) Women are therefore discriminated upon from, in most cases, acquiring formal education, mistreated and perpetually kept as house-help; the average Nigerian woman is seen as an available object for prostitution, forced marriage, street hawking, instrument of wide-range trafficking and a misfit in the society. Thus, the purported irrelevance associated with the status of women in society has merely reduced an average woman to an inferior commodity. Though there has been a notable change in the social structures of many families across the country in the new millennium, the patriarchy mentality is still consciously or unconsciously displayed especially by the males in the society. Consequently, this has to some extent affected the psyche of some Nigerian women as they reluctantly accept what the society has put in place since time immemorial so as not to either be seen as deviants or to avoid being marooned by the larger society. Gender Structure of the Nigerian Society Nigeria, a country in West Africa has a population of about 178.5 million people as at 2014. This population is scattered across 36 states and a federal capital territory, with the populace belonging to more than 100 ethnic groups. When it comes to the family structure of cultures in Nigeria, the Man is the head of the family. Allanana (2013) explained that in Nigeria, it is observed that the womanhood is reduced to a mere infidel and a second-class citizen, hence, there is the commonality of general belief system that the best place for women is in the ‘Kitchen’. This is a notion that is common among all the ethnic groups in the country. According to Aina (1998), from time immemorial, the Nigerian society has been a patriarchy society. While defining patriarchy, Asiyanbola (2005) said: It is a system of social stratification and differentiation on the basis of sex, which provides material advantages to males while simultaneously placing severe constraints on the roles and activities of females. In many Nigerian cultures, the woman is restricted from participating in some activities that are seen as strictly for men. For instance, participating in a wrestling contest or going on a hunting expedition. Allanana (2013) defined patriarchy as a system of social stratification and differentiation on the basis of sex, which provides material advantages to males while simultaneously placing severe constraints on the roles and activities of females. Asiyanbola (2005) explained this further: “Males are classed as having the following qualities: strength, vigor, virile/powerful courage, self-confidence and the ability to meet the outside world i.e. animal and human intruders head on and deal with it effectively. These qualities were reflected in the kinds of work that men engaged in. Men were responsible for much of what was thought of as ‘heavy’ labour.” This mentality denied many women in colonial and early post colonial Nigeria the This mentality denied many women in colonial and early post-colonial Nigeria the opportunity of benefitting from western education. Many parents had formed the opinion that the CINEJ Cinema Journal: Andrew Ali Ibbi 54 Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu woman will always end up in a man’s house and must do the bidding of her husband. Gender Structure of the Nigerian Society Even with the constraint of playing second fiddle, the Nigerian woman on close examination has been a strong force in the development of the family system in the country. Allanana (2013) explained that women constitute about half of the population of the Nigerian state and are known to play vital roles as mothers, producers, managers, community developers/organizers etc. Their contribution to the social and economic development of societies is also more than half as compared to that of men by virtue of their dual roles in the productive and reproductive spheres. CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films What Is Representation? According to Hall (1997), representation is the production of meaning through language. According to Hall (1997), representation is the production of meaning through language. The Oxford English Dictionary defines representation in two contexts: xford English Dictionary defines representation in two contexts: 1. To describe or depict something, to call it up in the mind by description or portrayal or imagination; to place a likeness of it before us in our mind or in the senses 2. To symbolize, stand for, to be a specimen of or to substitute for. For example, the red cap worn by elders in Igbo land is a representation of their titles in society. 55 However, while looking at it from the angle of film studies, representation can be seen as an attempt by the media to re-present some aspects of societal reality. Goodwill, Good and Godfrey (2007) aligned to this thought by saying “Film is a representational system that communicates concepts and feelings in such a way as to enable interpretation of their meanings.” A film may invite its audience to understand a preferred reading of it yet a viewer’s social positioning may influence their reading of a film. Stewart and Kowaltzke (2007) explain that a “media representation is a depiction, a likeness or a constructed image. A representation can be a single image, a sequence of images or a whole program, written words, spoken words or song lyrics.” It is obvious that the concept has a broader meaning than the simple definition given by the Oxford English Dictionary. Hall (1997) emphasized that representation is an essential part of the process by which meaning is produced and exchanged between members of a culture. Just like any other work of art, an element of representation is capable of drawing several interpretations. It therefore means that sometimes the intent of the movie maker is not meant to suggest a misrepresentation of a phenomenon but because the two step flow theory is active, the concept will be subjected to various interpretations by opinion leaders which could in the long run result in controversies. Representations invite audiences to understand them and agree with them in certain preferred ways. However, depending on the experiences of the audience, a single representation in a movie is bound to receive different interpretations. According to Stewart and Kowaltzke (2007), representations have a mode of address. CINEJ Cinema Journal: Andrew Ali Ibbi Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.ed What Is Representation? This implies that Hidden behind the apparent naturalness of the representation will be some assumptions about who you are. For example, if girls are portrayed as only belonging to the kitchen and can only play second fiddle to men, the possibility of the men feeling important in the house is high. CINEJ Cinema Journal: Andrew Ali Ibbi 56 Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu To them, the movie is addressing them as the lords over the women. In the same vein, some weak minded women may see themselves as being unfortunate in life thereby giving up on dreams they might have been building. An aspect of representation that is the crux of this paper is stereotype. This is seen as a judgmental type of representation. Stewart and Kowaltzke (2007) see stereotype as “A stereotype is an oversimplified, clichéd image, repeated so many times that it seems to have established a pattern. It is a highly judgmental type of representation.” Stereotypes are an extreme form of representation. They are constructed by a process of selection. Certain aspects are focused on and then exaggerated. This is in line with the views of Branston and Stafford (2006:241), “stereotypes are widely circulated ideas or assumptions about particular groups.” At the same time, an evaluation is made and the audience is invited to make a judgement, which is often based on prejudice. Repetition establishes stereotypes and over time allows them to appear natural and justified as part of societal norms. Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films Stereotypes in Films: A General Overview According to Branston and Stafford (2006), stereotyping has been a key concept in media studies, and is now perhaps too taken for granted. Many mistakes are made in using the term, which does not describe people or characters. It is easy to associate a particular character in a movie with a role that suites his personality in a movie. People find it difficult to distinguish between reality and fiction. A character in a horror movie stands the chance of suffering societal rejection as a result of a brutal role he/she has played in a movie. In an empirical study, Smith (2008: 12) was concerned that “females take up half the space in society, yet, especially in films aimed at children, they appear much less frequently than do males. Nevertheless, when they do make it onto the silver or small screen, their portrayals can 57 undermine their presence by being “hyper-attractive” or “hypersexual” and/or passive.” In trying to address this concern, Haralambos traced the genesis of gender stereotype representation to a verse in the Holy Bible (Genesis 3: 16) where God said: “I will greatly multiply thy sorrow and thy conception; in sorrow thou shalt bring forth children and thy desire shall be to thy husband and he shall rule over thee.” The following assumptions can be drawn from the quote above: undermine their presence by being “hyper-attractive” or “hypersexual” and/or passive.” In trying to address this concern, Haralambos traced the genesis of gender stereotype representation to a verse in the Holy Bible (Genesis 3: 16) where God said: “I will greatly multiply thy sorrow and thy conception; in sorrow thou shalt bring forth children and thy desire shall be to thy husband and he shall rule over thee.” The following assumptions can be drawn from the quote above: 2. Women are mothers and wives. 3. Women do the cooking, cleaning, sewing and washing. 4. They take care of men and are subordinate to male authority. hey are largely excluded from high status occupations and from positions of power Downing and Husband (2005) quoted Winant (1994) as saying that stereotype representation of gender and their likes are essentially metaphors for institutionalized social relationships that combine processes of exploitation and domination, on the one hand, with processes of subjection and representation, that is, with struggles over meaning and identity, on the other. Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu CINEJ Cinema Journal: Andrew Ali Ibbi Stereotypes in Films: A General Overview The authors are of the opinion that those responsible for gender stereotypes will first of all rely on a strong universal statement to establishes the reason for such representation. Going by this statement, it is obvious that the biblical verse quoted earlier will form the basis for misinterpretation resulting in justifying female stereotype representation in society. The mass media mirrors the society and since film is a mass medium, it is not an exception. Dutt (2014) aligned with this by saying: “Many cultural constructions, societal norms, fantasies and historical moments are conveyed and understood through films.” A clear indication that considering the importance of women in society, how they are represented in films should be taken seriously. According to Kord (2005), “films show us what we are, what we were, and what we could, should, or (do not) want to be. When at their best, they give birth to new visions of female CINEJ Cinema Journal: Andrew Ali Ibbi CINEJ Cinema Journal: Andrew Ali Ibbi 58 Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu strength and freedom. At their worst, they ridicule, denigrate, deny what real women have long achieved, and replaced it with spectres from the past.” Kuhn (1982) believes that women are increasingly used as visual accessories. Changes in film images have not always paralleled actual changes in society. Particularly with regard to the depiction of women, we can see how social values mediate between changes in the real world, the images that become available on television, and viewers’ choices of film images to watch. Women in films of the 1930s and 1940s seldom ventured outside of their socially prescribed roles as sweethearts, wives or mothers to the male hero. By providing a romantic interest for the hero, the woman served the function traditionally assigned to her gender (particularly in film) while allowing the male character to play out his own pre-ordained role (Stewart and Kowalztke, 2007: 40). According to Dutt (2014), “today’s on-screen women need to have it all, and then some; ‘the gorgeous Amazonian buttkicker’ with the great ass snug in super heroic spandex.” Pevere (2003) qualified this class of women as the “empowered” woman of corporate consumer society. To Ross (2006), women must be strong, aggressive, but still beautiful and sexy. CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films Stereotypes in Films: A General Overview Regrettably, this so-called tough woman is a ‘testament to a still male-dominant society’s own contradictory responses to women’s demands for equal treatment. Dutt (2014) points out that a duality is exhibited in most female characterizations in Hollywood (respect and rebellion, beauty and brains, power and submission, sexuality and timidity, and so on.) so that all viewers relate to and enjoy something. This position has been shared by many scholars around the world on the same subject matter and the Nigerian film industry is not an exception. CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films 59 Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu CINEJ Cinema Journal: Andrew Ali Ibbi Stereotype Representation of Women in Nigerian Movies The Nigerian film industry gained global recognition in the 2000s. It is one of the fastest growing film industries in the world. According to Adeshokan (2006), while reacting to reviews made on the Nigerian movie industry, “Very few of the commentaries bother to describe the films in any intelligible manner or even historicize them. Mere familiarity with the Nollywood label does not prepare anyone for films made in several Nigerian languages, nor does it draw attention to the vast use of video and digital technologies as markers of new social identities outside of filmmaking.” Nevertheless, Nollywood (as the industry is called) films have continued to enjoy patronage especially around the continent of Africa. This popularity has been further encouraged by the availability of different cable and satellite television platforms within the continent who have dedicated channels for Nollywood films. Adjudged as the third largest film industry in the world (Opeyemi, 2008), Nollywood has transformed over time from crude to professional filmmaking with a good number of its actors, directors, actors, producers and screenwriters now going into partnership with international movie producers. There is no disputing the fact that the media mirror’s society. This therefore means that representation in Nigerian films is a subtle suggestion to the world that this is exactly what is happening in Nigeria. Barber (1987) said “The views that ordinary people express may be false consciousness, but they are also their consciousness . . . conservative and misogynist, and sometimes frivolous . . . the people’s arts represent what people do in fact think, believe, and aspire to.” According to Bryce (2012) “Nollywood films, notably, care little for realism, beyond a love of surface: porticoed mansions, luxurious furnishings, gorgeous clothes, expensive cars, the proliferating signs of wealth by which they signal power and its attributes along with bodies, rich, CINEJ Cinema Journal: Andrew Ali Ibbi 60 Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu intact, and beautiful or poor, diseased, and abject.” There is no doubt that the industry has tried in its own way to project Nigeria in good light. Though the women have made a name for themselves in the Nigerian film industry, there have been lamentations from media scholars over the stereotype representation of women in Nigerian films. Stereotype Representation of Women in Nigerian Movies Okome (1997) as quoted by Shaka and Uchendu (2012) lamented that since the release of Living in Bondage the thematic preferences of Nollywood movies are based on the notions of inherited stereotyping of women perpetuated by patriarchy. Adeshokan (2006) admits that Nigeria is suffering from an image problem in the international community. The more reason why film makers should encourage positive stereotypes of women in films. This will help in boosting the image problem that Adeshokan (2006) was talking about. Having said this, scholars, especially feminists are of the opinion that the film industry has continued to adopt the patriarchal Nigerian society where the woman must be a submissive housewife to her husband, irrespective of whatever way he chooses to behave in society. Some noticeable stereotype representation of women in Nigerian films are as described below: CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu The girl next door The girl next door This is the stereotype of the sweet and trusting young woman who is portrayed as the ideal marriage prospect. This is the dream of every man. After messing around with several women, every mother in Nigerian films will tell her son to marry a ‘decent’ girl, preferably a virgin. Hence the man will go about searching for that ‘decent’ girl. In the Igbo film Nwa Mary, Edu Brazil that had messed around with another girl comes after Rachael Okonkwo, a religious girl that has preserved herself according to the dictates of the Bible. Sometimes, this search for a decent girl by mothers for their sons usually leads them into getting girls who will pretend to be good girls. As it is in Native Fowl where Clems Ohamezie’s mother was preserving the wayward Nuella Njubigbo (who pretended to be a virgin) for her son. The femme fatale This deadly woman uses her sexuality to destroy men in order to further her own ends; it is not usually love she is after. This is represented in many films like Aki the Blind where Ebube Nwogwu, the new wife of Amaechi Muonaguor uses her power as a woman to manipulate the rich man. Despite belonging to a cult, he obeyed every instructions she gave him. Similarly, in the Three Generals, Patience Ozokwor, in one of her roles as General has a set of girls that she sends to men to extract information and rob them. CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films 61 Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu CINEJ Cinema Journal: Andrew Ali Ibbi The career woman The career woman Since the 1980s the stereotype of the career woman has become increasingly common. She is successful but she can also be ruthless. In most cases, despite her level of success, a man will be needed to help in taking care of her emotional problems. This can be seen in movies like Anita starring Jackie Appiah, a successful lawyer and a faithful wife. The housewife This is the most favored stereotype in Nigerian films, the housewife exists only to support her husband and children. In line with the patriarchal nature of the society, a good wife by Nigerian standards is one that is totally submissive to the husband in his struggles in life. This spans beyond just taking care of the house or the kitchen as many men will say a woman’s place is in the kitchen. Out of the little she gets from the husband, she engages in petty trading to support the household. In most cases, even in her struggles, it is common to see the husband accusing her of engaging in prostitution to get money. Even with the accusations, the husband takes the money she has saved for the upkeep of the family to go after liquor and other women. In Baby Dance With Tears starring Mercy Johnson, she had to marry a man that she felt she was indebted to because he did her a favor. She was so supportive in the man’s business that even when she was pregnant, she was compelled to go out and dance until she had a miscarriage. Gold diggers and trophy wives These stereotypes perpetuate the myth that women marry for money and are reliant on men to support them. This can be seen in films like The Prince and the Slave where Patience Ozokwor in her bid to ensure that her daughter got married to the prince out schemed Eve Essin who the prince was in love with. This is a common representation in many films that involve a prince or a son of a wealthy man looking for a wife to settle down with. The gold diggers can come in two forms, sometimes it is the greedy mother that will force the daughter in to marrying the rich man as the case mentioned above in other times, it is the girl that will maneuver her way into the man’s life using whatever means possible. An example is in the movie, Prince Apart where Chacha Eke played into the hands of Uche Odoputa, the younger brother of her fiancé, Kenneth Okonkwo, just because she thought the former will be crowned king. CINEJ Cinema Journal: Andrew Ali Ibbi CINEJ Cinema Journal: Andrew Ali Ibbi 62 CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu The object of ritual This stereotype portrays women as the best options for rituals by rich men and even the gods. Young girls, virgins in most cases are usually the targets. If virgins are not targeted, it will be either the university campus girls or any woman the rich man can lure in to his trap. In the film, Agafe apart from the detailed sexual content of some scenes, the character Frank Artus spent money on young girls and used them as sacrificial lambs. Similarly, the movie, Seven Rivers had scenes of a rich man who deceived girls to come to his apartment where they are used for sacrifice. The object of barter This represents women as objects that can be used for exchange by their parents when indebted to a rich man and cannot pay. This is seen in the Yoruba movie, Labake Omo Oko where Faithia Balogun had to be married to a man against her wish just because her father is indebted to the man. In some cases, it is as a result of the parent’s greed for material things that they will 63 Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu compel the daughter to marry the man she does not like. An example is the Igbo film Nne Ifeadigo where Patience Ozokwor pushed her daughter, Nuella Njudigbo to marry a drug baron. Objects of sexploitation This stereotype portrays women as easy targets for sexual exploitation by men. In most cases, it will be the boss that will be exploiting the house girl. As in Baby Dance with Tears where Mercy Johnson was constantly harassed by her aunt’s husband. Her resistance put her in the bad books of her aunt. In Labake Omo Oko, Faithia Balogun while escaping from her matrimonial home as a result of maltreatment from her cowives, sought refuge in the house of a family acquaintance who in the middle of the night raped her and when the wife of the man eventually caught them, Faithia was asked to leave. CINEJ Cinema Journal: Andrew Ali Ibbi Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu Prostitutes This stereotype is common among young city girls trying to earn a living, especially campus girls. It suggests that the easy way for a woman to hustle in the city for better lifestyle is either to be sleeping around with different men or to serve as a mistress to a man who will service her needs even when she knows he is married. This is well expressed in films like Blackberry Babes. An indication that girls can use their bodies in exchange for money. Second choice This stereotype suggests that the girl child is less important in society when compared to the male child. At the point of conception, the family of the husband will be expecting the wife to give birth to a male. It makes it look as if the power to give birth to male children lies with the woman alone. It suggests that giving birth to girls is not a good option if a woman must be loved by her in-laws. In mother In-law, Camila Mberekpe was maltreating her son’s wife for not giving him a male child. In her desperation to get a male grandson, she had to marry a girl from the village for her son without his knowledge. This impression has come to affect the psyche of many Nigerian women in real life to the extent that the moment they conceive, they will be praying for a baby boy The witch This stereotype portrays women especially old women as witches whose mission is to deny the progress of young men and women. In Twins in Sorrow, Patience Ozokwo hated her son Clems CINEJ Cinema Journal: Andrew Ali Ibbi 64 Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu Ohamezie so much that she kept trying to kill him until she finally succeeded, after reducing him to a pauper. This is one out many examples of Nigerian films. Ohamezie so much that she kept trying to kill him until she finally succeeded, after reducing him to a pauper. This is one out many examples of Nigerian films. CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu Husband killer This stereotype portrays women especially wives of rich men as those who will eliminate the husbands just to inherit his wealth. It is common in Nigerian films to see the wife being accused of killing her husband. In most cases, the woman is made to take some oaths and perform some CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films 65 rituals that are harmful to her health. For example, in Widow’s Fate, Queen Nwokoye was accused of killing her husband and for that reason subjected to several wicked treatment engineered by her brother in law, Chiwetalu Agu, who is indeed the culprit. Bryce (2012) saw a potential danger in this trend. “Nollywood, by constantly returning us to traditional notions of the good, submissive wife and nurturing mother, does not represent the reality of women’s position in Nigeria and the manifold aspects of their self-realization.” Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu CINEJ Cinema Journal: Andrew Ali Ibbi Conclusion Although representation has been a subject of media studies for a very long time, Stewart and Kowaltzke (2007) posited that “After nearly 40 years of modern feminism, more than 30 per cent of advertising still portrays women as slim, blond bimbos less than 30 years old.” Within the Nigerian society, it is a major area of debate as various groups who feel misrepresented come up with forums to find ways of changing the situation. Bryce (2012) explains one of such forums. “ In 2010, a two-day forum, ‘Nollywood and the Dynamics of Representation,’ took place in Lagos, calling on participants to correct certain negative impressions created in our movies on the womenfolk who, it was claimed, come as wicked, manipulative, loose in morals, diabolic, and inferior to the men.” After this forum, several such forums have taken place in different dimensions. In the de facto taking over of responsibility for national identity by cinema since the 1990s, what is at stake is as much economic as cultural, as much about material power as about representation. This is because cinema is both an industry implicated in wealth-generation and a cultural medium that reaches deep into the domestic space of consumers for whom an elite literature is inaccessible. As a result, questions arise about the role and status of Nollywood as a CINEJ Cinema Journal: Andrew Ali Ibbi 66 Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu “national” cinema. (Bryce, 2012: 74). A devotion of quality time to research on the cultures in the country and a reflection of the present trend existing in the country will help in a more balanced approach to representation of women in Nigerian films. Stereotypes can be both positive and negative. It is time Nigerian filmmakers started looking the way of positive stereotype representation of Nigerian women in films, instead of continuous inundation of the market with negative stereotype representation. Volume 6.2 (2017) \| ISSN 2158-8724 (online) \| DOI 10.5195/cinej.2017.166 \| http://cinej.pitt.edu CINEJ Cinema Journal: Stereotype Representation of Women in Nigerian Films Recommendations The representation of women is an area that needs to be further researched upon. Filmmakers must try to reflect the current situation in society while bearing in mind the fact that society is dynamic. So many positive things about women have been going on around the country. These things are capable of bringing good scripts that will portray women in good light. There is no denying the fact that such issues reflected in Nigerian films like prostitution, gold diggers husband killers are truly things that happen in society. But it should be noted that the more the project these things on a daily basis, the more members of the audience will see them as the right thing to do, thereby increasing the chances of their flourishing. Good research will help in taking the minds of the audience away from such negative stereotypes and focus them on other issues happening in the society. Women should be involved in film production as directors and producers instead of allowing men to dominate the industry. Their own side of the story will always give the type of representation they want. This trend is beginning to happen as many female producers and directors are beginning to be involved in the Nollywood project. People like: Funke Akindele, Stephanie Okereke Linus, Rita Dominic, Uche Jombo among others have worked on award winning movies as producers and directors. Having more of them in the industry will turn negative 67 representation into positive ones that will make the society to treat the women as equals and sisters in progress. BIBLIOGRAPHY Adeshokan, A (2006). ‘Issues in the New Nigerian Cinema.’ Black Camera. Vol 21. No.1: 6 Aina, I. Olabisi (1998) “Women, culture and Society” in Amadu Sesay and Adetanwa Odebiyi (eds). Nigerian Women in Society and Development. Ibadan: Dokun Publishing House. Allanana, M. G. (2013). Patriarchy and Gender Inequality in Nigeria: The Way Forward. European Scientific Journal. Vol 9, 17: 115-144. Amobi, I (2013). 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https://openalex.org/W2505664412	https://journals.eco-vector.com/jowd/article/download/1327/947	Russian	null	Instrumental methods of diagnosis of ectopic pregnancy	Žurnalʺ akušerstva i ženskihʺ boleznej	2,015	cc-by	8,111	■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. По данным автора, гипотония матки наблюдалась только у 40 % женщин с ЭБ, а у 52 % женщин, имевших трубную беременность, контрастом на- полнялись обе трубы [48]. Пионером визуализации эктопической бе- ременности был Klaus K., который в 1908 году с помощью рентгенологического метода уста- новил диагноз переношенной брюшной бере- менности [30]. Прежде диагноз устанавлива- ли на основании клинико-анамнестических и объективно-пальпаторных данных. Поскольку в 98 % случаев эктопическая беременность (ЭБ) имеет трубную локализацию, при которой обычно она прерывается в первом триместре, то в 80 % случаев этот диагноз устанавливали на стадии разрыва маточной трубы, сопрово- ждавшегося яркой клинической картиной боле- вого и гиповолемического шока и внутреннего кровотечения [29]. По данным автора, гипотония матки наблюдалась только у 40 % женщин с ЭБ, а у 52 % женщин, имевших трубную беременность, контрастом на- полнялись обе трубы [48]. Для улучшения визуализации контуров об- разования в малом тазу и определения их разме- ров I. F. Stein et al. (1927) при рентгенологическом исследовании предложили использовать искус- ственный пневмоперитонеум [51]. р у [ ] По данным C. Weinberg et al. (1963), сочета- ние пневмопельвиографии с ГСГ увеличивает диагностические возможности каждого из мето- дов при диагностике трубной беременности, по- скольку их использование позволяет не только определить расширенную тень маточной трубы и расширение ее трубного угла, но и определить нарушение прохождения контраста по маточной трубе в случае нахождения в ней элементов плод- ного яйца [53]. р [ ] С целью более ранней диагностики ЭБ R. Dy roff (1926) предложил использовать метод гисте- росальпингографии (ГСГ) у женщин, имеющих подозрение на трубную локализацию плодного яйца до 12 недель беременности. Автор описал комплекс признаков патогномоничных для диаг ностики в этот срок: гипотоничная полость матки при отсутствии в ней плодного яйца, отсутствие тени трубы или дефект наполнения в расширен- ной трубе, расширение трубного угла матки с по- раженной стороны [23]. Однако K. F. Schultze Gunter (1939), проведя оценку этих признаков ме- тодом ГСГ у 25 женщин, имевших трубную бере- менность, сопоставил их с данными лапаротомии и не подтвердил их диагностическую значимость. С 80‑х годов XX столетия на первое место вы- ходят неионизирующие методы визуализации: ультразвуковая диагностика (УЗД), магнитно- резонансное исследование (МРИ) [2, 27]. Эхографию для диагностики ЭБ впервые при- менили M. Kobayashi et al. (1969). УДК: 618.31-07 УДК: 618.31-07 Обзоры Обзоры © T. M. Ishutina D. O. Ott Research Institute for Obstetrics and Gynecology, Saint Petersburg, Russia ■ The article presents a literary review of the capabilities of modern methods of instrumental diagnostics (x-ray, ultrasound, magnetic resonance research) with the assessment of their significance in the diagnosis of ectopic pregnancy with a brief historical digression. Инструментальные методы диагностики эктопической беременности © Т. М. Ишутина ФГБНУ «НИИ АГиР им. Д. О. Отта», Санкт-Петербург ФГБНУ «НИИ АГиР им. Д. О. Отта», Санкт-Петербург ■ В статье представлен литературный обзор современных методов инструментальной диагностики (лучевая, уль- тразвуковая диагностика, магнитно-резонансное исследование) с оценкой их значимости в диагностике эктопиче- ской беременности. ■ Ключевые слова: эктопическая беременность; ультразвуковая диагностика; магнитно-резонансное исследование; цветное допплеровское картирование; эластография. ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. М. Стыгар (1981) описали эктопически расположенное плодное яйцо как «симптом кольца, венчика» — кистозное образо- вание овальной формы, с повышенной плотности ободком. Этот симптом в совокупности с нали- чием свободной жидкости в Дугласовом кармане авторы считали патогномоничным для эктопиче- ской беременности [4]. Однако при 100 % специ- фичности он обладал низкой чувствительностью (от 5 до 29,1 %, по данным различных авто- ров) [3, 7, 12]. Так трубную беременность не уда- валось установить и локализовать плодное яйцо в полости матки ТА-методом раннее 6,5 недели менструального срока. Точность трансабдоми- нальной эхографии при выявлении ЭБ, по дан- ным В. Н. Демидова, Б. И. Зыкина (1990), не пре- вышала 25–30 % [3]. ТАУЗИ ЭБ в основном базировалась на симптоме отсутствия плодного яйца в полости матки, а не на визуализации при- даткового образования, что служило поводом для направления пациенток на диагностическую ла- пароскопию, при которой диагноз не всегда под- тверждался [29]. р К концу 1980‑х — началу 1990‑х гг. согласно публикациям того времени информативность ультразвуковой диагностики (УЗД) ЭБ при ТАУЗИ достигла 60–70 % [3, 20, 45]. Прорывом в ранней диагностике ЭБ стало вне- дрение в клиническую практику трансвагиналь- ной эхографии (ТВУЗИ). К началу 1990‑х годов большинство ученых придерживается мнения, что только ТВУЗИ позволяет с высокой степенью точ- ности и в относительно ранние сроки обнаружить эктопическую нидацию плодного яйца. К пре- имуществам данного метода А. Н. Стрижаков, А. И. Давыдов и соавт. (1998) относят отсутствие необходимости в специальной подготовке па- циенток, высокую разрешающую способность трансвагинальных датчиков, обеспечивающих идентификацию паталогического расширения маточных труб (с 8–10 мм). Плодное яйцо в по- лости матки стало возможно обнаружить на неде- лю раньше, чем при ТАУЗД [8]. T. S. Mehta et al. (1999) установили, что при ЭБ особенности эхо- структуры эндометрия отсутствуют [37]. По дан- ным ряда исследований [6, 9, 18], установлено, что утолщенный до 12–24 мм эндометрий с мел- кокистозными включениями встречается лишь у 14–27,8 % женщин с ЭБ, а у 33 % из них толщи- на эндометрия не превышает 3 мм. Кроме того, у 20 % женщин, имеющих ЭБ, в полости матки будет определяться так называемый «pseudosac» (ложное плодное яйцо), образованное секретом децидуального эндометрия или участками его от- слойки [18]. Дифференциальная диагностика ис- тинного и ложного плодного яйца основывалась на том, что истинное плодное яйцо должно, как правило, соответствовать сроку беременности, иметь округлую правильную форму, окруже- но 2–4 мм толщиной гиперэхогенным кольцом С целью улучшения диагностики ЭБ В. Н. Де мидов и Б. И. ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. Авторы раздели ли ее акустические признаки на маточные (увели- чение размеров матки, отсутствие в полости матки плодного яйца, появление в миометрии диффуз- ных сигналов с высокой плотностью) и внема- точные (визуализация в области придатков матки ISSN 1684–0461 ТОМ LXIV ВЫПУСК 5/2015 ОБЗОРЫ ОБЗОРЫ 78 ятным признакам (точность диагностики 78 %): увеличение тела матки и визуализация около нее небольшого кистозного образования (плодное яйцо) с характерным эхопозитивным венчиком; наличие свободной жидкости (кровь) позади мат- ки и в латеральных каналах живота, визуализация отдельных аморфных эхосигналов (сгустки кро- ви) в жидкости позади маточного пространства, сочетание свободной жидкости и образования без четких контуров с гетерогенной внутрен- ней структурой рядом с маткой. Среди возмож- ных признаков ЭБ (точность диагностики — 14 %): увеличение тела матки, визуализация в позади-маточном пространстве свободной жид- кости, не содержащей дополнительных эхострук- тур, наличие образования жидкостной, сме- шанной или плотной неоднородной структуры с неровными или нечеткими контурами в области придатков матки [3]. патологических образований без четких контуров с неоднородной структурой, а также эктопически расположенного эмбриона) [31]. Исследования проводили в В‑режиме, трансабдоминально кон- вексным датчиком при наполненном мочевом пузыре (ТАУЗИ). По мере развития эхографии и усовершенствования парка ультразвуковых сканеров критерии, предложенные M. Kobayashi et al. (1969), модифицировали и дополняли. Так, Б. И. Зыкин и А. М. Стыгар (1981) описали эктопически расположенное плодное яйцо как «симптом кольца, венчика» — кистозное образо- вание овальной формы, с повышенной плотности ободком. Этот симптом в совокупности с нали- чием свободной жидкости в Дугласовом кармане авторы считали патогномоничным для эктопиче- ской беременности [4]. Однако при 100 % специ- фичности он обладал низкой чувствительностью (от 5 до 29,1 %, по данным различных авто- ров) [3, 7, 12]. Так трубную беременность не уда- валось установить и локализовать плодное яйцо в полости матки ТА-методом раннее 6,5 недели менструального срока. Точность трансабдоми- нальной эхографии при выявлении ЭБ, по дан- ным В. Н. Демидова, Б. И. Зыкина (1990), не пре- вышала 25–30 % [3]. ТАУЗИ ЭБ в основном базировалась на симптоме отсутствия плодного яйца в полости матки, а не на визуализации при- даткового образования, что служило поводом для направления пациенток на диагностическую ла- пароскопию, при которой диагноз не всегда под- тверждался [29]. патологических образований без четких контуров с неоднородной структурой, а также эктопически расположенного эмбриона) [31]. Исследования проводили в В‑режиме, трансабдоминально кон- вексным датчиком при наполненном мочевом пузыре (ТАУЗИ). По мере развития эхографии и усовершенствования парка ультразвуковых сканеров критерии, предложенные M. Kobayashi et al. (1969), модифицировали и дополняли. Так, Б. И. Зыкин и А. ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. (2007), не подтверждает разрыва маточных труб, так как кровь обычно вы- текает из фимбриального отдела маточной трубы как при прогрессировании трубной беременно- сти из разрушенных в результате инвазий ворсин хориона мелких сосудов, так и при трубном вы- кидыше [7]. Если уровень эхогенной жидкости в полости малого таза, оцененной при ТАУЗИ, достигает дна матки или определяется в маточно- пузырном кармане, или в подпеченочном, или под- диафрагмальном пространстве, гемоперитонеум считается значительным (более 300–400 мл) и тре- бует неотложной хирургической помощи [31]. D. Jonathan et al. (2010) предлагают использовать метод ТВУЗИ как метод выбора в диагностике ЭБ [27]. Однако если этим методом не получе- но достаточной информации, то рекомендуют использовать ТАУЗИ для осмотра поддиафраг- мального пространства, Морисонова кармана и возможной внематочной локализации плодного яйца [27, 29]. и расположено эксцентрично за счет погружения в слой эндометрия [7]. Кроме того, Р. М. Doubilet и D. Benson (2010) установили, что наличие жид- кости в полости матки чаще встречается при ран- них сроках маточной беременности, чем при эк- топической ее локализации [22]. [ ] Проводились работы, посвященные сопо- ставлению эхографической картины с интраопе- рационными данными. Их задача заключалась в выявлении ультразвуковых симптомов, харак- терных для разных видов течения и осложения трубной беременности [11]. Так, И. А. Озерская и Н. К. Есаян (2007), сравнив ультразвуковые сим- птомы при разрыве маточной трубы и трубном вы- кидыше, пришли к выводу об отсутствии надеж- ных критериев их дифференцирования. При этом предположили, что эхографическое изображение, вероятно, зависит не столько от типа прерывания трубной беременности, сколько от давности пре- рывания, степени выраженности и длительности кровотечения, срока беременности, наличия или отсутствия спаечного процесса [7]. у р ц [ ] Pereira P. P. et al. (2009) сопоставили эхосим- птомы ампулярной беременности (трубное коль- цо и эктопическое плодное яйцо с живым эмбри- оном) с данными гистологического заключения о глубине инвазии трофобласта в стенку маточной трубы [43]. По данным авторов, при 1-й степени инвазия трофобласта ограничивалась слизистой, при 2‑й — трофобласт внедрялся в мышечный слой и при 3‑й — полностью инфильтрировал стенку маточной трубы. По заключению авторов, визуализация эктопического плодного яйца с жи- вым эмбрионом, в 82,1 % случаев соответство- вала 3‑й степени инвазии трофобласта в стенку маточной трубы. При выявлении эхосимптома «трубное кольцо» наиболее часто (41,3 %) опре- делялась 1‑я стадия инвазии трофобласта в стен- ку маточной трубы [43]. Эти выводы, совпавшие с результатами других исследователей (A. Natale et al. (2003), F. R. Cabar et al. ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. Зыкин (1990) разработали ряд кос- венных эхопризнаков: р 1. Наличие придаткового образования сложной структуры, образуемого в результате трубного разрыва или выкидыша. р р 2. Отсутствие плодного яйца в полости матки. 3. Увеличение размеров матки меньшее по срав- нению с размерами предполагаемого срока беременности (при отсутствии патологии матки). ) 4. Утолщение эндометрия (децидуальная реакция). 5. Обнаружение ложного плодного яйца в поло- сти матки. 6. Выявление свободной жидкости в полости ма- лого таза [3]. Авторы предложили актуальную по настоящее время классификацию эхокартины внематочной беременности (ВБ), основанную на совокупности диагностических признаков, имеющих абсолют- ное, вероятное или предположительное значение. Абсолютным признаком ЭБ (точность диагнос тики 100 %) считали эктопически расположен- ное плодное яйцо с живым эмбрионом. К веро- ТОМ LXIV ВЫПУСК 5/2015 ТОМ LXIV ВЫПУСК 5/2015 ISSN 1684–0461 79 ОБЗОРЫ мелкодисперсную взвесь и сгустки крови в случае их присутствия в жидкости Дугласова кармана. Наличие эхогенной свободной жидкости, по дан- ным A. C. Fleischer et al. (1990), D. N. Nyberg et al. (1991), встречается у 28–56 % женщин с ЭБ и по- ложительно коррелирует с данными об объеме гемоперитонеума, полученными интраопераци- онно [25, 40]. Однако этот симптом, по мнению И. А. Озерской и соавт. (2007), не подтверждает разрыва маточных труб, так как кровь обычно вы- текает из фимбриального отдела маточной трубы как при прогрессировании трубной беременно- сти из разрушенных в результате инвазий ворсин хориона мелких сосудов, так и при трубном вы- кидыше [7]. Если уровень эхогенной жидкости в полости малого таза, оцененной при ТАУЗИ, достигает дна матки или определяется в маточно- пузырном кармане, или в подпеченочном, или под- диафрагмальном пространстве, гемоперитонеум считается значительным (более 300–400 мл) и тре- бует неотложной хирургической помощи [31]. D. Jonathan et al. (2010) предлагают использовать метод ТВУЗИ как метод выбора в диагностике ЭБ [27]. Однако если этим методом не получе- но достаточной информации, то рекомендуют использовать ТАУЗИ для осмотра поддиафраг- мального пространства, Морисонова кармана и возможной внематочной локализации плодного яйца [27, 29]. мелкодисперсную взвесь и сгустки крови в случае их присутствия в жидкости Дугласова кармана. Наличие эхогенной свободной жидкости, по дан- ным A. C. Fleischer et al. (1990), D. N. Nyberg et al. (1991), встречается у 28–56 % женщин с ЭБ и по- ложительно коррелирует с данными об объеме гемоперитонеума, полученными интраопераци- онно [25, 40]. Однако этот симптом, по мнению И. А. Озерской и соавт. ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. (1992), чувствительность ис- пользования режима ЦДК для диагностики ЭБ еще выше и составляет 95 % по сравнению с 54 %, когда использовался только В‑режим [41]. Подобные же результаты подтверждает W. Lilyan et al. (2007): чувствительность метода повышается с 82,4 до 93,8 %, а специфичность с 42,9 до 55,6 %, точ- ность с 75,6 до 85,4 % [36]. По мнению J. S. Pellerito et al. (1992), если не использовать ЦДК, до 16 % ЭБ может быть пропущено [41]. 3. «Симптом бублика», или трубное кольцо, об- разовано расширенной маточной трубой с ис- тонченной стенкой вокруг «пустого» плодного яйца. Он встречается приблизительно в 20 % случаев, имеет 97,8 % ППЗ. у 4. «Эктопическое плодное яйцо» — плодное яйцо в проекции придатков, содержащее жел- точный мешок и/или эмбрион, в 10 % случаев с сердцебиением, встречается в 20 % случаев ЭБ со 100 % ППЗ [29]. Вместе с тем E. Kirk et al. (2014) считают, что такой высокий процент диагностики (90–92 %) может быть достигнут специалистами высоко- го уровня и на аппаратах экспертного класса, в противном случае процент диагностики ЭБ по данным первоначального УЗИ будет состав- лять — 74 % [29]. У 8–31 % женщин на ранних сроках локализацию плодного яйца по данным УЗИ установить не удается, из них у 7–20 % будет впоследствии диагноз внематочной беременно- сти [7, 29]. [ , ] Использование дополнительной опции ультра- звуковых сканеров (цветное допплеровское карти- рование (ЦДК) и импульсно-волновая (ИВ) доп- плерометрия) началось с середины 1980‑х годов, когда A. Kurjak исследовал кровоток в сосудах органов малого таза у женщин при различных па- тологических состояниях [33]. С 1990 по 1995 г. целый ряд авторов публикуют данные по иссле- дованию возможностей ЦДК в диагностике экто- пической беременности [3, 8, 21, 33]. Было выяв- лено, что особенностями кровотока эктопически расположенного трофобласта в режиме ЦДК является выраженная яркость цветовых сигна- лов и хаотическая их разбросанность в преде- лах эхогенной зоны придаткового образования. В случае эктопически расположенного плодно- го яйца трофобластический кровоток в режиме ЦДК при беременности более 4 недель опреде- ляется в виде замкнутого «огненного кольца». По данным ряда авторов, зона гиперваскуляри- зации придаткового образования в режиме ЦДК определяется в 80–92 % случаев ЭБ [17, 20, 21]. Отсутствие визуализации трофобластического кровотока в проекции придаткового образования может быть обусловлена малыми сроками бере- менности (диаметр плодного яйца 10 мм) или гибелью плодного яйца и отслойкой трофобла- ста [5, 6]. ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. (2006)), важны для выбора как тактики ведения (консервативная, выжидательная, хирургическая), так и вида опе- ративного пособия (тубэктомия или органосохра- няющие методики) при ЭБ [17, 38, 43]. К концу XX началу XXI столетия, благодаря возросшей доступности ультразвуковых ска- неров с высоким разрешением, более 80 % ЭБ диагностируют в начале первого триместра бе- ременности еще до разрыва плодовместилища, и более чем 50 % — до появления клинических симптомов [29]. Современная классификация эхосимптомов ЭБ, предложенная E. Kirk, C. Bottomley, T. Bourne (2014) позволяет в 90–92 % случаев ЭБ диагно- стировать по данным первичного ТВУЗИ: 1. Наличие неоднородного внеяичникового не кис тозного придаткового образования, субстатом которого является деформированная расши- ренная маточная труба с элементами отсло- ившегося, чаще погибшего, плодного яйца и сгустками крови. Этот признак встречает- ся приблизительно в 60 % случаев с частотой положительного прогностического значения (ППЗ) 88,6 %. ) р Нет единого мнения относительно значимости обнаружения свободной жидкости в Дугласовом кармане при ЭБ. В. Н. Демидов и Б. И. Зыкин (1990), D. N. Nyberg et al. (1991) считают, что жидкость в Дугласовом кармане определяется в 31 % случаев ЭБ, а ее наличие не всегда отража- ет разрыв или трубный аборт, т. к. свободная жид- кость в Дугласовом кармане может определяться и при физиологической беременности [3, 40]. Однако современные ультразвуковые сканеры по- зволяют различить более высокую эхогенность, 2. «Симптом капли» — выявление сферического полостного образования в виде трубы, суб- стратом которого является гематосальпинкс. Этот симптом имеет чувствительность 84 % и специфичность 99 %, 96 % ППЗ при 96 % ППЗ и 95 % ОПЗ в диагностике внематочной беременности. ТОМ LXIV ВЫПУСК 5/2015 ISSN 1684–0461 ОБЗОРЫ ОБЗОРЫ 80 ли этот показатель использовать в качестве диф ференциально-диагностического критерия истин ного и ложного плодного яйца [21]. Кроме того, в случае затруднения визуализации желтого тела при ТВУЗИ определение типичного кровотока в стенках желтого тела помогает его идентифика- ции. Последнее имеет значение для локализации зоны поиска эктопически расположенного тро- фобласта, в 85 % случаев расположенного ипси- латерально с желтым телом. В целом ЦДК не по- могает дифференцировать кровоток желтого тела или хориона эктопического плодного яйца, по- скольку характер и спектры их кривых скоростей кровотока (КСК) обычно бывают почти идентич- ны. Вместе с тем установлено, что комплексное применение трансвагинальной эхографии и ЦДК повышало информативность диагностики ЭБ по сравнению с использованием осмотра только в В‑режиме с чувствительностью 87 % по срав- нению с 71 % соответственно [21]. По данным J. S. Pellerito et al. ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. Asteria et al. (2008) доказали эффек- тивность оценки эластичности ткани на осно- ве распределения цвета, наложенного на изо- бражения в В‑режиме [14, 44]. Н. А. Воронцова, В. Е. Гажонова, Т. А. Чернышенко (2013) исследо- вали клиническую значимость соноэластографии в ранней диагностике внематочной беременности. Авторы описывают типичную для эктопического плодного яйца эластографическую картину: тип «голубого глаза» — округлого высокоплотного об- разования, расположенного между маткой и яич- ником, картировавшегося синим цветом в центре, окруженного четким высокоэластичным ободком красного цвета на фоне окружающих его эластич- ных тканей. Средний внутренний диаметр трубно- го образования, по данным авторов, в этих случаях составлял 3,5 ± 0,9 см [1]. У 9,8 % женщин, имею- щих неопределенные данные УЗИ в В‑режиме, в режиме ЭГ определялся тип изображения «го- лубой глаз». По данным Н. А. Воронцовой и со- авт. (2013), чувствительность ультразвукового исследования при применении соноэластографии в диагностике ЭБ составила 100 % [1]. В 2007 году N. M. Koji Waki и T. M. Takeshi Matsumura иссле- довали диагностические возможности количе- ственного показателя эластичности ткани — ко- эффициент деформации (SR). Они показали, что независимо от напряжения ткани, коэффициент деформации демонстрирует постоянные свой- ства, а его значение увеличивается по мере роста коэффициента упругости (SR) [32]. Hai-ling Wang et al. (2012) доказали, что с помощью коэффици- ента деформации можно достоверно повысить чувствительность с 78 до 80 % и специфичность с 87 до 92 % метода соноэластографии в диагно- стике злокачественных узловых образований щи- товидной железы [26]. До настоящего времени возможности количественной оценки эластично- сти ткани при эктопически расположенном плод- ном яйце не исследованы. Еще одним методом визуализации, преиму- ществом которого при его использовании стало отсутствие ионизирующего излучения, являет- ся магнитно-резонансное исследование (МРИ). Этот метод все чаще используется для оценки причин острой боли в животе у беременных [42]. По данным J. R. Leyendecker et al. (2004), повсе- местное оснащение больниц МР-томографами увеличивает возможность участия МРИ в диагно- стике ЭБ. Тем более что МРИ не уступает и даже превосходит КТ по возможностям обзора без ис- пользования контрастных средств и контрастиро- ванию жидкостьсодержащих полостей и объем- ной реконструкции [35]. Однако в отличие от УЗД МРИ применяется недавно и ее эффективность при ЭБ ранних сроков не изучена. р р р у S. A. Russell et al. ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. В качестве диагностических критериев эктопической трофобластической васкуляризации были предложены численные значения индек- са резистентности (ИР) — менее 0,40–0,45 при пульсационном индексе (ПИ) — менее 0,70 в со- судах трофобласта [33]. E. H. Dillon et al. (1990) также определили, что в сосудах трофобласта скорость кровотока не менее 21 см/с и предложи- Метод ЦДК, по мнению ряда авторов, не толь- ко повышает достоверность диагностики экто- пической беременности, но позволяет оценивать инволюцию плодного эктопического яйца или его элементов на фоне медикаментозной тера- пии [29, 34, 36]. Особенно это важно при меди- каментозной химиотерапии редких локализаций эктопического трофобласта (шеечной, шеечно- перешеечной, в рубце после кесарева сечения). А также помощь ЦДК неоценима при опреде- лении глубины инвазии трофобласта в стенку матки при интерстициальной трубной, угло- вой, в рудиментарном роге матки, в рубце по- сле кесарева сечения, шеечной и шеечно-пере шеечной ЭБ [5, 6, 8, 34]. Еще одним ультразвуковым методом, получив- шим признание при диагностике очаговых образо- ваний в различных органах на основании степени их упругости, оцененной по степени искажения ультразвуковой волны в цвете, является эластогра- фия (ЭГ) [10]. Несмотря на то, что о первых теоре- тических предпосылках эластографии сообщали с конца 80‑х годов XX века, первичные результаты клинического применения данной методики поя- вились лишь с конца 1990‑х [1]. Эластичность тка- ней отображается разными цветами на обычном ТОМ LXIV ВЫПУСК 5/2015 ISSN 1684–0461 81 ОБЗОРЫ ся магнитно-резонансное исследование (МРИ). Этот метод все чаще используется для оценки причин острой боли в животе у беременных [42]. По данным J. R. Leyendecker et al. (2004), повсе- местное оснащение больниц МР-томографами увеличивает возможность участия МРИ в диагно- стике ЭБ. Тем более что МРИ не уступает и даже превосходит КТ по возможностям обзора без ис- пользования контрастных средств и контрастиро- ванию жидкостьсодержащих полостей и объем- ной реконструкции [35]. Однако в отличие от УЗД МРИ применяется недавно и ее эффективность при ЭБ ранних сроков не изучена. экране В‑режима: более плотная структура тканей отображается оттенками синего, а легко сжимае- мые эластичные участки маркируются красной цветовой шкалой. По данным P. Chaturvedi et al. (1998), цвет выявленного в В‑режиме образования в режиме ультразвуковой эластографии варьиру- ет от красно-зеленого до преимущественно си- него по мере нарастания эхоплотности образова- ния. Также определяется класс цвета (эластотип) по шкале от 1 до 5 баллов с возрастанием балль- ности по мере сдвига от красного к синему цвету: 1 — красный, 2 — красно-зеленый, 3 — зеленый, 4 — зелено-синий и 5 — синий [19].T. Rago et al. (2007) и C. ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. Поэтому, учитывая вероят- ность осложнения течения беременности данной локализации разрывом плодовместилища важно обнаружить гематомы миометрия, признаки ин- вазии трофобласта в миометрий и гемоперитоне- ум [54]. По данным МРИ, нормальный миометрий имеет три различных слоя на Т2‑ВИ: гипоин- тенсивные наружный и внутренний слои и цен- тральный — гиперинтенсивный. Потеря непре- рывности контура внутреннего слоя миометрия может говорить об инвазии ворсин плацентарной ткани [54, 56]. Kataoka et al. (1999) считают МР-симптомом трубной беременности плодное яйцо в про- свете маточной трубы, которое определяется в виде кистозной структуры с утолщенной стен- кой, имеющей высокую интенсивность сигнала на Т2‑взвешенных изображениях (ВИ), характе- ризующуюся примыкающим к ней очагом свежей гематомы формы полумесяца средней или высокой интенсивности сигнала на Т1‑ВИ [28]. При этом четко дифференцированная отдельная структура в просвете маточной трубы, соответствующая плодному яйцу, наблюдается редко [54]. Чаще это деформированный кистозный компонент в струк- туре гетерогенных масс, находящихся в просвете маточной трубы, которая на отдельных изобра- жениях определяется как толстостенное кольцо, содержащее неоднородные массы с небольшими кистозными включениями [54]. При внутривен- ном введении контрастного вещества, по данным M. Nishino et al. (2002), определяется повышение интенсивности сигнала от стенки расширенной маточной трубы, а также компонентов, располо- женных в ее просвете [39]. Беременность в рудиментарном роге насту- пает при имплантации плодного яйца в пределах одного из рогов двурогой матки или матки с пе- регородкой, в зачаточном роге, сообщающимися или не сообщающимися с полостью матки [47]. По данным МРИ, при беременности в рудимен- тарном роге матки плодное яйцо или его элементы окружены слоем нормального миометрия и рас- положены вдоль латерального края нерудимен- тарного (доминирующего) рога. Доминирующий рог будет смещен вбок и по форме характеризо- ваться как «бананообразная матка» [49]. р [ ] МРИ помогает уточнить трудно дифферен- цируемые между собой виды беременности: ин- терстициальную, в углу матки, в рудиментарном роге матки. Понимание различий между этими субъектами имеет важное клиническое значение, поскольку тактика ведения и лечения таких паци- енток отличаются [54]. При интерстициальной бе- ременности плодное яйцо имплантируется в наи- более проксимальном отделе маточной трубы, расположенном в миометрии. По данным МРИ, при интерстициальной беременности элементы плодного яйца в виде неоднородных гиперинтен- сивных масс на Т2‑ВИ определяются латеральнее угла полости матки [24]. Брюшная беременность возникает, когда плодное яйцо имплантируется вне матки, маточ- ных труб и яичников и составляет примерно 1 % от всех ЭБ. Показатель материнской смертности при данной локализации достигает 20 % [13]. Отсутствие миометрия вокруг плодного яйца может быть важным диагностическим призна- ком. ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. (1993) считают, что нали- чие по данным МРИ геморрагической жидкости в Дугласовом кармане или в другом месте в брюш- ной полости при отсутствии маточной беременно- сти у пациенток с положительными результатами теста на беременность имеет 93 % положительно- го прогностического значения для диагностики внематочной беременности [46]. Поэтому, обна- ружив данный МР-симптом у беременной, необ- ходимо включать диагноз эктопический беремен- ности в дифференциально-диагностический ряд вместе с такими заболеваниям, как геморрагиче- ская форма апоплексии яичника при кисте желтого тела, аномалия прикрепления плаценты, рефлюкс крови через маточные трубы при самопроизволь- ном аборте. У беременных рекомендуют вклю- чать в протокол исследования импульсные после- довательности Т1‑взвешенных изображений для выявлении продуктов крови. По данным M. L. Tamai (2007), гематосаль- пинкс будет наиболее частой находкой при труб- ной беременности, т. к. инвазия ворсинок тро- фобласта в стенку маточной трубы приводит к кровоизлияниям в просвет маточной трубы [52]. Гематосальпинкс верифицируют как расширение маточной трубы за счет жидкостного содержимо- го, имеющего высокий уровень интенсивности сигнала на T1‑ВИ [54]. A. Rex et al. (2012) указы- вают, что если в просвете расширенной за счет продуктов крови маточной трубы определяются неоднородные массы, то вероятность трубной бе- ременности значительно возрастает [54]. Авторы отмечают, что наличие гематосальпинкса у жен- щин с положительными результатами теста на бе- ременность при отсутствии плодного яйца в по- лости матки делают очень высокой вероятность трубной беременности даже при отсутствии чет- ко определяемого эктопически расположенного плодного яйца [54]. W. G. Bradley et al. (1993) опи- сали МР-изображения гематомы в головном мозге по стадиям ее развития [16]. J. Yoshigi et al. (2006) предложили использовать эту классификацию для описания гематосальпинкса при ЭБ [56] (табл. 1). Еще одним методом визуализации, преиму- ществом которого при его использовании стало отсутствие ионизирующего излучения, являет- ТОМ LXIV ВЫПУСК 5/2015 ISSN 1684–0461 ОБЗОРЫ ОБЗОРЫ 82 Стадия Время Производые гемоглобина T1 T2 Сверхострая <24 часов Оксигемоглобин Средняя Средняя Острая 1 до 3 дней Дезоксигемоглобин Длительная Короткая Подострая — ранняя 3 + дней Метгемоглобин Короткая Короткая Подострый — поздняя 7 + дней Метгемоглобин Короткая Длительная Хронический — средняя 14 + дней Гемохроматин Средняя Средняя Хронический — поздняя 14 + дней Гемосидерин Средняя Короткая Таблица 1 Стадии гематомы дел маточной трубы. Важно также, что при бере- менности в углу матки МРИ позволяет определить аномалию прикрепления плаценты за счет четкой визуализации инвазии ворсин хориона в миоме- трий [15]. Вместе с тем высокая интенсивность сигнала в миометрии вокруг плодного яйца, опре- деляемая на Т1‑ВИ, будет свидетельствовать о на- личии кровоизлияния. Литература Воронцова Н. А., Гажонова В. Е., Чернышенко Т. А. и др. 1. Клиническая значимость соноэластографии в ранней диагностике внематочной беременности. Кремлевская медицина. Клинический вестник. 2013; 1: 106–11. Грязнова И. М. Внематочная беременность. М.: Медици- 2. на; 1980. Демидов В. Н., Зыкин Б. И. Ультразвуковая диагностика 3. в гинекологии. М.: Медицина; 1990. Демидов В. Н., Зыкин Б. И. Ультразвуковая диагностика 3. в гинекологии. М.: Медицина; 1990. Дергачев А. И. Ультразвуковая диагностика заболева- 4. ний внутренних органов: справочное пособие. М.: РУДН; 1995. Медведев В. М., Алтынник Н. А. Эктопическая беремен- 5. ность. В кн.: Допплерография в гинекологии. Под редак- цией Зыкина Б. И., Медведева М. В. 1‑е издание. М.: РАВУЗДПГ, Реальное время; 2000:145–9. Озерская И. А., Агеева М. И. Ультразвуковая диагностика 6. внематочной беременности. Ультразвуковая и функцио- нальная диагностика. 2005; (2): 101–12. Озерская И. А., Есаян Н. К. Возможности ультразвуковой 7. диагностики в определении типа прервавшейся трубной беременности. Ультразвуковая и функциональная диаг ностика. 2007; (2): 51–9. Стрижаков А. Н., Давыдов А. И., Шахламова М. Н., Бело- 8. церковская Л. Д. Внематочная беременность. М: Медици- на; 1998. Таким образом, сложился определенный алго- ритм инструментальной диагностики эктопиче- ской беременности. Первично проводится ТВУЗИ органов малого таза с пристальным исследовани- ем области придатков матки, боковых отделов матки, прямокишечно-маточного пространства. При необходимости большего обзора для оценки объема гемоперитонеума или поиска брюшной ло- кализации плодного яйца прибегают к ТАУЗИ ме- тодом наполненного мочевого пузыря. Для боль- шей уверенности в диагнозе можно дополнить В‑режим ТВУЗИ ЦДК с ИВ‑допплерометрией и соноэластографией. В случае неинформатив- ности первичного УЗИ назначают его повторно в динамике с интервалом от 2 до 7 дней в зави- симости от менструального срока беременности на момент обращения, прибегают к биохими- ческим методам диагностики ЭБ, МРТ органов малого таза. Флоренсова Е. В., Апарцин М. С. Особенности эхострук- 9. туры полости матки при внематочной беременности. Эхо- графия. 2002; 3 (1): 66–70. Чуркина С. О. Возможности соноэластографии в гинеко- 10. логии. Автореф. дис. канд. мед. наук. М.; 2011. Шония М. Б. Диагностика внематочной беременности 11. методом эхографии. Акушерство и гинекология. 1984; 1: 45–48. Alemano M. Q., Brizzolara M., Viora E. Echografia e gravidan- 12. zaextrauterina. Minervegynecol. 1985; 37 (9): 483–8. Alto W. A. Abdominal pregnancy. Am. Fam. Physician. 1990; 13. 41 (1): 209–14. Asteria C., Giovanardi A., Pizzocaro A. et al. US-elastogra- 14. phy in the differential diagnosis of benign and malignant thy- roid nodules. Thyroid. 2008; 18: 523–31. Baldawa P. S., Chaudhari H. K. Angular ectopic pregnancy 15. «беременность неизвестной локализации» (БНЛ). Примерно у 10 % женщин, имеющих БНЛ, в по- следующем диагностируется ЭБ [29]. «беременность неизвестной локализации» (БНЛ). Примерно у 10 % женщин, имеющих БНЛ, в по- следующем диагностируется ЭБ [29]. Статья представлена А. А. Цыпурдеевой, ФГБНУ «НИИ АГиР им. Д. О. Отта», Санкт-Петербург ■ Key words: ectopic pregnancy; ultrasound; magnetic resonance imaging; color Doppler; elastography. Поскольку данный вид беременности может быть доношенным, то и обнаружить ее можно уже на достаточно поздних сроках, когда разме- ры плодного яйца значительно искажают анато- мию окружающих органов. При ТВУЗИ, ограни- ченном сонографическими окнами, диагностика брюшной беременности затруднена. При угловой локализации плодное яйцо им- плантируется в одном из боковых углов матки, медиальнее маточно-трубного перехода и круглой связки матки. По данным МРИ, плодное яйцо при беременности в углу матки будет полностью окружено миометрием, с возможным фокусным истончением, однако латеральнее плодного яйца всегда будет определяться интерстициальный от- ТОМ LXIV ВЫПУСК 5/2015 ISSN 1684–0461 ОБЗОРЫ 83 ОБЗОРЫ Хотя радиочастотное излучение магнитно- резонансных томографов является неионизирую- щим, оно может привести к нагреву ткани в зоне интереса [54]. Результаты нескольких исследова- ний на животных доказали тератогенность дей- ствия на плод сильных магнитных полей на ран- них сроках беременности [54, 55]. С учетом, что органогенез происходит на ранних сроках бере- менности, некоторые радиологи полагают, что надо строго обосновывать целесообразность по- казания для направления на МРИ пациенток с бе- ременностью первого триместра. Однако ни одно рандомизированное исследование не было про- ведено на людях и никаких вредных последствий для плода человека не было зарегистрировано до настоящего времени [54]. A. Rex et al. (2012) считали, что если риск для беременной требовал применения метода визуализации с ионизирую- щим излучением, то МРИ оправдано независи- мо от срока беременности [54]. До сих пор нет единого мнения о целесообразности применения контрастирования, в частности внутривенного введения гадолиния матери. Результаты иссле- дований на животных доказали отрицательное его воздействие на плод. Было показано, что га- долиний сразу после внутривенного введения его матери проникает через плацентарный ба- рьер и появляется в мочевом пузыре плода [50]. По мнению F. G. Shellock et al. (1999), разумным подходом было бы не вводить внутривенно га- долиний в качестве контрастного вещества, если выгода своевременной диагностики ЭБ не пере- весит потенциальный риск для плода [50]. Литература Zbl. Gyn. 1926; 50: 1798. Filhastre M., Dechaud H., Lesnik A., Taourel P. Interstitial 24. pregnancy: role of MRI. EurRadiol 2005; 15 (1): 93–5. Pellerito Js., Taylor K. J. W., Quedens-Case C., Ham- 41. mers L. 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Yoshigi J., Yashiro N., Kinoshita T., O’uchi T., Kitagaki H. Di- 56. agnosis of ectopic pregnancy with MRI: efficacy of T2-weigh ted imaging. Magn. Reson. Med. Sci. 2006; 5 (1): 25–32. Pereira P. P., Cabar F. R., Schultz R., Zugaib M. Association 43. between ultrasound findings and extent of trophoblastic inva- ISSN 1684–0461 ТОМ LXIV ВЫПУСК 5/2015 Ишутина Татьяна Михайловна — врач УЗД лаборатории физиоло- гии и патофизиологии плода с отделением ультразвуковой диагно- стики. ФГБНУ «НИИ АГиР им. Д. О. Отта». 199034, Россия, Санкт- Петербург, Менделеевская линия, д. 3. E‑mail: itm_@mail.ru. Ishutina Tatiana Mikhaylovna — physician ultrasound lab. physiology and pathophysiology of the fetus with ultrasound department. D. O. Ott Research Institute of Obstetrics and Gynecology, RAMS. 199034, St. Pe tersburg, Mendeleyevskaya Line, 3, Russia. E‑mail: itm_@mail.ru. ■ Адреса автора для переписки ■ Адреса автора для переписки Ишутина Татьяна Михайловна — врач УЗД лаборатории физиоло- гии и патофизиологии плода с отделением ультразвуковой диагно- стики. ФГБНУ «НИИ АГиР им. Д. О. Отта». 199034, Россия, Санкт- Петербург, Менделеевская линия, д. 3. E‑mail: itm_@mail.ru. ■ Адреса автора для переписки Ishutina Tatiana Mikhaylovna — physician ultrasound lab. physiology and pathophysiology of the fetus with ultrasound department. D. O. Ott Research Institute of Obstetrics and Gynecology, RAMS. 199034, St. References Pe tersburg, Mendeleyevskaya Line, 3, Russia. E‑mail: itm_@mail.ru. Ishutina Tatiana Mikhaylovna — physician ultrasound lab. physiology and pathophysiology of the fetus with ultrasound department. D. O. Ott Research Institute of Obstetrics and Gynecology, RAMS. 199034, St. Pe tersburg, Mendeleyevskaya Line, 3, Russia. E‑mail: itm_@mail.ru. ТОМ LXIV ВЫПУСК 5/2015 ISSN 1684–0461
https://openalex.org/W4200533432	https://research-information.bris.ac.uk/ws/files/308553215/Full_text_PDF_final_published_version_.pdf	English	null	Editorial: Governing Carbon Dioxide Removal	Frontiers in climate	2,021	cc-by	2,984	Bellamy, R., Geden, O., Fridahl, M., Cox, E., & Palmer, J. (2021). Governing Carbon Dioxide Removal. Frontiers in Climate, 3. https://doi.org/10.3389/fclim.2021.816346 Bellamy, R., Geden, O., Fridahl, M., Cox, E., & Palmer, J. (2021). Governing Carbon Dioxide Removal. Frontiers in Climate, 3. https://doi.org/10.3389/fclim.2021.816346 Bellamy, R., Geden, O., Fridahl, M., Cox, E., & Palmer, J. (2021). Governing Carbon Dioxide Removal. Frontiers in Climate, 3. https://doi.org/10.3389/fclim.2021.816346 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.3389/fclim.2021.816346 Link to publication record on the Bristol Research Portal PDF-document p PDF-document This is the final published version of the article (version of record). It first appeared online via Frontiers at https://doi.org/10.3389/fclim.2021.816346 .Please refer to any applicable terms of use of the publisher. INTRODUCTION Carbon dioxide removal (CDR), also known as negative emissions, greenhouse gas removal, or simply carbon removal, is in need of eﬀective governance. If governments are to deliver on the growing number of pledges to meet “net zero” and “net-negative” emissions targets, and if the world is to successfully limit global warming to “well-below 2◦C” compared to pre-industrial levels, then carbon dioxide (CO2) will need to be removed from the atmosphere. This is because, at the very least, residual greenhouse gas emissions from hard-to-transition sectors like agriculture will need to be compensated for. Furthermore, if the world were to overshoot 1.5◦, CO2 concentrations will need to be brought back down. The central questions for CDR governance therefore no longer concern whether CDR should be pursued, but how; which CDR methods should be pursued, to what extent, when, where, and by whom (Bellamy and Geden, 2019). Despite this, the governance frameworks and democratic processes that will be needed to responsibly incentivize, develop, and sustain CDR remain largely neglected not just by policymakers, but also by much of the academic research community as well. Rob Bellamy 1,2, Oliver Geden 2,3, Mathias Fridahl 4,5, Emily Cox 6 and James Palmer 2,7 1 Department of Geography, University of Manchester, Manchester, United Kingdom, 2 Institute for Science, Innovation and Society, University of Oxford, Oxford, United Kingdom, 3 German Institute for International and Security Affairs, Berlin, Germany, 4 Department of Thematic Studies—Environmental Change, Linköping University, Linköping, Sweden, 5 Centre for Climate Science and Policy Research, Linköping University, Linköping, Sweden, 6 School of Psychology, Cardiff University, Cardiff, United Kingdom, 7 School of Geographical Sciences, University of Bristol, Bristol, United Kingdom Keywords: climate policy, governance, carbon dioxide removal, greenhouse gas removal, negative emissions Governing Carbon Dioxide Removal Governing Carbon Dioxide Removal Governing Carbon Dioxide Removal Edited and reviewed by: Phil Renforth, Heriot-Watt University, United Kingdom Correspondence: Rob Bellamy rob.bellamy@manchester.ac.uk Edited and reviewed by: Phil Renforth, Heriot-Watt University, United Kingdom *Correspondence: Rob Bellamy rob.bellamy@manchester.ac.uk y Spurring demand for CDR not just from multiple policy angles, but also multiple policy scales, will require an approach that minimizes negative trade-oﬀs and identiﬁes potential co-beneﬁts (Cox and Edwards, 2019). Yet uncertainties around CDR eﬀectiveness, technical eﬃciency, scale, risks, and interactions with other policy objectives—both within and beyond the realm of climate governance—all demand careful consideration (Fridahl et al., 2020). Moreover, eﬀective CDR governance must also contend with conﬂicting interests and account for diverse and geographically varying societal values and knowledges in relation to technology appraisal and selection, policy instrument choice, and guiding principles (Bellamy, 2018; Bellamy et al., 2021). This Research Topic seeks to address such critical questions around CDR governance as it emerges: how is CDR framed and what are the governance implications? How can we account for societal values and knowledges in CDR governance? How do existing governance regimes relate to CDR and how might they be reformed? What new governance designs are needed? Are existing institutions and systems suitable for governing CDR? Specialty section: This article was submitted to Negative Emission Technologies, a section of the journal Frontiers in Climate Received: 16 November 2021 Accepted: 17 November 2021 Published: 08 December 2021 Specialty section: This article was submitted to Negative Emission Technologies, a section of the journal Frontiers in Climate University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ EDITORIAL published: 08 December 2021 doi: 10.3389/fclim.2021.816346 EDITORIAL REFORMING GOVERNANCE Turning to the current state of existing CDR governance, Schenuit et al. synthesize commonalities and diﬀerences in recent developments in CDR policy in eight OECD countries and the EU, using an analytical framework that draws on the multi- level perspective of sociotechnical transitions. They propose a typology of three varieties of emerging CDR policymaking: incremental modiﬁcation of existing national policy mixes; early integration of CDR policy that treats emission reductions and removals as fungible; and proactive CDR policy entrepreneurship with support for niche development. Fridahl et al. examine the extent to which existing international (UN), supranational (EU), and national (Swedish) climate policy instruments incentivize bioenergy with carbon capture and storage (BECCS). They ﬁnd that no instruments create suﬃcient demand-pull to cover operational expenditure, economic instruments provide only partial technology-push support, and regulatory instruments provide only partial clarity on environmental safeguards and responsibilities. They conclude that the existing policy mix requires substantial reform if BECCS is to contribute to Sweden’s or other EU Member States’ climate policy targets. Boettcher et al. explore the increasing attention paid to marine CDR, and in particular how developments within four intertwined knowledge systems and governance sectors—namely modeling pathways, climate policy, innovation, and international legal frameworks—could result in diﬀerent futures. In one future, hype around marine CDR delays decarbonization, while in another, reforms to research and governance practices seek co-beneﬁts between ocean protection, economy, and climate. Lezaun et al. review how the more speciﬁc climate policy of a 2050 net zero target in the UK is forcing the integration of two disparate policy domains—forestry and geoengineering— and a more explicit articulation of the role CDR is expected to play. Net zero, they argue, provides an opportunity to bring transparency and accountability to underlying tensions, such as around “natural” and “engineered” CDR, by making explicit the role of CDR and subjecting them to public debate. The lack of demand-pull instruments in the EU is further explored by Rickels et al. They observe that despite the emissions cap in the EU Emissions Trading System (ETS) becoming net negative in one of the central EU-wide net zero scenarios, no mechanism allows for the inclusion of CDR credits. They conceptually discuss economic, legal, and political challenges surrounding the integration of CDR credits into the EU ETS. At the US State level, Sanchez et al. FRAMING GOVERNANCE to the inclusion of CDR into country level climate policy goals. They oﬀer a number of recommendations, including expressing the intent for deliberation directly in Nationally Determined Contributions; embedding considerations of people in institutions responsible for shaping the roles that CDR will play; and ensuring correspondence between project level questions and country level targets. Lezaun proposes a framework for increasing local participation in the assessment of marine CDR in particular, to counter framings such as planetary scale geoengineering that obscure the local and site-speciﬁc nature of many marine CDR proposals. He argues that this must begin with expanding the range of actors and factors included in discussions, for example in marine spatial planning. Thinking about global inclusion, Healey et al. warn how CDR policies may be inequitable if they are seen to avoid or delay gross emission reductions, use natural resources at a scale that threatens food security, leave knowledge of CDR as a Global North monopoly, or leave the implications of CDR for development unexamined. The use of CDR, they argue, requires global agreement on reducing emissions and enhancing removals, equity in burden sharing, and an interdisciplinary eﬀort led by individual jurisdictions to create CDR portfolios that are matched to local needs. Otto et al. undertake a secondary analysis of interviews with German environmental NGO representatives to identify CDR narratives. They ﬁnd two stories that reﬂect dominant climate policy discourses around ecological modernization and civic environmentalism: that CDR is either a necessity or a risk to mitigation, respectively. Turning to the envisaged role of CDR in diﬀerent countries’ long-term low emission development strategies, Buylova et al. ﬁnd that national plans echo such discourses. They identify three possible visions for CDR: as a panacea that risks deterring mitigation, as a necessary fallback in case mitigation is not enough, and as a chimera in which CDR is illusory due to a lack of speciﬁc targets and plans. Asayama argues that the apparent paradox of CDR being essential but also a potential distraction has less to do with CDR itself than with the diﬃculties of escaping carbon lock-in. To better situate CDR in the challenge of rapid decarbonization, he argues, we should be asking how it can be used in alignment with a managed decline in fossil fuel production. Citation: Bellamy R, Geden O, Fridahl M, Cox E and Palmer J (2021) Editorial: Governing Carbon Dioxide Removal. Front. Clim. 3:816346. doi: 10.3389/fclim.2021.816346 December 2021 \| Volume 3 \| Article 816346 Frontiers in Climate \| www.frontiersin.org Editorial: Governing Carbon Dioxide Removal Bellamy et al. FRAMING GOVERNANCE Boettcher undertakes a sociology-of-knowledge discourse analysis of interviews with UK stakeholders working at the industry-policy interface, to explore the competing forms of knowledge shaping assumptions about appropriate governance instruments for CDR. She reveals three dominant knowledge types: political-realist, utilitarian-economic, and discourse- ethical; and highlights the need for further “opening up” of discursive diversity in the development of CDR governance. Castree draws attention to how metaphors in particular will help to govern future action on CDR by framing present-day understandings of a world to come, and in turn how we might responsibly steer the use of metaphors to avoid depoliticization, polarization, or oversimpliﬁcation. He argues for a “post normal” discourse on CDR, where high-stakes decisions made in the context of epistemic uncertainty are informed by clear reasoning among divergent actors and their values. REFORMING GOVERNANCE contemplate administrative changes to California’s Low Carbon Fuel Standard to further stimulate commercialization of promising low carbon INCLUSIVE GOVERNANCE Borth and Nicholson focus in on this question of public debate by arguing for a deliberative orientation when it comes December 2021 \| Volume 3 \| Article 816346 Frontiers in Climate \| www.frontiersin.org 2 Editorial: Governing Carbon Dioxide Removal Bellamy et al. to undermine the IPCC’s ambition of opening up its scientiﬁc work to include more diversity in the process of drafting reports, and potentially also inﬂuence the governance of CDR. Palmer and Carton then turn to examine how BECCS is evolving into “BECCUS”—bioenergy with carbon utilization and storage— seeing this as a “ﬁx” for fossil fuel capitalism predicated on reconﬁguring the relationship between climate change and energy use, and not simply as an attempt to make BECCS more economically viable. They call for CDR governance to adjudicate between conﬂicting ideas about the role of intensive energy use in future global sustainable development pathways. Finally, considering the wider systems in which CDR governance will emerge, Hall and Davis argue that critical social science should name and analyse the structural features of capitalism and their relation to CDR and its governance. They oﬀer three principles to assist with this: that CDR is likely to emerge within capitalism, which is crisis prone, growth dependent, and market expanding; that there are diﬀerent varieties of capitalism and this will aﬀect the feasibility of diﬀerent CDR policies; and that capitalism is ideologically and culturally maintained. and carbon negative fuels. They propose embracing up-to- date science regarding lifecycle greenhouse gas emissions; creating additional, targeted incentives through a volumetric technology carve-out or credit multiplier; and ensuring that the standard stimulates the best-performing fuels across a range of sustainability criteria. In terms of international reforms, Honegger et al. develop six functions which they argue are jointly needed for policy mixes to mobilize CDR in a way that is compatible with the Paris Agreement. These include clarity on the role for CDR; accelerating innovation for aﬀordable and reliable CDR options; ensuring public participation in decision making on CDR; transitioning from piloting to scaled operations; ensuring robust monitoring, reporting, veriﬁcation, and accounting; and preventing adverse impacts and maximizing co-beneﬁts for sustainable development goals. More generally, Carton et al. highlight that the obfuscation of emissions reductions by treating emissions and removals as equivalent is not the only problem of equivalence in CDR accounting. SUMMARY The articles in this Research Topic contribute critical knowledge on the framing, inclusiveness, reformation, creation, and systems of emerging CDR governance. These contributions will be invaluable for government, industry and civil society stakeholders seeking to understand, reform and expand governance for CDR. They also represent an important resource for researchers seeking to build upon the nascent questions raised herein. What framings are still missing from the CDR governance debate? How can we implement and evaluate more geographically inclusive CDR governance? How do implemented reforms and new CDR governance creations perform in practice, and what other decision-making processes and policy frameworks might still be needed? How does CDR governance impact and interact with other systems and mechanisms for climate change mitigation and adaptation, and with non-climate goals? And ﬁnally, how can institutions and economic systems be reformed to account for alternative perspectives and to embed principles of just governance? CREATING GOVERNANCE However, reforms can only go so far, and Zetterberg et al. oﬀer ﬁve possible models for creating new incentives and ﬁnancing for BECCS, using Sweden as an example. These include: government guarantees for purchasing BECCS outcomes; quota obligations on selected sectors; allowing BECCS credits to compensate for hard-to-abate emissions within the EU ETS; using private entities for voluntary compensation; and other states acting as buyers of BECCS outcomes to meet their mitigation targets. They conclude that successful implementation of BECCS will require a combination of several of these, implemented sequentially. Also looking at BECCS, Klement et al. argue that pulp and paper mills have potential for commercial roll-out of BECCS, and they seek to ﬁnd business-driven ways of incentivising BECCS within this industry. By projecting the costs and negative emissions related to BECCS from the pulp mill to typical consumer products, they show how BECCS can substantially reduce their carbon footprint, while only marginally increasing their cost. AUTHOR CONTRIBUTIONS RB wrote the editorial with input from all authors to edit and improve the ﬁnal text of the article. INCLUSIVE GOVERNANCE To ensure a just response to the climate crisis, they argue for the “undoing” of three additional problematic equivalences in carbon accounting: the equivalence of fossil and biotic forms of carbon; the equivalence of emissions and removals across diﬀerent geographies; and the equivalence of present and near-term mitigation actions and those projected in the distant future. Frontiers in Climate \| www.frontiersin.org ACKNOWLEDGMENTS Turning to the wider institutional contexts in which CDR governance is mediated, Hansson et al. analyse BECCS- related expert review comments and author responses on the Intergovernmental Panel on Climate Change (IPCC) Special Report on1.5◦C. They show that boundary work at the science- policy interface acts to deﬂect fundamental critiques of BECCS, particularly regarding the way in which it is presented as a viable technology at a grand scale. This, they argue, threatens RB acknowledges funding from the University of Manchester’s Presidential Fellowship scheme and the Natural Environment Research Council under grant NE/V013106/1 (CO2 Removal Hub, CO2RE). MF acknowledges funding from the Swedish Research Council Formas (grant 2019-01993 and 2019-01973) and the Swedish Energy Agency (grant 46222-1 and 51569-1). December 2021 \| Volume 3 \| Article 816346 Frontiers in Climate \| www.frontiersin.org 3 Editorial: Governing Carbon Dioxide Removal Bellamy et al. EC acknowledges funding from the Engineering and Physical Sciences Research Council under grants EP/S029575/1 (UK h C ) d / / ( l Zero project), and from the Leverhulme Trust under grant RC-2015-029. JP acknowledges funding from the University of Bristol’s Vice-Chancellor’s Fellowships scheme. Zero project), and from the Leverhulme Trust under grant RC-2015-029. JP acknowledges funding from the University of Bristol’s Vice-Chancellor’s Fellowships scheme. EC acknowledges funding from the Engineering and Physical Sciences Research Council under grants EP/S029575/1 (UK Energy Research Center) and EP/V011960/1 (Delivering Net Frontiers in Climate \| www.frontiersin.org REFERENCES 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. Bellamy, R. (2018): Incentivize negative emissions responsibly. Na. Energy 3, 532–534. doi: 10.1038/s41560-018-0156-6 Bellamy, R., Fridahl, M., Lezaun, J., Palmer, J., Rodriguez, E., Lefvert, A., et al. (2021): Incentivising bioenergy with carbon capture and storage (BECCS) responsibly: Comparing stakeholder policy preferences in the United Kingdom and Sweden. Environ. Sci. Policy 116, 47–55. doi: 10.1016/j.envsci.2020.09.022 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Bellamy, R., and Geden, O. (2019): Govern CO2 removal from the ground up. Nat. Geosci. 12, 874–876. doi: 10.1038/s41561-019-0475-7 Cox, E., and Edwards, N. (2019): Beyond carbon pricing: policy levers for negative emissions technologies. Clim. Policy 19, 1144–1156. doi: 10.1080/14693062.2019.1634509 Copyright © 2021 Bellamy, Geden, Fridahl, Cox and Palmer. 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. Fridahl, M., Hansson, A., and Haikola, S. (2020): Towards indicators for a negative emissions climate stabilisation index: problems and prospects. Climate 8, 75. doi: 10.3390/cli806 0075 December 2021 \| Volume 3 \| Article 816346 Frontiers in Climate \| www.frontiersin.org 4
W3098190271.txt	https://iopscience.iop.org/article/10.1088/2040-8986/aa5f5f/pdf	en		Optimised photonic crystal waveguide for chiral light–matter interactions	Journal of optics	2,017	cc-by	3,035	Journal of Optics You may also like PAPER • OPEN ACCESS Optimised photonic crystal waveguide for chiral light–matter interactions To cite this article: Ben Lang et al 2017 J. Opt. 19 045001 - Recent advances and progress in photonic crystal-based gas sensors Amit Kumar Goyal, Hemant Sankar Dutta and Suchandan Pal - Biomaterials in light amplification Jaroslaw Mysliwiec, Konrad Cyprych, Lech Sznitko et al. - Dispersion engineered slow light in photonic crystals: a comparison S A Schulz, L O’Faolain, D M Beggs et al. View the article online for updates and enhancements. This content was downloaded from IP address 51.159.168.146 on 21/05/2024 at 04:22 Journal of Optics J. Opt. 19 (2017) 045001 (4pp) https://doi.org/10.1088/2040-8986/aa5f5f Optimised photonic crystal waveguide for chiral light–matter interactions Ben Lang1, Ruth Oulton1 and Daryl M Beggs2 1 Quantum Engineering Technology Labs, H. H. Wills Physics Laboratory and Department of Electrical & Electronic Engineering, University of Bristol, BS8 1FD, United Kingdom 2 School of Physics and Astronomy, Cardiff University, Queen’s Buildings, The Parade, Cardiff CF24 3AA, United Kingdom E-mail: bl9453@bristol.ac.uk Received 14 November 2016, revised 16 January 2017 Accepted for publication 9 February 2017 Published 10 March 2017 Abstract We present slow-light photonic crystal waveguide designs that provide a ×8.6 improvement of the local density of optical states at a fully chiral point over previous designs. Keywords: polarisation singularities, photonic crystal waveguides, slow light (Some ﬁgures may appear in colour only in the online journal) realised [7]. In principle, there is no upper limit to the light– matter interaction enhancement. At the bandedge, the PhCWG has stopped modes with group velocity vg=0 and an inﬁnite density of optical states (the van Hove singularity [8]). Only the practicality of fabricating a perfect waveguide without defects prevents the use of these bandedge modes. As shown by our recent work, the case of an emitter placed at a C-point requires additional consideration. Timereversal symmetry dictates that all chiral components of the waveguide mode must vanish in the stopped light at the bandedge, and therefore no C-points can be supported. Modes in the slow-light regime resemble the bandedge mode, and thus contain less chirality. This forces a compromise between strong light–matter interactions (for which slow light is desirable, typically found near the bandedge) and making those interactions chiral (for which we want powerful circularly polarised ﬁelds, which become scarce near the bandedge). We found that the optimum local density of optical states (LDOS) at a C-point is found in modes with modest group velocities of vg<c/10 for the standard PhCWG design (the so-called W1 waveguide) [9]. Only a limited number of alternative nano-photonic designs for QDs have so-far been considered [10, 11]. In this paper we present alternative PhCWG designs that possess larger LDOS at the C-points. Our search for these designs begins with the archetypical W1 waveguide of one row of missing holes from an hexagonal lattice of holes with radius r=0.3a in a GaAs dielectric membrane. We form new designs by displacing each 1. Introduction Chirality of light in (nano-)photonic structures is proving to be a valuable resource [1–3]. In quantum optics, chirality couples the spin direction of electrons to the travel direction of light. This chiral light–matter interaction is at its most useful when the chirality reaches 100% at a singular position known as a C-point. A quantum dot (QD) placed at a C-point can display a spin-dependent unidirectional-emission [4] an attractive property for quantum optics, as it allows spinencoded static qubits to be converted to path-encoded ﬂying qubits. Further, ensembles of emitters coupled to a chiral waveguide can show remarkable entanglement properties [5, 6]. Photonic crystal waveguides (PhCWGs) present several unique beneﬁts to realising chirality-direction coupling. Firstly, the longitudinal component of the waveguide modes is large, meaning that C-points with 100% chirality are common, and moreover tend to occur in the high-index part of the waveguide, where a QD could be placed. Secondly PhCWGs support slow-light. Extending the beneﬁts of slowlight to the interaction of a QD at a C-point is attractive. Slow-light enhances the density of optical states allowing extremely bright sources and high collection efﬁciencies to be Original 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. 2040-8978/17/045001+04$33.00 1 © 2017 IOP Publishing Ltd Printed in the UK J. Opt. 19 (2017) 045001 B Lang et al Figure 1. (a) The refractive index proﬁle of a W1. The arrows indicate how D1 and D2 control the displacements of the innermost holes. O marks the origin of the in-plane coordinate system (x, y)=(0, 0). (b) Largest LDOS at a C-point as a function of (D1, D2), normalised to the largest LDOS at a C-point in a W1. The dotted line is a contour along which this ratio is unity. G on the colour bar indicates the value for the glide waveguide described in [10]. Figure 2. (a) 2d FDTD simulations of circular dipoles at the ideal points in the optimised and W1 waveguides. (b) Power radiated forwards, backwards and sideways: note the log scale. experimentally achievable values [22] and ﬁltering computational errors at the bandedge where ∣E∣2  0 and ng  ¥. Figure 1(b) presents the main results, showing the enhancement of the LDOS at C-points where they occur in a single-mode waveguide. The vast majority of designs show little or no enhancement over the standard W1 (i.e. the ratio of LDOS at the C-points is below or close to one), but there is a small region of the search space that show signiﬁcant enhancements. Of these, the best design identiﬁed has 8.61 times the C-point LDOS of a W1 for (D1, D2)=(−0.11, 0.15)a at a C-point at frequency 0.2791c/a and position (x, y)=(0.5, 1.170)a from the origin shown in ﬁgure 1(a). We repeated the calculations in 3d for a small subset of the best designs, and found good agreement with the 2d versions. However, a small shift of the optimal point was seen, to (D1, D2)=(−0.07, 0.14)a. Finite difference time domain simulations conﬁrming the unidirectional emission from the best identiﬁed C-points in the optimised and W1 waveguides are shown in ﬁgure 2(a) [15]. Our simulations are again 2d, using the same effective index approximation as above. Figure 2(b) shows the calculated power radiated in the forwards, backwards and sideways directions. The ∼×10 enhancement in LDOS is well replicated in these calculations. Emission into the backwards direction is suppressed by a factor of 106 in the W1 and 104 in the optimised design. In principle, emission in the backwards direction should be precisely zero for the correct location, polarisation and frequency of the dipole. However, away from this precise frequency, as the modes polarisation proﬁle changes, the former C-point becomes elliptical and allows emission into the backwards direction. When the light is slower i.e. a smaller group velocity vg=dω/dk the change of wavevector and therefore mode proﬁle is larger for a small change of frequency. Therefore slower light designs, such as the optimised waveguide, are likely to display a smaller bandwidth for high directionality and therefore the dip is not as well resolved in the simulation. For these source locations in these waveguides the suppression of the backwards emission is over 100:1 for bandwidths of ∼2×10−3 c/a and ∼4×10−4 c/a. Even in the hole in the ﬁrst row of holes closest to the waveguide a distance D1 towards the waveguide core, and the second row of holes a distance D2 (ﬁgure 1(a)). In this way the dispersion [12] and electric ﬁeld proﬁle [13] of the PhCWG can be modiﬁed. Modifying the hole positions is preferred to the radii as they are more accurately realised in electron-beam lithography [14]. The LDOS is a function of position and frequency and is proportional to the product ng∣E∣2 , where ng = ∣c vg∣ is the group index and ∣E∣2 is the electric ﬁeld intensity. We are interested in positions of unit directionality, where the directionality is deﬁned as the difference between power emitted by a spin transition (modelled as a circular pointdipole) into the forwards and backwards waveguide modes, normalised by the total power emitted into the waveguide. For a single-mode waveguide the directionality is simply given by the degree of chirality h = ∣S3∣ = 2∣Im (Ex*Ey )∣ ∣E∣2 . η=1 is typically not possible if the waveguide is multi-mode (it requires the extraordinary coincidence of C-points at the same position in all modes at the same frequency) and so multimode frequency regions are ignored. For each choice of (D1, D2) we search for C-points with η=1, and calculate the relative LDOS (ng∣E∣2 ) at each using a frequency domain eigensolver [16]. In each design (D1, D2), the C-point with the highest LDOS is compared to the C-point with the highest LDOS in the W1 design (D1, D2)=(0, 0). Our calculations use the effective index method [17], with neff=2.66, suitable for wavelengths around 900 nm and a GaAs membrane of thickness 100 nm. This 2d approximation shows good agreement with experiment over a wide range of circumstances [18, 19], although it has drawbacks: notably it tends to underestimate the group index near the bandedge [20] and naturally as a 2d method it misses 3d effects [21]. We treat values of ng>100 as 100 in the LDOS calculation, serving the dual purpose of focusing the search on 2 J. Opt. 19 (2017) 045001 B Lang et al latter case (the optimised waveguide) this is considerably larger than the linewidth of a QD (20 μeV∼4×10−6 c/a). The best designs presented here exploit slow light far from the bandedge by ﬂattening the dispersion. There is an element of brinkmanship to this, as ﬂattening the dispersion towards an inﬂection-like curve maximises the LDOS, but overshooting creates a multimode region that ruins the performance. This abrupt drop is visible in ﬁgure 1(b) on the lower side of the bright region. This motivates a minor digression. In principle a perfect inﬂection point in the dispersion appears able to support unidirectionality with an inﬁnite density of states. However even inﬁnitesimal perturbations (for example, from disorder) can break the inﬂection point into a local maximum and local minimum pair with a small separation. This local maximum/ minimum pair results in a multi-mode region with a slowlight mode allowing (strong) emission in both directions. Although such abruptness may at ﬁrst glance seem un-physical, it is a consequence of us considering only modes with real kx (propagating modes), equivalent to considering an inﬁnitely long PhCWG. Evanescent modes in a ﬁnite PhCWG always allow some light to tunnel from the dipole out of the waveguide in the wrong direction. Near an inﬂection point there exist weakly evanescent modes (with small imaginary kx) that can tunnel a long distance [23]. These weakly evanescent modes smooth out this transition in ﬁnite-length PhCWGs. In real-world samples, QDs are typically strain-grown in random locations [24] (although positioning methods are being developed [25]). Furthermore, the size and shape dispersion means that the resonant frequencies of the QDs are randomly distributed around the desired one. Experiments then typically proceed by testing a large number of samples, until a suitable one is found. In the above, we have calculated the performance of the PhCWG for an ideally placed QD pitched at the ideal frequency, but we are also interested in the yield: how many samples can we expect to test before ﬁnding a good QD positioned at or near a C-point. To answer this question we have also calculated the probability that a QD placed at a random location in the GaAs with a random frequency (selected uniformly from the bandwidth of the fundamental mode) will have η>0.9. Our calculations neglect positions with negligible LDOS and assume η<0.9 at multimode frequencies. Figure 3 shows the result of this calculation. The optimised waveguide identiﬁed above (white cross, ﬁgrue 3) has a poor yield (∼1%). However by consideration of ﬁgures 1(b) and 3 together PhCWGs with a desired compromise of yield and performance can be chosen. For example for (D1, D2)=(0.13, 0.13) the yield is 33% (for a W1 the yield is 24%) and the maximum LDOS is 4× greater than that in a W1. Nearby values offer a different trade-off between yield and LDOS enhancement. In conclusion we have identiﬁed modiﬁed PhCWG designs that promise signiﬁcant increases in chiral performance, with a ×8.6 enhancement of the LDOS. As the LDOS is a measure of the light–matter interaction strength, and is Figure 3. Calculated yield. The probability that directionality η exceeds 0.9 for randomly placed QDs. G has the same meaning as in ﬁgure 3. directly proportional to the emission rate and efﬁciency [16], our optimised design will allow fabrication of waveguides almost one order of magnitude brighter than using the standard W1 design. In the ﬁnal stages of this work we became aware of related work suggesting a modiﬁed glide-plane waveguide design for strong chiral interactions [26]. Our calculations suggest this design is excellent with >100 times LDOS enhancement at the best C-point compared to a W1 (see ﬁgure 1) and a ∼15% yield (see ﬁgure 3). Acknowledgments This work has been funded by the project SPANGL4Q, under FET-Open grant number: FP7-284743. RO was sponsored by the EPSRC under grant no. EP/G004366/1. DMB acknowledges support from a Marie Curie individual fellowship QUIPS. 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W2588812529.txt	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_S2_from_Myeloid_Cells_That_Impair_Immunotherapy_Are_Restored_in_Melanomas_with_Acquired_Resistance_to_BRAF_Inhibitors/22417425/1/files/39863535.pdf	en		Myeloid Cells That Impair Immunotherapy Are Restored in Melanomas with Acquired Resistance to BRAF Inhibitors	Cancer research	2,017	cc-by	85	Final Tumor Mass (grams) A 1.0 0.5 0.0 B Final Tumor Mass (grams) P=0.92 1.5 1.5 Untreated 120d BRAFi P=0.93 P=0.25 P=0.39 P=0.78 P=0.23 P=0.43 1.0 0.5 0.0 Untreated 4d 60d 120d BRAFi Supplementary Fig. S2: Tumor size comparisons between groups. (A) Final mass, in grams, of untreated and BRAFi-resistant tumors that were assessed by flow cytometry in Figs 2 and 3. (B) Final mass, in grams, of tumors submitted for microarray analysis as shown in Fig. 4. Statistical differences were calculated by unpaired t-test.
https://openalex.org/W3190331646	https://discovery.ucl.ac.uk/id/eprint/10132824/7/Schrag_1-s2.0-S1353802021002819-main.pdf	English	null	Digital health technology for non-motor symptoms in people with Parkinson's disease: Futile or future?	Parkinsonism & related disorders (Online)/Parkinsonism & related disorders	2,021	cc-by	10,360	Digital health technology for non-motor symptoms in people with Parkinson’s disease: Futile or future? Daniel J. van Wamelen a,b,c,, Jirada Sringean d, Dhaval Trivedi a,b, Camille B. Carroll e, Anette E. Schrag f, Per Odin g, Angelo Antonini h, Bastiaan R. Bloem c, Roongroj Bhidayasiri d,i, K. Ray Chaudhuri a,b, on behalf of The International Parkinson and Movement Disorder Society Non Motor Parkinson’s Disease Study Group a King’s College London, Department of Neurosciences, Institute of Psychiatry, Psychology & Neuroscience, London, United Kingdom b Parkinson’s Foundation Centre of Excellence at King’s College Hospital, Denmark Hill, London, United Kingdom c Radboud University Medical Centre; Donders Institute for Brain, Cognition and Behaviour; Department of Neurology, Nijmegen, the Netherlands d Chulalongkorn Centre of Excellence for Parkinson’s Disease & Related Disorders, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand e Faculty of Health, University of Plymouth, Plymouth, Devon, United Kingdom f Department of Clinical and Movement Neurosciences, University College London, London, United Kingdom g Division of Neurology, Department of Clinical Sciences, Lund University, Lund, Sweden h Movement Disorders Unit, Department of Neuroscience, University of Padua, Padua, Italy i The Academy of Science, The Royal Society of Thailand, Bangkok, Thailand A B S T R A C T Keywords: Parkinson’s disease Sensor Wearable Accelerometer Non-motor symptoms Keywords: Parkinson’s disease Sensor Wearable Accelerometer Non-motor symptoms Introduction: There is an ongoing digital revolution in the field of Parkinson’s disease (PD) for the objective measurement of motor aspects, to be used in clinical trials and possibly support therapeutic choices. The focus of remote technologies is now also slowly shifting towards the broad but more “hidden” spectrum of non-motor symptoms (NMS). Methods: A narrative review of digital health technologies for measuring NMS in people with PD was conducted. These digital technologies were defined as assessment tools for NMS offered remotely in the form of a wearable, downloadable as a mobile app, or any other objective measurement of NMS in PD that did not require a hospital visit and could be performed remotely. Searches were performed using peer-reviewed literature indexed data bases (MEDLINE, Embase, PsycINFO, Cochrane Database of Systematic Reviews, Cochrane CENTRAL Register of Controlled Trials), as well as Google and Google Scholar. i Results: Eighteen studies deploying digital health technology in PD were identified, for example for the mea surement of sleep disorders, cognitive dysfunction and orthostatic hypotension. In addition, we describe promising developments in other conditions that could be translated for use in PD. i Conclusion: Unlike motor symptoms, non-motor features of PD are difficult to measure directly using remote digital technologies. Nonetheless, it is currently possible to reliably measure several NMS and further digital technology developments are underway to offer further capture of often under-reported and under-recognised NMS. Corresponding author. King’s College London, Department of Basic and Clinical Neuroscience, The Maurice Wohl Clinical Neuroscience Institute, Denmark Hill, London, SE5 9RT, United Kingdom. E-mail address: Daniel.van Wamelen@kcl.ac.uk (D.J. van Wamelen). Parkinsonism and Related Disorders xxx (xxxx) xxx Contents lists available at ScienceDirect Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis Parkinsonism and Related Disorders xxx (xxxx) xxx Contents lists available at ScienceDirect Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis https://doi.org/10.1016/j.parkreldis.2021.07.032 E-mail address: Daniel.van_Wamelen@kcl.ac.uk (D.J. van Wamelen). 3.2. Orthostatic hypotension Monitoring and measurement of NMS in PD is complicated by the physiology of certain NMS which makes it difficult to objectively mea sure them, not only from a digital health perspective. Similarly, dedi cated non-motor scales are not always able to detect and correctly identify NMS [9], also bearing in mind these scales measure patient or clinician reported outcomes and not physiological processes. To date, the only relatively well-explored NMS aspect of PD in which wearable technology has been deployed, is the use of actigraphy for circadian and sleep disorders [10]. However, in recent years attempts have been made to address the objective assessment of some other NMS in PD. In addition to the dual challenge posed by first objectively measuring NMS and then transforming this into digital health outcomes, there is the lack of clear guidelines and criteria for the selection, technical validation, and clin ical validation of novel digital endpoints [11]. The use of technology-based digital devices should aim to supplement objective measures as well as the more commonly used subjective measures, including scales and questionnaires, in determining presence and severity of symptoms in the clinical management of PD beyond research projects [12,13]. In this narrative review we aim to describe the state of the art of digital health technology for objective assessment of NMS in PD. Among Parkinson’s-related cardiovascular abnormalities, ortho static hypotension (OH) is the most common and one of the better described conditions with an overall prevalence ranging from 10% to 70%. OH is multifactorial in origin, as it can be iatrogenic, but also an intrinsic feature of PD occurring early in the course of the disease [15], as well as in the prodromal phase [16], with neuropathological studies confirming the presence of pathological alpha-synuclein deposits in central and peripheral regulatory nuclei [17]. In addition to OH, supine hypertension and nocturnal hypertension are other common problems in PD and detection is required as OH is associated e.g. with white matter laesions [18]. The gold standard for OH diagnosis is a standard blood pressure measurement in the supine and upright position or a head-up-tilt test in hospital settings, and for detection of supine or nocturnal hypertension the use of 24-h home monitoring [19]. Scaling down the standard de vices used for this, would enable blood pressure measurement in a home environment. For example, Hellman et al. 1. Introduction Digital health technology covers a broad area of technical applications and includes devices worn on the body (in the form of watches, bracelets or devices embedded in clothing), smartphones, sensors embedded into a patient’s home (for example in smart beds) or analysis of regularly used appliances, such as computer keyboards. Ideally, such remote The use of digital health technology, some in combination with connected devices (e.g. smartphones), is on the rise to improve the management of medical conditions, including Parkinson’s disease (PD). * Corresponding author. King’s College London, Department of Basic and Clinical Neuroscience, The Maurice Wohl Clinical Neuroscience Institute, Denmark Hill, London, SE5 9RT, United Kingdom. E-mail address: Daniel.van_Wamelen@kcl.ac.uk (D.J. van Wamelen). Available online 31 July 2021 Received 21 May 2021; Received in revised form 26 July 2021; Accepted 28 July 2021 y 1353-8020/© 2021 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/). 1353-8020/© 2021 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/). 1353-8020/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY licens 1353-8020/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://cre Please cite this article as: T, Parkinsonism and Related Disorders, https://doi.org/10.1016/j.parkreldis.2021.07.03 Parkinsonism and Related Disorders xxx (xxxx) xxx D.J. van Wamelen et al. measurement techniques should be non-disruptive to the person’s normal lifestyle. Specifically, the technology should allow for use any where without barriers, and should be feasible for use both at home and within the community. Digital technologies theoretically provide an opportunity to monitor people for extended periods of time, provided that participants comply with extended use, which has been a vexing issue in most studies thus far. Additionally, digital technologies poten tially allow for care to be delivered at home and in the community, thereby limiting the need for hospital visits [1]. Importantly, these technologies enable us to passively collect data in the background, and these data are therefore not influenced by rater or patient bias, which is often the case with subjective patient diaries [2]. Another concern with existing approaches is the attentional compensation which is typically associated with in-clinic sessions while patients know they are being observed [3]. 2. Methods We performed a review of digital health technology relating to NMS occurring in PD. The objectives of this review were to summarise the different types of digital technology currently in use to measure NMS in PD, as well as those technologies that hold promise for future use in PD, and to inform future non-motor research and health policies. The aim of this review was to provide a narrative overview and viewpoint of digital health technologies for NMS in PD, rather than a systematic review. Further developments are likely to be expected to take on the form of smart watches. Several of such watches are in existence and are capable of measuring heart rate, blood pressure, and oxygen saturation. Currently, however, it would seem that only heart rate meets accuracy guidelines, but not the other vital sign measurements [22,23]. The search strategy included the terms ‘Parkinson’ or ‘Parkinson’s’ combined with ‘gyroscope’, ‘accelerometer’, ‘technology’, ‘app’ or ‘wearable’. The search was not limited by date, language, or study design. The searches were performed using peer-reviewed literature indexed databases (MEDLINE, Embase, PsycINFO, Cochrane Database of Systematic Reviews, Cochrane CENTRAL Register of Controlled Trials), as well as Google and Google Scholar by the first and second authors and 3. Results An overview of the 18 studies with identified technologies measuring NMS and wearable outcomes associated with NMS is provided in Table 1 and examples are given in Fig. 1. 3.2. Orthostatic hypotension reported continuous non-invasive arterial pressure monitoring in PD in patients who had documented OH compared to those without OH [20]. They showed that the wearable device was capable of reproducing findings made in a hospital setting, which was considered the ‘gold standard’ in this study. Vallelonga et al. compared ambulatory blood pressure monitoring (ABPM) for OH with office blood pressure measurement [21] to test the diagnostic accuracy of ABPM-based hypotensive episodes (Hypo-ep) and hypotension during periods of wakefulness (Hypo-aw). They showed a diagnostic accuracy of 87.6% to detect OH, suggesting that remote monitoring of blood pressure is feasible although the large device and monitor size are limitations. 3.1. Overall non-motor symptoms Currently, no objective measurements of general non-motor burden is available. Even though certain apps have used the availability of pa tients scoring self-reported outcome measures [14], these are still sub jective and come with the limitations of patient completed diaries. 3.2. Orthostatic hypotension 1. Introduction In fact, these benefits have already led to the invention and testing for usefulness and reliability for many devices in PD and neurocognitive disorders [4], some of which have already made their way into clinical practice [5]. However, in the case of PD, the focus of digital technology has been almost exclusively on the motor symptoms associated with this disease, with devices measuring tremor, bradyki nesia, wearing-off, dyskinesia, gait patterns and falls [6,7]. Attempts have even been made to measure rigidity objectively [8]. Non-motor symptoms (NMS), which are pivotal to the natural history of PD and which are a crucial determinant of the patient’s and caregiver’s quality of life, have thus far received little attention as a target for objective measurements through digital technology, although the introduction of commercial systems to record heart rate, ECG and blood oxygen satu ration has raised the interest from both patients and healthy subjects. conflicts were resolved with the senior authors of the manuscript. Reference lists of identified manuscripts were used to identify any other relevant studies. The final search was performed in December 2020. For the purpose of this review, digital technology was defined as digital assessment tools for NMS offered remotely in the form of a wearable, downloadable as a mobile app, or any objective digital measurement of NMS in PD that did not require a hospital visit and could be performed at home. Inclusion criteria were: 1) studies addressing digital health technology in people with Parkinson’s disease, and 2) measurement and/or monitoring of non-motor symptoms. Exclusion criteria were: 1) digital technologies in populations other than people with Parkinson’s disease, and 2) measurement and/or monitoring of motor symptoms. 3.3. Sleep dysfunction Sleep dysfunction, such as onset and maintenance insomnia, are a common feature occurring in PD although with wide range estimate 2 Table 1 Remote monitoring devices for non-motor symptoms Eye Movement Sleep Behavioural Disorder; PDSS: Parkins Mental State Examination. Non motor symptom Study Remote monitoring devic Orthostatic hypotension and cardiovascular Hellman et al., 2015 [20] Scaled down wearable blood pressure monitor Vallelonga et al., 2019 [21] Scaled down wearable blood pressure monitor Sleep Nass and Nass 2008 [74] Actigraphy (triaxial wrist- worn device) Perez-Lloret et al., 2009 [29] Actigraphy (triaxial wrist- worn device) Stavitsky et al., 2010 [75] Actigraphy (triaxial wrist- worn device) Naismith et al., 2010 [30] Actigraphy (triaxial wrist- worn device) Maglione et al., 2013 [10] Actigraphy (triaxial wrist- worn device) Louter et al., 2014 [31] Actigraphy (triaxial wrist- worn device) Gunn et al., 2014 [76] Actigraphy (triaxial wrist- worn device) Klingelhoefer et al., 2016 [32] Triaxial wrist- worn device Excessive daytime sleepiness Kotschet et al., 2014 [33] Triaxial wrist- worn device Bolitho et al., 2013 [77] Actigraphy (triaxial wrist- worn device) Cognition Bolitho et al., 2013 [77] Actigraphy (triaxial wrist- worn device) Bloem et al., 2019 [47] Triaxial sensor protocol D.J. van Wamelen et al. Parkinsonism and Related Disorders xxx (xxxx) xxx D.J. van Wamelen et al. Table 1 Remote monitoring devices for non-motor symptoms in Parkinson’s disease. Abbreviations: OH: orthostatic hypotension; PD: Parkinson’s disease; RBD: Rapi Eye Movement Sleep Behavioural Disorder; PDSS: Parkinson’s disease sleep scale; PSG: polysomnography; NMSQ: Non Motor Symptoms Questionnaire; MMSE: Min Mental State Examination. Non motor symptom Study Remote monitoring device Number of participants and duration of monitoring Validation against Measure used and findings Orthostatic hypotension and cardiovascular Hellman et al., 2015 [20] Scaled down wearable blood pressure monitor •52 participants (31 with OH and 21 without) In clinic blood pressure measurements Continuous non-invasive arterial blood pressure. Similar proportion of participants with OH for both devices •One-off session of 3 min Vallelonga et al., 2019 [21] Scaled down wearable blood pressure monitor •113 participants In clinic blood pressure measurements Continuous non-invasive arterial blood pressure. Two or more episodes with ≤15 mmHg systolic drop 75% diagnostic accuracy for OH. Excessive daytime sleepiness 3.3. Sleep dysfunction One or more episodes 93% specificity for OH •24 h blood pressure monitoring Sleep Nass and Nass 2008 [74] Actigraphy (triaxial wrist- worn device) •17 participants with PD None Activity measure between participants with PD and controls differ significantly, particularly at nighttime •69 healthy controls •3 days and nights Perez-Lloret et al., 2009 [29] Actigraphy (triaxial wrist- worn device) •71 participants with PD PDSS Significant moderate correlations between actigraphy and PDSS domains •21 healthy controls •7 days and nights Stavitsky et al., 2010 [75] Actigraphy (triaxial wrist- worn device) •30 participants with PD Sleep diary Scores on subjective sleep correlated moderately with actigraphy-derived estimates, but only in participants with PD •14 healthy controls PDSS •7 days and nights Epworth Sleepiness Scale Naismith et al., 2010 [30] Actigraphy (triaxial wrist- worn device) •22 participants with PD REM Sleep Behaviour Questionnaire Participants with RBD had higher number of activity bouts than those without RBD •2 weeks Scales for Outcomes in Parkinson’s disease (SCOPA) Sleep Maglione et al., 2013 [10] Actigraphy (triaxial wrist- worn device) •61 participants with PD PSG Significant moderate correlations between actigraphy and polysomnography measures for sleep time and wake after sleep onset •1 night Louter et al., 2014 [31] Actigraphy (triaxial wrist- worn device) •45 participants with PD PSG For predicting RBD 95 wake bouts per night on actigraphy had 95.5% specificity, 20.1% sensitivity 85.7% positive predictive value compared to PSG •1 night Gunn et al., 2014 [76] Actigraphy (triaxial wrist- worn device) •95 participants with PD Digit Span Backwards subtest (raw score) of the Wechsler Adult Intelligence Scale-III (WAIS-III) Working memory and verbal memory consolidation significantly associated with sleep efficiency •48 healthy controls •2 weeks Klingelhoefer et al., 2016 [32] Triaxial wrist- worn device •60 participants with PD Parkinson’s Disease Sleep Scale Percentage of time spent immobile correlated moderately with sleep/fatigue questions of the NMSQ and with PDSS •6 days and nights Excessive daytime sleepiness Kotschet et al., 2014 [33] Triaxial wrist- worn device •68 participants with PD Epworth Sleepiness Scale Percentage of time spent immobile was significantly higher in those with ESS score ≥10 •6 days and nights Bolitho et al., 2013 [77] Actigraphy (triaxial wrist- worn device) •85 participants with PD Epworth Sleepiness Scale Daytime inactivity measures did not correlate with ESS •21 healthy controls •6 days and nights Cognition Bolitho et al., 2013 [77] Actigraphy (triaxial wrist- worn device) •85 participants with PD Cambridge Neuropsychological Test Automated Battery Mini Mental State Examination Participants with excessive sleepiness had a trend towards poorer working memory •21 healthy controls •6 days and nights Bloem et al., 2019 [47] Triaxial sensor protocol •650 participants with PD •2 years MDS-UPDRS part I Not available Montreal Cognitive Assessment Phonemic and semantic Fluency Brixton Spatial 15 Words Test Benton Judgment of Line Orientation Letter Number Sequencing Continuous non-invasive arterial blood pressure. •96 participants with PD •One-off session Weiss et al., 2019 [45] Body fixed sensor •96 participants with PD •One-off session 3.3. Sleep dysfunction Two or more episodes with ≤15 mmHg systolic drop 75% diagnostic accuracy for OH. One or more episodes 93% specificity for OH Activity measure between participants with PD and controls differ significantly, particularly at nighttime Significant moderate correlations between actigraphy and PDSS domains Scores on subjective sleep correlated moderately with actigraphy-derived estimates, but only in participants with PD Scores on subjective sleep correlated moderately with actigraphy-derived estimates, but only in participants with PD Percentage of time spent immobile correlated moderately with sleep/fatigue questions of the NMSQ and with PDSS Percentage of time spent immobile was significantly higher in those with ESS score ≥10 Daytime inactivity measures did not correlate with ESS Participants with excessive sleepiness had a trend towards poorer working memory Timed up-and-go strategies were not related to cognitive function (continued on next page) 3 Parkinsonism and Related Disorders xxx (xxxx) xxx Table 1 (continued) Non motor symptom Study Remote monitoring device Number of participants and duration of monitoring Validation against Measure used and findings van Uem et al., 2018 [44] Home-based sensor •47 participants with PD •3 days Significant associations between % sedentary and active episodes and MMSE, also with number of sedentary bouts Wu et al., 2018 [46] Actigraphy (triaxial wrist- worn device) •35 participants with PD MMSE Less stable day-to-day rest-activity rhythm was associated with poorer executive, visuospatial, and psychomotor functioning, but not memory •7–10 days Neuropsychiatric testing Impulse control disorder Evans et al., 2014 [39] Triaxial wrist- worn device •25 participants with PD Questionnaire for Impulsive- Compulsive disorder (QUIP) Number of medication acknowledgements correlated significantly with QUIP scores •6 days and nights Urinary Sringean et al., 2016 [36] Stationary homebased sensor •19 participants with PD (and their partners) Amount of times getting up at night for toilet visits Sensor measured the amount of times that participants got out of bed; significant discrepancy between partner reported and objective measure •1 night Gastrointestinal Kyritsis et al., 2021 [78] Triaxial wrist- worn device •21 participants with PD Time to move food from plate to mouth Objective measure for time taken •7 healthy controls D.J. van Wamelen et al. Objective measure for time taken Figure 1. Examples of non-motor areas in Parkinson’s disease where wearable and remote technologies have been investigated and non-motor areas where further developments are expected based on technologies available in other conditions. Abbreviations: GI: gastrointestinal; GPS: global positioning system; ADL: activities of daily living. 3.4. Impulse control disorder Impulse control disorders (ICD) are characterised by the inability to assert self-control in emotions and behaviours, leading to compulsive and/or impulsive actions that harm oneself or others. The pathways involved in ICD have been implicated in rapid eye movement sleep behaviour disorder, constipation, and cognitive impairment [37]. Despite it being an important problem in PD [38], only one study has examined the role of wearables in ICD. Using a 3-axial accelerometer device, Evans and colleagues showed that ratings of ICD, in the form of Questionnaire for Impulsive-Compulsive Disorder in Parkinson’s Disease-Rating Scale (QUIP-RS) scores, was strongly positively associ ated with the number of acknowledgements per intake of oral medica tion in a small cohort of 25 PD patients. The intake was recorded by having the patient record the intake of medication by pressing a button on the watch; for this simple procedure it would not be necessary to have a tri-axial accelerometer or similar device but given the ease with which this can be recorded it would be easy to add it as a feature to any wearable device. This is also one of the few studies looking at wearable outcome measures that do not rely on motor markers to serve as a sur rogate, but instead relied on assessing ICD severity by the amount of times a patient acknowledged the intake of medication using a paradigm embedded in the wearable device use [39]. Recently, Mirelman and colleagues showed that nocturnal move ments, including turning and being in upright position and walking during nocturnal period, were correlated to dysexecutive patterns using Trail Making Test scores which were inversely correlated to the number of rotations during the night. In addition, it was shown that Non-Motor Symptoms Scale scores were also inversely associated with turning in bed at night and nocturia [35]. In addition, circadian disruption, inde pendent of sleep, has been investigated as a marker for cognition, and a decreased day-to-day rest-activity rhythm, as measured by actigraphy, was associated with poorer executive, visuospatial, and psychomotor functioning. Interdaily stability of circadian patterns predicted 14% of the variation occurring in executive function, psychomotor and visuo spatial performance in a cohort of 35 PD patients [46]. Further developments in the objective capture of ICD could involve the analysis and quantification of internet use as it has been suggested these are linked in PD patients. More specifically, Wu et al. 3.5. Cognition variability. However, as actigraphy records bouts of activity, there are difficulties in distinguishing Rapid Eye Movement Behaviour Disorder (RBD) from awake time using actigraphy. Several studies have reported the use of actigraphy to detect RBD [30,31], in which PD patients with RBD had significantly higher numbers of bouts of activity, scored as “awake”, on actigraphy. When compared to an RBD questionnaire, the actigraphy outcomes had low sensitivity for detecting RBD. In recent years many forms of digital technology have been devel oped to measure cognitive performance. Although many of these tech nologies have not been tested in people with PD, there are a number of studies in Alzheimer’s disease. Due to its nature, digital health tech nologies assessing cognition range from wearable sensors to phone apps covering various aspects of cognitive function, such as the “Remote Assessment of Disease and Relapse – Alzheimer’s Disease” (RADAR-AD), using remote monitoring to actively and passively measure cognitive and affective biomarkers [34]. Examples of how cognitive performance can be measured include the passive use of Global Positioning System (GPS) movement trajectories or deviation from navigation tools for spatial navigationa and memory, or the more active use of phone apps to measure performance on gamified/virtual reality tests as a measure for planning skills and task completion [34]. Other examples include, but are not limited to, app based technologies to assess spatial navigation, memory and other cognitive functions, phone app measurements using the performance on gamified/virtual reality tests for planning skills, smart home sensors for opening/closing of doors and window as a measure for daily activities, and speech analysis for dysnomia (https:// mezur.iohttps://altoida.com) [34]. Tri-axial accelerometers are similar to actigraphy but are able to better quantify the amount of motor activity. Using such a device, Klingelhoefer et al. reported that periods of immobility (periods during which no significant motor activity was detected) correlated well with PD Sleep Scale (PDSS) [32] although the association between wearables sensor output and motor activity captured by Hauser diary data was poor, perhaps supporting the benefits of objective rather than subjective (diary-based) outcomes. For daytime sleepiness, Kotschet et al. showed that the immobility measure of a tri-axial accelerometer was correlated with high Epworth Sleepiness Scores (ESS) [33]. i Future developments in this field will be likely to draw from the experience gained in other fields of neurology, including Alzheimer disease. 3.5. Cognition Examples of remote sleep measurement that are likely to make their way into PD are mobile phone sleep trackers, bed- or under- mattress fitted sensors (‘smart beds’), and wearable electroencephalo gram headbands [34], but these have yet to be validated for use in PD. Especially ‘smart beds’ could prove useful and some evidence has already been presented that they are able to quantify nocturnal move ments, including turning, being in upright position and walking during nocturnal period, in PD [35]. The usefulness of such sleep measurements is contained in its objectiveness, instead of patient and carer (sleep) diaries, which come with inherent inaccuracies and can be discrepant between patients and carer [36]. p In PD, several studies have shown that gait parameters measured by conventional wearable triaxial sensors could be useful as an indirect indicator of cognitive decline, although further studies are required to assess confounders for the observed associations, and ideally such monitoring should move away from indirect markers of cognitive per formance. A UK based study measured gait parameters using a single axis sensor and reported specific patterns for dementia typical for PD, dementia with Lewy bodies, and Alzheimer’s disease [41]. Terashi and colleagues have recently shown that in a cohort of 106 Japanese non fluctuating PD patients the gait acceleration amplitude showed a mod erate positive association with Mini Mental State Examination (MMSE) scores [42]. This latter relation remained significant after the correction for UPDRS postural instability and gait disorder scores. The same group of researchers reported similar findings in drug-naïve PD patients, where daily physical activity was moderately positively associated with Frontal Assessment Battery and Behavioural Assessment of Dysexecutive Func tion scores. Significant associations were not present for other cognitive assessments, including MMSE, Beck Depression Inventory and Stark stein’s apathy scores [43]. In another study, in a cohort of 47 German PD patients, higher levels of daily activity measured with a back worn 3D accelerometer were associated with better cognitive scores [44]. How ever, Weiss and colleagues were unable to demonstrate a relation be tween timed up-and-go assessment, as measured by a body-fixed sensor, in PD patients with cognitive outcome measures (MoCA and MMSE) [45]. D.J. van Wamelen et al. Parkinsonism and Related Disorders xxx (xxxx) xxx 3.3. Sleep dysfunction Figure 1. Examples of non-motor areas in Parkinson’s disease where wearable and remote technologies have been investigated and non-motor areas where further developments are expected based on technologies available in other conditions. Abbreviations: GI: gastrointestinal; GPS: global positioning system; ADL: activities of daily living. regarding prevalence (20% up to 80%) reported in PD cross-sectional subjective assessments [24]. The data related to its progression during the course of the disease are heterogeneous, but nonetheless sleep problems have a high impact on quality of life in PD and are often missed in outpatient settings [25–27]. actigraphy, containing tri-axial accelerometers, quantifies the amount of motor activity during daytime and night-time, as a measure of sleep and physiological diurnal patterns, and was approved by FDA to measure limb activity associated with movement during sleep for physiologic applications [10]. Several studies have shown the usefulness of actig raphy in PD and this has been recently reviewed by Zampogna et al. [28]. Examples of the use of actigraphy include the findings by e.g. Perez-Lloret et al. reported that actigraphy data in PD significantly correlated with PD Sleep Scale (PDSS) and patient-completed diary data related to sleep quality and daytime somnolence [29]. Maglione and colleagues [10] reported a positive correlation of night-time actigraphy for the assessment of sleep quality and quantity in PD with PSG showing that actigraphy may be useful as a measurement for total sleep time, sleep efficiency, and wake time after sleep onset although with some Polysomnography (PSG) remains the gold standard for monitoring or diagnosing sleep dysfunction in PD, but is expensive, not widely avail able and requires patients to stay in bespoke facilities overnight. Wearable sensors would offer an alternative as a simpler, cheaper, and probably effective strategy. Nocturnal accelerometers are already in relatively wide-spread use for sleep monitoring and are used as an outcome measure in research settings. These do not include EEG re cordings, which are required for direct assessment of sleep function, but only indirectly measure sleep-associated movements. Wrist worn 4 3.7. Gastrointestinal As with any other symptom, the objective measurement and digital monitoring of NMS in PD requires a consensus to evaluate the quality and usefulness of digital devices, including but not limited to clinical utility, user experience, and governance for collection [62]. An addi tional difficulty, unlike the case with motor symptoms, is posed by the fact that NMS are often difficult to measure. Several suggestions have been made towards a standardised approach for digital healthcare technology and one of the most recent ones, by Goldsack and colleagues, includes a three-step approach: (1) verification (systematic evaluation by manufacturers), (2) analytical validation (valuation of processed data and testing on human subjects), and (3) clinical validation (evaluation of identification, measurement, and prediction of meaningful clinical, biological, physical, functional state, or experience in the specified context of use) [62]. From the available evidence on digital health technology for NMS, studies identified here would fall into category 3, but only with regards to the identification element of this category. In addition, it should be noted that some evidence presented here is based on indirect markers for specific NMS based on motor outcomes. None theless, such markers (mostly bradykinesia and dopaminergic by default) of NMS based on wearable and objective motor measures, might be a good starting point to improve at least some NMS in PD. As an example, the number of acknowledgements per intake of oral medica tion appears to be strongly correlated to repetitive behaviour which is typically seen with ICD [39]. In addition, arguments have been made to suggest that it is not necessary to have exact measurements of a NMS in question, as long as the symptom of interest can be related to an outcome and lead to a management pathway with therapeutic intervention after identification [63,64]. Focusing on what correlates best to subjective patient disability, rather than subjective assessments as many in-clinic assessments may be biased due to e.g. attentional bias [3]. Gastrointestinal dysfunction in PD affects the whole gastrointestinal tract and can be observed in each stage of the disease, from the pro dromal to the advanced phases [50]. Especially constipation is a com mon feature with an overall prevalence ranging from 11 to 83%, and is considered as one of the earliest symptoms in PD and a risk factor for the development of this condition [51]. 4. Discussion Currently very few objective measures for the multifaceted NMS of PD exist. As such, it is not surprising to find that studies have tried to objectify and quantify these symptoms in the form of wearable sensors (Table 1). Nonetheless, few studies have looked at deploying wearable and remote technology for the measurement and monitoring of NMS in PD, but some technologies used to measure NMS in other conditions hold promise for future use in PD (Fig. 1). 3.4. Impulse control disorder showed that PD patients with ICDs had a relative increased tendency towards excessive use of the Internet compared to those without ICDs as well as healthy controls [40]. However, in this study the information on internet use was gathered through means of a face-to-face structured interview and all participants had specifically consented to this, in addition to the self-reported documentation of type of websites they spend most time on whilst online. This is likely to be a limiting factor in the further devel opment of objective measures for ICD based on Internet usage, as well as the clear privacy-related aspects. A prospective, longitudinal, single-centre cohort study known as the ‘Personalized Parkinson Project’, is aiming to address the usefulness of a highly advanced smartwatch (including 3-axial accelerometers, a goni ometer, a barometer, and sensors for skin temperature, environmental light and sound) in a large cohort who will wear the watch for up to 3 years. This study also includes a wide battery of neuro-psychological 5 5 Parkinsonism and Related Disorders xxx (xxxx) xxx D.J. van Wamelen et al. assessments that will be measured annually as outcome in 650 partici pants. This study is currently underway, and the results are awaited [47]. assessments that will be measured annually as outcome in 650 partici pants. This study is currently underway, and the results are awaited [47]. home setting, providing remote monitoring. In this study patients had an inert registration device mounted in their home which picked up data from a body-fixed sensor collecting quantitative nocturnal movements of PD patients and comparing these outcomes to patients and spouse reported outcomes. Here it was shown that nocturia, measured by the number of times a patient got out of bed during the night, could be effectively picked up by the sensors, supported by sleep diaries [36]. 3.6. Depressive symptoms Even though no studies appear to exist looking at any direct rela tionship between depressive symptoms and measurable wearable sensor outcomes in PD, two studies have shown some evidence for associated changes that might be indicative of depressive symptoms. One study reported that PD patients with depressive symptoms showed a more pronounced systolic blood pressure drop on head up tilting and a higher prevalence of orthostatic hypotension compared to non-depressed pa tients even though there were no 24 h BP recordings performed in either group [48]. Similarly, PD patients with a geriatric depression score of five or over had higher adjusted levels of systolic, diastolic, and mean blood pressure dipping in addition to nocturnal high systolic pressure, and the presence of moderate to severe depressive symptoms was inversely associated with systolic dipping in a regression model [49]. Even though such measurements could be arguably used to capture depressive symptoms, it may be better to rely on more direct measures rather than surrogate markers as these often rely on associations and could be confounded by many factors, such as disease duration and age. Future monitoring of urinary (and gastrointestinal) NMS in PD could be achieved by further developing and validating the use of ‘smart toi lets’. Such toilets are capable of calculating urine flow rate and volume, classifying stool according to the Bristol stool form scale, and able to identify individual users through their fingerprint and the distinctive anoderm features [61]. Such devices could hold great potential in measurement and classification of urinary NMS in PD. 3.7. Gastrointestinal Also, other gastrointestinal prob lems, including dysphagia, are common in the later stages of PD with a major impact on quality of life and a pivotal role in prognosis (morbidity and mortality) in advanced PD [52,53]. There are no validated direct measures of function of gastrointestinal dysfunction in PD, although such efforts are underway to test digital health technology measuring these symptoms in PD. In fact, recently a smart belt for such symptoms has been developed and has received a CE mark, showing the device meets European standards on safety, health or environmental requirements (https://ec.europa.eu/research/participan ts/documents/downloadPublic?documentIds=080166e5b77ec i f58&appId=PPGMS). The smart belt is composed of five sensors (four acoustic and one for electrogastrogram) and an elastic band. The device records bowel sounds (using the acoustic sensors) and the electrical activity generated by the muscles’ contractions triggered by stomach and intestines during digestion (electrogastrogram sensor). To date, no results have been published regarding the use of this belt and its out comes in PD. Currently also other efforts are being made to measure other gastrointestinal and associated symptoms in PD, including impulsive eating disorders, appetite-related NMS, such as weight change, as well as dysphagia. Here, “plate-to-mouth” time, measured through a tri-axial wrist-worn sensor, has already been validated as an objective measure for eating behaviour in PD [54]. Once devices and digital technology have been developed and vali dated for NMS in PD, a further distinction needs to be made to define how this technology is to be used in clinical practice. Here, the example set by the National Institute for Health and Care Excellence (NICE) in the United Kingdom can serve as a guide towards classification of this kind of technology for the use in PD. The NICE guidelines propose a three- tiered system to classify digital health technologies, where evidence tiers are cumulative which means that a technology must meet all standards in both previous tier(s) and its own tier. In tier 1 the digital technology has no measurable patient outcomes but provides services to the health and social care system; in tier 2 the technology informs about a condition, provides simple monitoring, or allows two-communication References between patient and health care professional; and in tier 3 the tech nology additionally allows for preventative changes, self-management, treatment, active monitoring, impact calculator, or diagnosis of a con dition [65]. The majority of the current studies that have looked at remote monitoring for NMS in PD would classify as tier 2 and further efforts are needed to move to tier 3 where NMS outcomes can be measured directly and trigger an intervention. At this stage, current limitations to the objective measurement of NMS, including recall bias affecting outcomes depending on the clinical scales or diaries used for addressing the NMS [2,66,67], will have been overcome. [1] S.A. Lowe, G. ´Olaighin, Monitoring human health behaviour in one’s living environment: a technological review, Med. Eng. Phys. 36 (2) (2014) 147–168. [2] M K E b D R K li B K H K C Th F P i i G P V Di J [2] M.K. Erb, D.R. Karlin, B.K. Ho, K.C. Thomas, F. Parisi, G.P. Vergara-Diaz, J. F. Daneault, P.W. Wacnik, H. Zhang, T. Kangarloo, C. Demanuele, C.R. Brooks, C. N. Detheridge, N. Shaafi Kabiri, J.S. Bhangu, P. Bonato, mHealth and wearable technology should replace motor diaries to track motor fluctuations in Parkinson’s disease, NPJ Digit Med 3 (2020) 6. [3] V. Robles-García, Y. Corral-Berganti˜nos, N. Espinosa, M.A. J´acome, C. García- Sancho, J. Cudeiro, P. Arias, Spatiotemporal gait patterns during overt and covert evaluation in patients with Parkinson’s disease and healthy subjects: is there a hawthorne effect? J Appl Biomech 31(3) (2015) 189–194. Other factors that need to be overcome when it comes to objective digital monitoring of NMS is compliance, especially given the fact that PD results in motor and cognitive impairment which may be relevant for at least some remote and wearable technologies. So far, limited evidence has shown that compliance levels with wearable technology is relatively high for PD patients. Cohen et al. showed that compliance rates were reduced by 30% over a 6-month period [68], confirmed by another study where the 6-month compliance rate was 62–68% in two cohorts of 953 participants in total [69]. The main predictors for adherence appear to be caregivers’ burden and patients’ self-rated health status [70]. The study by Cohen et al. References also showed an interesting observation in that daily smartwatch data streaming patterns peaked around mid-day and dropped sharply in the evening hours [68], perhaps indicative of a physiological circadian pattern [71]. [4] A. Channa, N. Popescu, V. Ciobanu, Wearable Solutions for Patients with Parkinson’s Disease and Neurocognitive Disorder: A Systematic Review, Sensors, Basel, 2020, 20(9). [5] R. Pahwa, F. Bergquist, M. Horne, M.E. Minshall, Objective measurement in Parkinson’s disease: a descriptive analysis of Parkinson’s symptom scores from a large population of patients across the world using the Personal KinetiGraph®, J Clin Mov Disord 7 (2020) 5. [6] A. Zampogna, I. Mileti, E. Palermo, C. Celletti, M. Paoloni, A. Manoni, I. Mazzetta, G. Dalla Costa, C. P´erez-L´opez, F. Camerota, L. Leocani, J. Cabestany, F. Irrera, A. Suppa, Fifteen Years of Wireless Sensors for Balance Assessment in Neurological Disorders, Sensors, Basel, 2020, 20(11). [7] A.L. Silva de Lima, T. Smits, S.K.L. Darweesh, G. Valenti, M. Milosevic, M. Pijl, H. Baldus, N.M. de Vries, M.J. Meinders, B.R. Bloem, Home-based monitoring of falls using wearable sensors in Parkinson’s disease, Movement Disorders 35 (1) (2020) 109–115. [8] Z. Wu, X. Jiang, M. Zhong, B. Shen, J. Zhu, Y. Pan, J. Dong, P. Xu, W. Zhang, L. Zhang, Wearable sensors measure ankle joint changes of patients with Parkinson’s disease before and after acute levodopa challenge, Parkinson’s Dis. 2020 (2020) 2976535. Examples on the use of wearable sensor outcomes to guide treatment decisions are already available, albeit mostly involving motor outcomes. One such study showed that the use of a tri-axial wrist worn device for monitoring motor outcomes had an additional benefit on the increase in On-time after pharmacist-led medication review in 27 patients with PD [72]. At the same time the use of the wearable sensor in this study did not improve non-motor outcomes, although it should be noted that only a dichotomous outcome measure, the Non-Motor Symptoms Question naire, was used and treatment decisions were based on motor outcomes only [72]. One could imagine, however, that if wearable sensor out comes, as outlined in this review, are used to identify NMS that would otherwise go unrecognised, this would have a great impact on treatment and quality of life. Examples include depressive symptoms and cognitive impairment, which are not only debilitating in themselves, but are moreover independent risk factors for non-adherence [73], and recog nition of these symptoms could additionally lead to motor outcome improvements. 5. Conclusions Mathias, S.R. Raj, D. Robertson, P. Sandroni, I. Schatz, R. Schondorff, J. M. Stewart, J.G. van Dijk, Consensus statement on the definition of orthostatic hypotension, neurally mediated syncope and the postural tachycardia syndrome, Clin. Auton. Res. : official journal of the Clinical Autonomic Research Society 21 (2) (2011) 69–72. Parkinsonism and Related Disorders xxx (xxxx) xxx D.J. van Wamelen et al. References [9] D.J. van Wamelen, P. Martinez-Martin, D. Weintraub, A. Schrag, A. 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W4379157627.txt	https://journal.jfpublisher.com/index.php/jsh/article/download/316/362	en		WOUND CARE WITH MOIST WOUND DRESSINGS FOR HEALING DIABETIC GANGRENE: SYSTEMATIC REVIEW	Lux Mensana	2,023	cc-by	3,908	LUX MENSANA Volume 2, Issue 2, 2023 Page. 137-146 Original Research Article WOUND CARE WITH MOIST WOUND DRESSINGS FOR HEALING DIABETIC GANGRENE: SYSTEMATIC REVIEW 1) Ary Dwi Wuryani 1)* IIK Strada Indonesia, Jl. Manila No.37, Tosaren, Pesantren, Kediri City, East Java, Indonesia 64133 *Corresponding Author, E-mail : sutrisnoarik@gmail.com ABSTRACT Introduction. Wound care with moist wound dressings is an effective and beneficial option for treating diabetic ulcers which often result in healing of old wounds that end in amputation. Writing this literature review aims to identify the effectiveness of the use of wound care with diabetic gangrene moist wound dressing. Method. The literature review writing method goes through several stages: creating questions and keywords namely MESH (Medical Subject Heading), identification, eligibility, selection of article inclusions, screening, and assessment. The selection process listed in the literature framework resulted in 4 articles. Articles are summarized and assessed using critical appraisal according to the research design in each article. Result&Analysis. The wound care method with moist wound dressings has been proven to significantly help the healing process of diabetic gangrene wounds. The wound care method with the principle of moisture balance, is more effective than conventional methods. Discussion. Modern dressing wound care has been proven to help heal diabetic wounds effectively, this is evidenced by the mechanism of the average decrease in the occurrence of the wound dressing process and an increase in the quality of life of diabetic ulcer wound patients after being treated with modern wound dressing methods. Keywords: Diabetic Gangrene, Moist Wound Dressings, Wound Care. INTRODUCTION The data and information center of the Diabetes mellitus (DM) is a health problem that needs to be treated thoroughly, uncontrolled DM can cause metabolic complications or long-term vascular complications, namely microangiopathy and macroangiopathy. Diabetics are also prone to wound infections in the feet, ineffective wound treatment increases the incidence of complications from diabetic gangrene (Buchori, Sukron and Wahyudi, 2020). Ministry of Health of the Republic of Indonesia (Kemenkes) explained that in 2021 the most common complication in Diabetes Mellitus cases is neuropathy, which is 54%, where neuropathy decreases sensation in the distal area of the foot so that there is a high risk of foot gangrene and can even lead to infection to amputation (Suprihatin and Purwanti, 2021). Wounds that arise spontaneously or due to trauma can cause open wounds 137 capable of producing gangrenous gas amputation events can be avoided with resulting in osteomyelitis (Fitria et al., proper foot care (Santoso, Rahayu and 2017). Patients with gangrene can be Irawan, 2022). treated by improving peripheral perfusion. Efforts are made wounds to Good perfusion helps in transporting gangrenous oxygen and blood to damaged tissues thus mechanical control consists of resting the helping to grow new tissues (Hidayat, foot, avoiding pressure loads on the 2017). wound area, which heal activity on include: the foot Previous wound care management facilitating the spread of infection, using using conventional methods is with pillows on the feet while lying down to methods that do not make the wound area prevent abrasions on the heels, decubitus moist by using gauze attached to the mattresses. wound. This is what usually makes gauze mechanism will stick to the wound and make the developed as an effort to heal diabetic newly grown cells will be damaged during gangrene the causing dressings. Modern wound care uses discomfort at the change of client modern dressings with moist and closed dressing. For this reason, it is necessary to wound care techniques or known as moist choose the right wound care method to wound dressings. Moist wound dressings speed up the wound dressings process. is a method to maintain wound moisture Currently, wound care has undergone by many developments, one of which is the materials so that wound dressings, tissue method of wound care with modern growth can occur naturally. Fast and dressings, namely by maintaining a moist precise wound dressings without causing wound environment to maintain tissue pain during treatment can improve the fluid loss and cell death (Handayani, quality of life of patients with diabetic 2016). Proper wound care is an effort to gangrene while metabolic control is the help speed up the wound dressings control process so it needs to continue to be hypertension, developed (Ose, Utami and Damayanti, electrolyte disorders, anemia, impaired 2018). Diabetic wounds that do not heal kidney function, comorbidity infections in are a risk factor for infection and a leading the lungs; wound control consists of cause of amputation and death. But debridement and necrotomy, dressing, diabetes experts estimate that 1/2 to 3/4 drugs to accelerate healing, if necessary next wound treatment, The that wounds using vascular is currently is Moist moisture-retaining of other control factors, being wound dressing such as: hypercholesterolemia, 138 by operative measures; Infection control results; 3) The research subjects were such as: adequate diabetic gangrene patients; 4) open access antibiotics adapted to culture examination, and full paper discussing wound dressing. empirical to The exclusion criteria for this study were: aerobic, 1) articles in languages other than English infections and Indonesian; 2) systematic reviews; 3) elsewhere; educational control, including Research data is incomplete or not the patient and family, explanation of the available. Search articles using online disease, diagnostic and therapeutic action databases (PubMed, Google Scholar and plans, risks to be experienced and Scopus). The process of searching and prognosis; Vascular control, for example, filtering articles uses the Prism chart is modifying risk factors in the form of (chart 1). Articles included in this study smoking cessation, medical therapy in the must meet the inclusion criteria and have form of medication and complementary been reviewed using critical appraisal in therapy accordance with the research design of administration of therapy multiorganism, according anaerobic, overcoming systemic (Naziyah, Suharyanto and Fauziyah, 2022) each article (CASP Checklists, 2023). METHOD AND ANALYSIS RESULT The research design is a systematic review. Article searches used the PubMed Characteristics of the Research Subject, online database and Google scholar and there are a total of 828 articles searched Scopus. this from online databases PubMed, Google systematic review were articles published Scholar and Scopus using the keywords from 2011 to 2020. The search process "moist was carried out by the authors in the gangrene", and "wound care"". There are a period January-February 2023. In the total of 4 articles that fit the inclusion process articles, criteria and are processed in qualitative researchers used the keywords "moist synthesis. The characteristics of each wound dressing", "diabetic gangrene", and article included in the qualitative synthesis "diabetic gangrene". injury cure". The are described in table 1. inclusion criteria for this study were: 1) discuss wound care with moist wound articles explaining wound care with moist dressing techniques are found in several wound dressing in diabetic gangrenous online databases, especially on PubMed, wound dressing 2) original research Google Scholar and Scopus, but study that The of articles searching used for in wound dressing", "diabetic Articles that 139 Conducted a search using appropriate keywords Google Scholar 640 PubMed 160 (Medical Subject Heading) Specifics in the last 5 years Scopus 28 828 Article (2011-2020) 560 Article Specifics in the last 3 years (2018-2020) 102 Article Results with Inclusion criteria 17 Article Results of Literature articles to be analyzed 4 Article Figure 1. Literature review Search Framework articles that specifically discuss wound category of wound regeneration as many care as 15 respondents (100%). with moist wound dressing techniques for wound dressing in diabetic The second article showed that the gangrene patients are still limited. This physical domain in the intervention group review summarizes the results of research before on several subjects. The subject consisted treatment obtained an average difference of diabetic gangrene patients The first of -0.72, meaning that between before and article shows that based on the results of after there was an average increase of data analysis of the wound dressing 0.72. The psychological domain in the process intervention experienced by respondents and after group modern before dressing and after undergoing modern dressing treatment in modern dressing treatment obtained an the conventional average difference of -0.83, meaning that treatment in the control group with 15 between before and after there was an respondents, it can be seen that the wound average increase of 0.83. The social dressing process in respondents after domain in the intervention group before modern dressing In the intervention group and after modern dressing treatment with healthy tissue as many as 8 obtained an average difference of -0.61, respondents (53.3%), wound regeneration meaning that between before and after as many as 7 respondents (46.7%) and in there was an average increase of 0.61. intervention and the conventional control group with the 140 Table 1. Results of Literature Reviews No Author (year) Title Purpose Method & Sample Interven tion Research results Conclusion 1. Subandi and Sanjaya (2020) The Effectivenes s of Modern Dressing Against the Wound Healing Process of Type 2 Diabetes Mellitus Analyze the effectiveness of modern dressings on the healing process This study used the Quasy Experimental design with a Pre-Postest With Control Group approach &; a sample number of 15 people with type 2 diabetes Provides modern interventi on dressings on the wound healing process of type 2 diabetes. This study showed that there were differences in pre- and postwound scores in the treatment group and in the dick group Modern dressings have effectiveness against the process . 2. Situmora ng and Yazid (2021) Wound Care with Modern Dressing on the Quality of Life of Diabetic Ulcer Patients at Asri Wound Care Center Medan Helfrida Knowing the effect of wound care with modern dressings on the quality of life of diabetic ulcer patients at Asri Wound Care Center Medan Helfrida Correlation and sample descriptive research, namely 30 respondents with diabetic ulcers who went to Asri Wound Care Center Medan with total sampling technique Providin g wound care with modern dressings to the quality of life of diabetic ulcer patients This study shows that there is a significant difference in improving quality of life before and after wound care using modern dressing at Asri Wound Care Center Medan Wound care with modern effective dressings improves the quality of life of diabetic ulcer sufferers at Asri Wound Care Center Medan Helfrida 3. Sitohang and Harahap (2019) The Effect of Using Modern Dressings on the Diabetic Wound Healing Process at the Asri Wound Care Center Clinic in Medan Knowing the Effect of Using Modern Dressings on the Diabetic Wound Healing Process at the Asri Wound Care Center Medan Clinic This study used an analytic descriptive design with the design "One Group pretest posttest" & a sample of 30 respondents with diabetes mellitus who were on an outpatient basis at the Asri Wound Care Center Clinic Providin g modern dressing (Foam) in the process of healing diabetic wounds at the Asri Wound Care Center Clinic The results in the study indicate that there is an effect of modern wound dressings on the healing process of diabetes mellitus wounds and that treatment must be carried out routinely according to the wound care schedule. There is a decrease in the average wound healing process before and after using modern dressings 4. Rukmi (2018) The Effect of Modern Dressing Implementat ion on the Quality of Life of Diabetic Ulcer Patients Knowing the Effect of Modern Dressing Implementati on on the Quality of Life of Diabetic Ulcer Patients This study has a preexperimental design with a one-group pre-post-test design & a sample of 17 respondents from Griya Clinic Wound Care Center Providin g wound care with modern dressings on the Quality of Life of Diabetic Ulcer Patients The results showed that there was a significant difference between the quality of life before and after Wound care was performed, with an average change in quality of life score of 13 points Wound care with modern dressings can improve quality of life, as seen from the increase in the results of calculating the quality of life in patients with diabetic ulcers. 141 The environmental domain in the intervention after repair after modern treatment of dresing. modern dressing treatment obtained an Likewise, the quality of life score was average difference of -0.44, meaning that obtained between before and after there was an treatment the average was 65.88 and after average the modern dressing treatment the average researchers concluded from the results of became 78.76. So researchers concluded the research that has been done, there is an that there is an effect of wound care with effect of wound care with modern modern dressings in improving the quality dressing methods on the quality of life of of life of diabetic gangrene patients. diabetic group increase gangrene before of 0.44. patients and So it can be concluded that there is wound So in before modern dressing the intervention group. DISCUSSION The third article shows that the average wound dressing process before The strict setting of criteria on the and after the use of modern dressings method greatly affects the number of decreases. Where the average before is articles obtained. The determination of the 34.5 and after 26.9. the average difference article taken initially by entering the word was 7.6 with a difference of 5.9 to 9.9 lock for each variable that has been (95% the selected according to MESH (Medical difference). So the researchers concluded Subject Heading) then a search is carried that there was an average decrease in the out using google scholar After seeing that wound dressing process before the use of the number of articles obtained is limited, modern dressings. the criteria for taking further articles, confidence interval of The fourth article shows that the specific in the last 5 years, the results characteristics of the wound condition obtained are still too broad to determine before modern dressing treatment are 2nd articles that can be used. Because it is felt degree (58.8%), with a yellow base that the results obtained are still too much (41.2%), a large amount of exudate to be determined, then specified again in (70.8%) and positive signs of infection the last 3 years. The results of articles (64.7%). While the characteristics of the obtained wound condition after modern dressing keywords and specific in the last 3 years treatment are 2nd degree (58.8%), with a are taken and analyzed which ones meet red base (88.2%), moderate exudate the inclusion criteria and can be used as (58.8%) and no signs of infection (0%). articles to be used, with reference to from searches by entering 142 articles related to wound care applies advance dressing. However, the interventions using modern dressings for patient will determine the material / diabetic wound dressing. After lowering dressing to be applied because this is the criteria in the form of research related to financing. Wound care given to methods, finally 4 articles were obtained. patients should be able to improve the The results are in line with the results of process the research in the article, the results of (Handayani, 2016). of wound development the study generally state that the method The treatment provided is to of wound care with modern dresing has provide warmth and moist environment indeed proven to be significantly able to (moist) to the wound. Moist conditions on help the wound dressing process through the wound surface can enhance the the mechanism of the average occurrence process of wound development, prevent of wound dressing process and increasing tissue the quality of life of diabetic gangrene (Handayani, 2016). Other studies also wound patients after treatment with state that a humid environment can modern dresing methods. Wound dressing accelerate the inflammatory response, is influenced by several factors Some of resulting the factors that affect wound dressing are (Nabila, Efendi and Husni, 2013). immunological or immune status, blood dehydration in In a and faster cell humid cell death. proliferation atmosphere cell sugar levels, wound rehydration and metabolism will be better because more washing, nutrition, blood albumin levels, water, oxygen available. The moist mood effect can supply and vascularization (Kartika, 2015). nutrients, and vitamins are prevent tissue dehydration, cell death, The method of wound care that is accelerate angiogenesis, increase the developing today is using the principle of breakdown of dead tissue and fibrin, and moisture balance, which is said to be more reduce pain during medication (Nabila, effective methods. Efendi and Husni, 2013). Another type of Wound care using the principle of modern dressing, Ca Alginate, contains moisture balance is known as the modern Ca can help stop bleeding. Then there is method of dressing (Kartika, 2015). hydrocellulose which is able to absorb than conventional In its implementation, wound care twice as much liquid as Ca Alginate. Next to patients in this wound care practice is a hydrocolloid that is able to protect uses the concept of modern wound care from water and bacterial contamination, with the principle of moisture balance and can be used for primary and secondary 143 dressing. The use of modern types of the author considers that the use of dressings is adapted to the type of wound. modern dressing mamang is very good to For wounds with a lot of exudate, a be used in the healing process of diabetic dressing material is chosen that absorbs gangrene, especially like the experience liquids such as foam, while for wounds experienced by the author himself in that have begun to grow granulation, gel is treating gangrene patients, Where the given to create a moist atmosphere that treatment will help accelerate wound dressing infection needs to be done repeatedly by (Kartika, 2015). The results of research changing the bandage and cleaning the from Subandi and Sanjaya (2020) stated wound area so that the wound remains that there is still a diabetic wound rate in clean so that it can help the wound the 15%. dressing process This is certainly directly not proportional to the principle of modern understand the existence of proper wound dressing care, namely moisture balance. care and some people choose conventional Where the results that can be found when wound care because it is easy to obtain the principle of moisture balance is tools done applied in the treatment of gangrenous independently, while this technique has wounds repeatedly, it will make it easier quite a lot of negative impacts such as for nurses to open the bandage and high risk of infection, quick dry dressing, prevent damage to newly grown tissue due risk of causing new wounds and odorous to the attachment of tissue in a dry dressings. In contrast to modern dressings dressing as commonly experienced in that rely on moisture for the wound conventional wound care. community Indicating and that as much people materials, still can as do be carried out in preventing dressing process with the excess of Articles about the implementation absorbing eskudat well, not smelling, of wound care with modern dressings on effective hospital treatment. Therefore, the diabetic wound dressing are still not treatment method must be in the nature of much, but the evidence found from the maintaining moisture and maintaining article is strong enough because the warmth in the wound. Modern treatment articles displayed are published articles methods have a working principle by from good, official literature and have maintaining moisture and warmth of the been peer reviewed before publication wound area (Handayani, 2016). The quality and evidence presented in the Based on the article about modern article are quite strong, it's just that further dressing that has been described above, research is still needed with more human 144 samples and to prove the effectiveness of economical, non-addictive, can be given implementing wound care methods with at any time and have minimal side effects modern dressings on diabetic wound for the patient. With so few research dressing in human samples. Although the results using the best research methods number of articles looking at the effect of conducted on humans, further research modern dresing wound care interventions with better quality will greatly assist the on diabetic wound dressing is still small, process of developing modern wound modern dresing wound care interventions dressing methods to be practiced in have great opportunities to be practiced in Indonesia. This study can be corrected as clinical settings, a guide in providing wound care methods especially in Indonesia. This condition is with modern dressings for healing diabetic supported by the many advantages of wounds and community wound care with modern dressings more economical, easy to use, does not cause addiction, can be used at any time and has no side effects when given to patients who do diabetic wound care. Further research needs to be done on more human samples in different countries with different cultural characteristics. The selection of wound care sites with modern dressings has an important role because there are nerve pathways so it needs to be a concern so that wound care with modern dressings can work more optimally CONCLUSION The results of the literature review show that wound care with modern dressings is proven to be able to help heal diabetic wounds. The materials used in the treatment of wounds with modern REFERENCES Buchori, A., Sukron, S. and Wahyudi, J. T. 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https://openalex.org/W4387405963	https://www.researchsquare.com/article/rs-3400408/latest.pdf	English	null	The triangle congruence axiom that led to ellipse collinearity	Research Square (Research Square)	2,023	cc-by	4,044	The triangle congruence axiom that led to ellipse collinearity Peter Csiba (  csibap@ujs.sk ) Selye János University Peter Vajo Selye János University 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. Abstract This paper aims to prove the assumption that the center of the circle containing two vertices of a complete quadrilateral, along with the center of the conic section formed by the two diagonal points of the quadrilateral (with the third diagonal point being ﬁxed), are collinear with the midpoint of the segment deﬁned by the intersection of their common tangent lines. The common tangent lines are drawn from the two remaining vertices of the complete quadrilateral. Furthermore, a general theorem can be deduced from this proof, asserting that when a conic section is considered instead of a circle, the centers of the two conic sections and the midpoint of the section formed by the intersections of their common tangent lines are collinear. The assumptions were discovered and proved using the dynamic mathematics software GeoGebra, which oﬀers many visual tools such as the Show Trace or Locus tool, the CAS View, and the 3D calculator. These tools were employed to provide analytic proof. Consequently, a correlation has been identiﬁed that converts a projective correspondence into an aﬃne property. Peter Csiba1† and Peter Vajo1† Peter Csiba1† and Peter Vajo1† 1Department of Mathematics, J. Selye University, Bratislavsk´a cesta 3322, Kom´arno, 945 01, Slovakia. Peter Csiba1 † and Peter Vajo1† 1Department of Mathematics, J. Selye University, Bratislavsk´a cesta 3322, Kom´arno, 945 01, Slovakia. 1Department of Mathematics, J. Selye University, Bratislavsk´a cesta 3322, Kom´arno, 945 01, Slovakia. 1Department of Mathematics, J. Selye University, Bratislavsk´a cesta 3322, Kom´arno, 945 01, Slovakia. Corresponding author(s). E-mail(s): csibap@ujs.sk; Contributing authors: vajo.peter@student.ujs.sk; †These authors contributed equally to this work. Keywords: conic section, tangent lines, center of a conic section, midpoint, collinearity The triangle congruence axiom that led to ellipse collinearity Peter Csiba1† and Peter Vajo1† 1 Introduction This section provides a comprehensive overview of several essential concepts and sig- niﬁcant ﬁndings discussed in the article[1]. The exploration of triangle congruences has been a subject of study for numerous decades, and its signiﬁcance remains enduring. 1 Beginning with Rigby’s[2] research in the 1980s, followed by Donnelly’s[3][4] investiga- tion into triangle congruences within the context of the absolute plane, and concluding with Schellenberg’s[5] focus on the pedagogical implications of triangle axioms. While the following triangle congruences suﬃce to demonstrate triangle congruence: While the following triangle congruences suﬃce to demonstrate triangle congruence: SSS. Two triangles are congruent iﬀtheir corresponding sides are equal. ASA. Two triangles are congruent iﬀtwo pairs of corresponding angles, and the sides between them, are equal. ASA. Two triangles are congruent iﬀtwo pairs of corresponding angles, and the sides between them, are equal. SAS. Two triangles are congruent iﬀtwo pairs of corresponding sides, and the angles between those sides, are equal. AAS. Two triangles are congruent iﬀtwo pairs of corresponding angles, and one non-includes sides, are equal. SsA. Two triangles are congruent iﬀtwo pairs of corresponding sides, and the angles opposite the longest sides, are equal. RHS. Two right-angled triangles are congruent iﬀtheir corresponding hypotenuses, and one sides, are equal. (where S, A, s, R, and H represent, respectively, the side, angle, shorter side, right- angle, and hypotenuse of a triangle), the same cannot be stated for condition sSA. This condition’s ambiguity has perked the interest of other researchers as well [6][7]. Condition sSA holds for two triangles, if their two pairs of corresponding sides, and the angles opposite the shorter sides, are equal. As shown in article[1], this produces two distinct triangles ABC and A′B′C′. Each of these triangles has two sides c, b and c′, b′ respectively and an angle opposite one of the sides (which may be the smaller), such that c = c′ and b = b′ and γ = γ. If the two triangles are superimposed so that one (the smaller) of their sides, c and c′, coincides, the two identical (A = A′ and B = B′) and two non-identical vertices (C and C’) deﬁne a complete quadrilateral. Figure 1 depicts this construction. See the works [8][9] for a more in-depth look at complete quadrilaterals and projective geometry, and [10] about projective geometry and conic sections. Theorem 1. One of the diagonal points (D) of the complete quadrilateral ABCC’ will remain unchanged, while the other two (M, M) will describe a hyperbola when the angle (γ) deﬁning the triangle is altered. 1 Introduction (1) where if Θ = (2ayc −c2 −1)λ −c2 + 1, then (1) h1 = Θ2, h2 = (4a2 xc2 + c4 −2c2 + 1)λ2 + (2c4 −2)λ + c4 −2c2 + 1, h3 = −4axcλΘ, h4 = 4axc2λΘ, h5 = −4c(2a2 xc2 + ayc3 −ayc −c2 + 1)λ2 −4c(ayc3 −ayc + c2 −1)λ, h6 = (4a2 xc4 + 4a2 yc4 −4a2 yc2 −4ayc3 −c4 + 4ayc + 2c2 −1)λ2 +2(2ayc3 + c4 −2ayc −2c2 + 1)λ −c4 + 2c2 −1. ( (2) (2) h4 = 4axc2λΘ, h5 = −4c(2a2 xc2 + ayc3 −ayc −c2 + 1)λ2 −4c(ayc3 −ayc + c2 −1)λ, h6 = (4a2 xc4 + 4a2 yc4 −4a2 yc2 −4ayc3 −c4 + 4ayc + 2c2 −1)λ2 +2(2ayc3 + c4 −2ayc −2c2 + 1)λ −c4 + 2c2 −1. when K is a circle with the coordinate system’s origin [0,0] as its center and the radius r = 1. Let D be a ﬁxed y-axis point with coordinates (0, 1/c), where c ̸= 1 and c ̸= 0. Moreover, let A(ax, ay) be an arbitrary point, and since B is on the line AD with equation (ayc −1)x −axcy + ax = 0, then B(λax, λay + (1 −λ)(1/c)), where λ is ﬁxed and λ ̸= 0, λ ̸= 1. ̸ , ̸ Read more about conic sections in [13][14]. The article’s[1] Corollary 7 explains that lines tangent to circle K at points A and B are also tangent to conic section H, and it will simplify our construction and increase the likelihood of noticing connections if we view the structure as a circle and a conic section with common tangent lines that intersect at point A and B. 1 Introduction [ ] Using condition sSA, if triangles ABC and A′B′C′, where c = c′, b = b′ and γ = γ are superimposed in such way that sides c and c′ coincides, then: Theorem 1. One of the diagonal points (D) of the complete quadrilateral ABCC’ will remain unchanged, while the other two (M, M) will describe a hyperbola when the angle (γ) deﬁning the triangle is altered. 2 2 Fig. 1 Two distinct triangles with the locus of points M and M as a hyperbola Fig. 1 Two distinct triangles with the locus of points M and M as a hyperbola See the proof for Theorem 1 in article [1] under Theorem 3. See the proof for Theorem 1 in article [1] under Theorem 3. Generalization of Theorem 1 leads to the following theorem: Theorem 2. Assume that A, B, and D are ﬁxed collinear points. Consider K to be a circle, C to be an arbitrary point on the circle, and C’ to be the other intersection of the secant CD with the circle. The diagonal point D of the complete quadrilateral ABCC’ is ﬁxed, but the other two (M,M) describe a conic section if C runs along the circle, and the coeﬃcients of the general equation for this conic section can be calculated. See the proof for Theorem 2 in article [1] under Theorem 7. Fig. 2 The conic section indicated by GeoGebra’s Locus tool Fig. 2 The conic section indicated by GeoGebra’s Locus tool 3 This concept may be derived from GeoGebra by utilizing the Show trace and Locus options on the diagonal points as points C and C′ move along the circle. Moreover, by locating additional points on the conic section, we can use GeoGebra’s Conic through 5 points tool to construct the conic section. It is known as the Braikenridge-Maclaurin Construction to construct a conic section from ﬁve points lying on it. Read more about this in [11][12]. [ ][ ] Article[1] deﬁned the equation of H Article[1] deﬁned the equation of H h1x2 + h2y2 + h3xy + h4x + h5y + h6 = 0. 2 Results Intriguingly, the line deﬁned by the center points of circle K and conic section H appears to intersect line segment AB at its midpoint. 4 Fig. 3 (a)The line deﬁned by the center points intersects line segment AB at its midpoint (b)GeoGebra visually veriﬁed this assumption, as it remains true in modiﬁed constructions as well Fig. 3 (a)The line deﬁned by the center points intersects line segment AB at its midpoint (b)GeoGebra visually veriﬁed this assumption, as it remains true in modiﬁed constructions as well In other words: Theorem 3. The centers of circle K and conic section H and the midpoint of the line segment deﬁned by the intersections of their common tangent lines are collinear. Using GeoGebra’s interactive feature and modifying the structure appear to strengthen this assumption. For proving this, however, an analytical approach would be highly eﬀective. We will use the coordinates provided in the aforementioned article[1] to establish proof for our theorem. Proof. Let the center of circle K serve as our coordinate system’s origin, with coordi- nates [0,0] and r = 1. Let D be a ﬁxed y-axis point with coordinates (0, 1/c), where c ̸= 1 and c ̸= 0. Moreover, let A(ax, ay) be an arbitrary point, and since B is on the line AD with equation (ayc −1)x −axcy + ax = 0, then B(λax, λay + (1 −λ)(1/c)), where λ is ﬁxed and λ ̸= 0, λ ̸= 1. In this case, the equation for H is (1), where (2). Using vectors to demonstrate collinearity seems reasonable, but in order to do that, we need to ﬁnd the coordinates of the center point of conic section H and the mid- point of line segment AB. Note that we already know the coordinates of the center of circle K, as the origin [0, 0] is known. Using the origin as the common initial point of the two vectors and having K and S as terminal points, parallelism and dependence can be proven, which is suﬃcient for proving the collinearity of the centers of circle K and conic section H and the midpoint of line segment AB. Let K be the midpoint of line segment AB. Since we know the coordinates of A and B, the coordinates of K can be calculated. 2 Results The coordinates of point K are K = ax + λax 2 ; ay + λay + (1 −λ) 1 c 2 (3) (3) 5 5 Determining the coordinates of the center of the conic section H proves to be a more challenging task. The center of this conic section may not be centered around the origin, nor may its axes lie along the x- and y-axes. The standard form of the equation of a central conic section is produced by translating and rotating the conic section so that its center lies at the origin of the coordinate system and its axes coincide with the coordinate axes. This is comparable to stating that the coordinate system’s center is shifted and the coordinate axes are rotated to satisfy these criteria. Read more about translating and rotating coordinates in [15]. Due to the complexity of our coeﬃcients, however, it becomes less beneﬁcial to employ this approach. The notion of determining the coordinates of the center of conic section H based on its equation alone is similar to the bulletproof midpoint calculation that we used to determine the coordinates of point K. If we knew the coordinates of the antipodal points of conic section H, we could calculate the coordinates of its center, since the line segment joining the antipodal points must pass through the center of the conic section, and it is the midpoint of this line segment. By using the intercept theorem, the antipodal points are produced by parallel intersecting lines to the conic section. Looking at the case when the parallel intersecting lines are vertical tangent lines (perpendicular to the x-axis) to H, we can determine the coordinates of these tangent (antipodal) points from the general conic section equation. See more about intersecting lines and conics in [16]. Let’s substitute p for x (x = p), as x has a concrete value when working with vertical tangent lines to equation (1). Solving the quadratic equation for y yields the following roots: y1,2 = −h3p −h5 ± p −4h1h2p2 + h2 3p2 + 2h3h5p −4h2h4p −4h2h6 + h2 5 2h2 (4) (4) Since tangent lines only meet at one point (the tangent point), the discriminant, which is the quadratic equation under the root, must equal zero. 2 Results Given that the expression under the root equates to zero y1,2 = −h3p −h5 2h2 (5) (5) We must solve the quadratic equation −4h1h2p2+h2 3p2+2h3h5p−4h2h4p−4h2h6+ h2 5 when x = p = 0 to know when y takes on these values. −4h1h2p2 + h2 3p2 + 2h3h5p −4h2h4p −4h2h6 + h2 5 = 0 (6) (6) p1,2 = h3h5 −2h2h4 ± 2 p −4h1h2 2h6 + h1h2h2 5 + h2 3h2h6 −h3h2h4h5 + h2 2h2 4 4h1h2 −h2 3 (7) (7) Hence, when x=0 6 y1,2 = −h3x1,2 −h5 2h2 (8) (8) 2h2 2 We can summarize the coordinates of the antipodal points of conic section H. The coordinates of the antipodal points are: We can summarize the coordinates of the antipodal points of conic section H. The coordinates of the antipodal points are: [x1; y1] = " h3h5 −2h2h4 + 2 √ D 4h1h2 −h2 3 ; −h3x1 −h5 2h2 # (9) (9) and [x2; y2] = " h3h5 −2h2h4 −2 √ D 4h1h2 −h2 3 ; −h3x2 −h5 2h2 # (10) (10) where where D = −4h1h2 2h6 + h1h2h2 5 + h2 3h2h6 −h3h2h4h5 + h2 2h2 4 (11) (11) Let S serve as the center of conic section H. Knowing that the center of conic section H is the midpoint of the line segment created by the antipodal points, the coordinates of this midpoint may now be calculated as the arithmetic mean of the coordinates of points [x1; y1] and [x2; y2]. S = h3h5 −2h2h4 4h1h2 −h2 3 ; −2h1h5 + h3h4 4h1h2 −h2 3 (12) (12) Substituting the value of the coeﬃcients from 2 yields the coordinates of the conic section’s center point: S = 2axc2λ2 + 2axc2λ c2λ2 + 2c2λ + c2 −λ2 + 2λ −1; 2ayc2λ2 + 2ayc2λ −2cλ2 + 2cλ c2λ2 + 2c2λ + c2 −λ2 + 2λ −1 (13) (13) These are the coordinates of the center of conic section H. These are the coordinates of the center of conic section H. Now we have the coordinates for all three points: the midpoint of line segment AB, the center of conic section H, and the center of circle K. → → Let’s deﬁne the vectors → OK and → OS, where both vectors have the origin as their initial point and have diﬀerent K and S terminal points. 2 Results −−→ OK and −→ OS vectors are deﬁned as follows: −−→ OK = ax + λax 2 ; ay + λay + (1 −λ) 1 c 2 . (14) −→ OS = 2axc2λ2 + 2axc2λ c2λ2 + 2c2λ + c2 −λ2 + 2λ −1; 2ayc2λ2 + 2ayc2λ −2cλ2 + 2cλ c2λ2 + 2c2λ + c2 −λ2 + 2λ −1 (15) −−→ OK = ax + λax 2 ; ay + λay + (1 −λ) 1 c 2 . (14) (14) −→ OS = 2axc2λ2 + 2axc2λ c2λ2 + 2c2λ + c2 −λ2 + 2λ −1; 2ayc2λ2 + 2ayc2λ −2cλ2 + 2cλ c2λ2 + 2c2λ + c2 −λ2 + 2λ −1 (15) (15) Since, strictly speaking, vectors can be deﬁned as having only the properties direc- tion and magnitude, we can deﬁne parallel vectors as vectors with the same or opposing 7 direction and variable magnitude. Hence, two vectors are parallel if and only if they are scalar multiples of each other.→ → → → If there exists a k such that −→ OS = k ∗−−→ OK, then −−→ OK and −→ OS vectors are parallel and k = −→ OS/−−→ OK for both x and y coordinates. GeoGebra not only played a signiﬁcant role in noticing our assumption, but it also helped to visually verify it and create ﬁgures for the article, as well as precisely validate some highly complex computations. The GeoGebra ﬁles of all the constructions and computations are available at [17]. Theorem 4. The centers of conic section K and conic section H and the midpoint of the line segment deﬁned by the intersections of their common tangent lines are collinear. The question is whether this relationship can be demonstrated in a more general form for arbitrary conic sections as opposed to a circle. Since the aﬃne transformation preserves collinearity and ratios of distances, it is possible to prove this for ellipses obtained by stretching a circle; despite this, for hyperbolas and parabolas, the proof and the resulting values are too complex to be done analytically. Fig. 4 The line deﬁned by the center points of the two conic sections appears to intersect the AB segment at its midpoint Fig. 2 Results 4 The line deﬁned by the center points of the two conic sections appears to intersect the AB segment at its midpoint However, we can ﬁnd a three-dimensional transformation that includes the proof for Theorem 3. Proof. We select a point outside of this plane as the center of projection and another plane that contains the line AB. Consequently, the image of the circle (as depicted in 8 Figure 5, there is a correspondence between which points correspond to which images in the other plane) will be an arbitrary conic section (it can be an ellipse, a parabola, or a hyperbola), and because the line AB is ﬁxed, the midpoint of the line AB and the midpoint of their image are the same in the projection, so the same relationship holds true here. Figure 5, there is a correspondence between which points correspond to which images in the other plane) will be an arbitrary conic section (it can be an ellipse, a parabola, or a hyperbola), and because the line AB is ﬁxed, the midpoint of the line AB and the midpoint of their image are the same in the projection, so the same relationship holds true here. Figure 5 depicts the three-dimensional structure formed by intersecting planes. Line AB serves as the intersection line, while point P serves as the center of projection. The circle is projected onto an inclined plane, resulting in an ellipse where the same collinearity concept can be proved. Fig. 5 Collinearity in a three-dimensional space Fig. 5 Collinearity in a three-dimensional space Collinearity is a quality that is preserved under aﬃne transformations, and it can be observed that every ellipse may be obtained as an aﬃne transformation of a circle. In our case, it is noteworthy that the process of projective transformation results in a metric proof. Moreover, this observation provides evidence for the distinct properties exhibited by ellipses in terms of the collinearity of their centers and the point of intersection of their common tangent lines. Declarations • Funding: “This research was funded by VEGA grant (Vedeck´a Grantov´a Agent´ura MˇSVVaˇS SR) number 1/0386/21.” • Conﬂict of interest: The authors declare no conﬂict of interest. • Ethics approval: Not applicable • Consent to participate: Not applicable • Consent for publication: Not applicable • Availability of data and materials: Not applicable • Code availability: Not applicable • Authors’ contributions: Not applicable • Funding: “This research was funded by VEGA grant (Vedeck´a Grantov´a Agent´ura MˇSVVaˇS SR) number 1/0386/21.” • Funding: “This research was funded by VEGA grant (Vedeck´a Grantov´a Agent´ura MˇSVVaˇS SR) number 1/0386/21.” • Conﬂict of interest: The authors declare no conﬂict of interest. • Ethics approval: Not applicable • Consent to participate: Not applicable • Consent for publication: Not applicable • Availability of data and materials: Not applicable • Code availability: Not applicable • Authors’ contributions: Not applicable 3 Conclusion Through an examination of the unique characteristic of the side-Side-angle (sSA) tri- angle congruence axiom presented in this paper, novel theorems about the centers of conic sections and the intersection points of their common tangent lines were formu- lated and then proven. Initially, we have established the veriﬁcation of our assumption that the center of the circle on which two vertices of a complete quadrilateral lie and the center of the conic section described by the two diagonal points of the complete 9 quadrilateral (the third diagonal point is ﬁxed) are collinear with the midpoint of the section deﬁned by the intersection of their common tangent lines. The general theorem for such collinearity is derived by looking at not a circle but conic sections, and it states that the centers of the two conic sections and the midpoint of the section deﬁned by the intersection of their common tangent lines are collinear. The presence of collinearity is not universal in the examination of conic sections and their common tangent lines. For instance, the center points of two touching ellipses and the inter- section of their two common tangent lines are not collinear. Nevertheless, this general theorem establishes that this distinctive property is indeed present in conic sections and the section deﬁned by the intersection of their common tangent lines. The article highlights that the phenomena and relationships discussed can be eﬃciently demonstrated utilizing interactive geometric software like GeoGebra. Con- sequently, these demonstrations can be utilized to engage students as well as aspiring or current mathematics teachers. Furthermore, it is important to note that the math- ematical apparatus employed in the proofs does not exceed the standard curriculum of geometry, mathematical analysis, and algebra courses used in mathematics teacher education. Supplementary information. The GeoGebra ﬁles—constructions and computa- tions included—serve as supplementary ﬁles to the article. They may be accessed online at [17]. [2] Rigby, J.F.: Axioms for absolute geometry. 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https://openalex.org/W2055615655	https://www.arkat-usa.org/get-file/29079/	English	null	Photocatalytic oxidation of dihydropyrimidinones using titanium dioxide suspension	ARKIVOC	2,009	cc-by	3,923	Keywords: Titanium dioxide, photocatalytic, photooxidation, dihydropyrimidinone Keywords: Titanium dioxide, photocatalytic, photooxidation, dihydropyrimidinone ARKIVOC 2009 (x) 255-264 ARKIVOC 2009 (x) 255-264 General Papers E-mail: manas@mail.yu.ac.ir E-mail: manas@mail.yu.ac.ir Abstract Abstract Photocatalytic oxidation has been used for the oxidation of some ethyl 3,4-dihydropyrimidin- 2(1H)-one-5-carboxylates to their corresponding ethyl pyrimidin-2(1H)-one-5-carboxylates using a TiO2/O2 system under UV irradiation by a 400 W high pressure mercury lamp in acetonitrile. The results revealed that the order of photocatalytic activity for photooxidation was TiO2 (anatase) > TiO2 (rutile). The effects of some other physicochemical parameters such as amount of photocatalyst, pH, solvent and time of irradiation were studied. The pyrimidinones were attained from the related dihydropyrimidinones after 2-4.5 h. The results showed the photo stability of this type of compound. Department of Chemistry, Yasouj University, Yasouj 75918-74831, Iran Department of Chemistry, Yasouj University, Yasouj 75918-74831, Iran Introduction 3,4-Dihydropyrimidin-2(1H)-ones (DHPMs) belong to an important class of heterocyclic compounds that have attracted interest due to their pharmacological and biological properties, such as antihypertensive, calcium channel blocking, alpha-1a-antagonism, neuropeptide Y(NPY) antagonism, antitumor, antibacterial, and antiinflammatory activities.1-6 Oxidation of DHPMs to pyrimidine-2(1H)-ones is relevant to MKC-442, a HEPT(1-[(2- hydroxyethoxy)methyl]-6-(phenylthio)thymine) analogue which is in clinical trials, and similar compounds are also expected to inhibit the HIV virus.7 Several nucleosides containing 5- substituted pyrimidine moiety have been shown to inhibit the growth of murine mammary carcinoma virus.8 Pyrimidine-cores with extended π-systems have interesting fluorescent properties and similar compounds are useful in the development of advanced electronic and photonic materials.9 Furthermore, it is of interest to synthesize structurally diverse pyrimidines by the oxidation of DHPMs.10 ©ARKAT USA, Inc. ©ARKAT USA, Inc. ISSN 1551-7012 Page 255 Page 255 ARKIVOC 2009 (x) 255-264 General Papers In contrast to the easy oxidation of typical Hantzsch dihydropyridines methods,11-13 the dehydrogenation of 3,4-dihydropyrimidin-2(1H)-ones is nontrivial.14,15 Oxidants such as HNO3,16 PCC,17 chloranil,17 KMnO4/clay,17 DDQ,18 Co(NO3)2-6H2O/K2S2O8,19 electrochemical oxidation20 and Pd/C21 as well as sono-thermal oxidation22 have been examined. None of these oxidations are efficient, some use excessively corrosive or harmful reagents, strong reaction conditions, or present difficulties in product isolation, and/or mostly low yields. Therefore, an alternative procedure is needed. The potential of heterogeneous photocatalysis in chemistry is now a well-established procedure.23 The strong oxidizing power of the photogenerated holes of semiconductors (large band gap material), the chemical inertness and resistance to both photocorrosion and decomposition reactions, which plague other band gap materials (e.g., Si, GaAs, GaP, Inp, CdS, etc.), low cost and wide availability in addition to the nontioxicity of TiO2 (anatase and rutile) and zinc oxide have made them superior photocatalysts.24 Several articles and reviews have been written on the use of semiconductor oxides as photocatalysts.23-30 With illumination of a semiconductor photocatalyst such as TiO2 by photons whose energy is equal to or higher than their band-gap energy (for anatase, Eg = 3.23 eV), absorption of these photons occurs and the bulk of electron–hole pairs generate. These electron–hole pairs dissociate into free photoelectrons in the conduction band e- CB and photoholes in the valence band h P+ VB (equation 1). TiO2* (e- CB .... Introduction h+ VB) hυ TiO2 (1) (1) Some of the photoelectrons and photoholes can reach the surface of the photocatalyst and then an electron transfer proceeds towards adsorbed acceptor molecules and positive photoholes are transferred to adsorbed donor molecules. The photohole transfer corresponds to the cession of an electron by donor molecules to the photocatalyst. A chemical acceptor species can be photocatalytically reduced by eCB only if the conduction band potential of the photocatalyst is more negative than the redox potential of the acceptor species. In the same way, a chemical donor species can be photocatalytically oxidized by h BVB only if the valence band potential of the photocatalyst is more positive than the redox potential of the donor species. Both reactions should occur simultaneously because electroneutrality has to be maintained.31,32 Some of the photoelectrons and photoholes can reach the surface of the photocatalyst and then an electron transfer proceeds towards adsorbed acceptor molecules and positive photoholes are transferred to adsorbed donor molecules. The photohole transfer corresponds to the cession of an electron by donor molecules to the photocatalyst. A chemical acceptor species can be photocatalytically reduced by eCB only if the conduction band potential of the photocatalyst is more negative than the redox potential of the acceptor species. In the same way, a chemical donor species can be photocatalytically oxidized by h BVB only if the valence band potential of the photocatalyst is more positive than the redox potential of the donor species. Both reactions should occur simultaneously because electroneutrality has to be maintained.31,32 In continuation of our previous studies on photochemical reactions,33-37 we report here the photocatalytic oxidation of DHPMs. To the best of our knowledge this is the first report of the oxidation of 3,4-dihydropyrimidin-2(1H)-ones using photocatalytic system. ISSN 1551-7012 ©ARKAT USA, Inc. Page 256 Page 256 ARKIVOC 2009 (x) 255-264 ARKIVOC 2009 (x) 255-264 General Papers Results and Discussion Our investigation showed us that UV irradiation is nesessary for the effective progress of the oxidation reactions and, without the selected oxidant and oxygen, oxidation did not occur. With this preliminary result, the optimization of important operational parameters was performed in the photooxidation reaction of 3,4-dihydropyrimidin-2(1H)-ones (Scheme 1). NH N H H R EtO2C H3C O N N H R EtO2C H3C O TiO2, hυ CH3CN, RT 1 2 Scheme 1 2 The type of photocatalyst yp p y TiO2 (anatase or rutile) (40 mg) was used for the photocatalytic conversion of 1 mmol of 5- ethoxycarbonyl-6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one or 5-ethoxycarbonyl-6- methyl-4-(4-methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one in acetonitrile with oxygen bubbling, under irradiation. The time required for completion of the oxidation was used to compare the photocatalytic activity of semiconductor oxides. As shown in Table 1, titanium dioxide (anatase) > titanium dioxide (rutile) in progressing the oxidation reaction. Perhaps, the higher oxygen uptake quantum yields and slower recombination of photogenerated electron/hole couples are responsible for the better photocatalytic activity of TiO2 anatase with respect to TiO2 rutile. Table 1. The effect of photocatalyst type on the photocatalytic oxidation of DHPMs Type of catalyst Entry R Anatase Rutile Time (h) Yields (%)a Time (h) Yields (%) 1 C6H5 3 90 5 50 2 4-CH3OC6H4 3 80 6 60 aIsolated yields Table 1. The effect of photocatalyst type on the photocatalytic oxidation of DHPMs Type of catalyst Entry R Anatase Rutile Time (h) Yields (%)a Time (h) Yields (%) 1 C6H5 3 90 5 50 2 4-CH3OC6H4 3 80 6 60 aIsolated yields ble 1. The effect of photocatalyst type on the photocatalytic oxidation of DHPMs The amount of photocatalyst Using the better photocatalyst, the optimum amount of it required for photocatalytic oxidation was investigated. Thus, oxidations were run using various amounts of TiO2. As shown in Table 2, oxidation times were decreased by increasing the photocatalyst quantity, then reached the Page 257 ©ARKAT USA, Inc. ©ARKAT USA, Inc. ISSN 1551-7012 Page 257 ARKIVOC 2009 (x) 255-264 General Papers lowest time of completion and finally remained constant at a value of 40 mg. It is interesting to note that this phenomenon has been observed previously in other photocatalytic reactions.38-41 lowest time of completion and finally remained constant at a value of 40 mg. It is interesting to note that this phenomenon has been observed previously in other photocatalytic reactions.38-41 This can be rationalized in terms of availability of active sites on the TiO2 surface and the poor penetration of photoactivating light into the suspension. The availability of active sites increases with the suspension of photocatalyst loading, but the light penetration and hence the photoactivated volume of the suspension shrinks. Moreover, the increase in the time of oxidation at higher photocatalyst loading may be due to deactivation of activated molecules by collision with ground state molecules. Shielding by TiO2 may also take place (equation 2). TiO2 * + TiO2 → TiO2+ TiO2* (2) TiO2 * + TiO2 → TiO2+ TiO2* (2) (2) Where TiO2 * is the TiO2 with active species adsorbed on its surface and TiO2 is the deactivated form. Table 2. The effects of photocatalyst amounts of Titanium dioxide on the oxidation of typical 3,4- dihydropyrimidin-2(1H)-onea Entry Amount of TiO2 (mg) Time (h) Yields (%)b 1 10 6 85 2 20 4.5 88 3 40 3 90 4 60 4 86 5 80 5.5 84 aTio2 (anatase) as photocatalyst and 5-ethoxycarbonyl-6-methyl-4-phenyl-3,4- aTio2 (anatase) as photocatalyst and 5-ethoxycarbonyl-6-methyl-4-phenyl-3,4- dihydropyrimidin-2(1H)-one as typical DHPMs were used. bIsolated yields. Effect of pH bThe typical DHPMs were converted to the corresponding ethyl pyrimidin- 2(1H)-one-5-carboxylate. cIsolated yields. Effect of pH The potentials of both valence and conduction bands of TiO2 follow a pH dependence, according to equations 3 and 4, that show decreasing 59 mV per pH unit and consequently, the ability of electrons and holes to participate in redox processes is determined by the pH of the medium.42 These equations are associated with TiO2 in anatase form at 25 °C. ECB = -0.05 - 0.059 pH (3) EVB = 3.15 - 0.059 pH (4) (3) (4) (4) The effects of varying of pH from 3-11 are summarized in Table 3. From the table we see that pH 7 was optimum in the presence of TiO2 (anatase). It seems that, since TiO2 usually has an isoelectric point of charge at a pH about ~7, its surface will gain a positive charge at pHs lower than ~7 via protonation (equation 5) and a negative charge when the material is suspended in a The effects of varying of pH from 3-11 are summarized in Table 3. From the table we see that pH 7 was optimum in the presence of TiO2 (anatase). It seems that, since TiO2 usually has an isoelectric point of charge at a pH about ~7, its surface will gain a positive charge at pHs lower than ~7 via protonation (equation 5) and a negative charge when the material is suspended in a Page 258 ©ARKAT USA, Inc. ©ARKAT USA, Inc. ISSN 1551-7012 Page 258 ARKIVOC 2009 (x) 255-264 General Papers solution with pHs higher than ~7 via deprotonation (equation 6), respectively. At pHs lower than ~7, both the titled compounds and TiO2 surface are present mostly in positively charged and protonated form and therefore repel each other. At pH 7, both the DHPMs and photocatalyst surface are mostly in neutral and in an un-protonated form and therefore the substrate molecules are more readily adsorbed on to the photocatalyst surface and the DHPMs oxidation reaction is favored.43 TiOH + H+ ⇆ TiOH2 + (5) TiOH + OH- ⇆ TiO- + H2O (6) (6) Table 3. Effect of pH on the photocatalytic oxidation of DHPMsa Table 3. Effect of pH on the photocatalytic oxidation of DHPMsa Entry pH Time (h) Yields (%)c 1 3 6 85 2 5 4.5 87 3 7 3 90 4 9 4 86 5 11 7 88 aTio2 (anatase) as photocatalyst and 5-ethoxycarbonyl-6-methyl-4-phenyl- 3,4-dihydropyrimidin-2(1H)-one as typical DHPMs were used. Photocatalytic oxidation of dihydropyrimidinones In the optimum conditions, the photocatalytic reactions (Scheme 1) proceed efficiently in high yields. The results are summarized in Table 4. DHPMs with electron withdrawing substituents take longer reaction times than those with electron donor substituents. ISSN 1551-7012 ©ARKAT USA, Inc. Page 259 Page 259 ARKIVOC 2009 (x) 255-264 General Papers Table 4. Photocatalytic oxidation of 3,4-Dihydropyrimidin-2(1H)ones with TiO2 / O2 syste TiO2 (anatase) TiO2 (rotile) Entry Productsa Time (h) Yields (%)b Time (h) Yields (%) mp oC 1 N H N O EtO2C Me 3 90 5 50 133-13422 2 N H N O EtO2C Me Cl 2.5 95 5 57 180-182 3 N H N O EtO2C Me OCH3 3 85 6 50 151-15322 4 N H N O EtO2C Me Cl Cl 2 96 4.5 55 185-186 5 N H N O EtO2C Me NO2 4.5 80 8 45 190-19244 6 N H N O EtO2C Me NO2 4 85 6 35 152-15422 7 N H N O EtO2C Me CH3 2 95 4 53 136-13822 talytic oxidation of 3,4-Dihydropyrimidin-2(1H)ones with TiO2 / O2 system 3 3 96 4 5 6 ISSN 1551-7012 Page 260 ©ARKAT USA, Inc. ISSN 1551-7012 Page 260 Page 260 ARKIVOC 2009 (x) 255-264 General Papers Table 4. Continued 8 N H N O EtO2C Me Cl 2.5 85 5 40 165-16744 9 N H N O EtO2C Me Br 2 95 4.2 49 167-168 10 N H N O EtO2C Me OCH3 2 80 4 43 121-12322 a Characterized by spectral analysis and comparison with authentic samples.22,44 b Yields refer to isolated and purified products. 8 N H N O EtO2C Me Cl 2.5 85 5 40 165-16744 9 N H N O EtO2C Me Br 2 95 4.2 49 167-168 10 N H N O EtO2C Me OCH3 2 80 4 43 121-12322 a Characterized by spectral analysis and comparison with authentic samples.22,44 b Yields refer to isolated and purified products. 8 N H N O EtO2C Me Cl 2.5 9 N H N O EtO2C Me Br 2 10 N H N O EtO2C Me OCH3 2 Me 9 2 Me 10 a Characterized by spectral analysis and comparison with authentic samples.22,44 b Yields refer to isolated and purified products. aValues refer to yield(%)/time(h for A, B, C and min for D) ratios; Conclusions The reported work demonstrates that using the TiO2/O2 photocatalytic system for dehydrogenation of 3,4-dihydropyrimidin-2(1H)-ones enhances the reaction rate compared to thermal oxidation. The results also show that TiO2 in anatase form is more effective than its rutile form in the oxidation. Easy reaction progress, moderate reaction times and good to excellent yields are some advantages of this oxidative method. Comparative results To demonstrate the potential of our new approach, the presently obtained experimental results and the data acquired with the other methodologies are compared in Table 5. The yield/time ratios of the present method are better or comparable with others. Table 5. Comparison of some our results with those reported in the litraturea Entryb Ac B C 1 90/3 83/1 92/11 2 95/2.5 - - 3 85/3 81/1 92/7 5 80/4.5 80/1 - 6 85/4 - 90/27 7 95/2 - 90/7 8 85/2.5 85/1 90/5 Table 5. Comparison of some our results with those reported in the litraturea aValues refer to yield(%)/time(h for A, B, C and min for D) ratios; b The entries refer to those in Table 4; b The entries refer to those in Table 4; cA: Our method; B: CAN (3 eq.), NaHCO3 (5 eq.), aq. Acetone, argon atm., –5 °C;44 C: K2S2O8, aq. CH3CN, 70 °C, Ultrasonic.22 ©ARKAT USA, Inc. ©ARKAT USA, Inc. ISSN 1551-7012 Page 261 Page 261 ARKIVOC 2009 (x) 255-264 ARKIVOC 2009 (x) 255-264 General Papers Experimental Section General Procedures. Chemicals were purchased from Merck, Fluka and Aldrich chemical companies. The commercially available TiO2 powders were anatase in crystalline form with a surface area about 50 m2 /g and primary particle size of 30 nm and rutile with approximate 0.2 micron in size and surface area of about 14.747 m2/g. 3,4-Dihydropyrimidin-2(1H)-ones were prepared according to the reported procedures.45 Reactions were monitored by TLC. The products were isolated and identified by comparison of their physical and spectral data with authentic samples. IR spectra were recorded on FT-IR 680-Jasco-instrument model. 1H NMR data were obtained on 300 MHz DPX-Brucker model. General procedure for the photocatalytic oxidation of 3,4-dihydropyrimidin-2(1H)-ones Titanium dioxide (40 mg, TiO2) was added to a solution containing a DHPM (1 mmol) in 20 mL acetonitrile through which oxygen was bubbling. The mixture was stirred at room temperature with irradiation by UV light (400 W high pressure mercury lamp) for the appropriate time (2- 6 h). After completion of the reaction, as monitored by TLC (CCl4: EtOAc), the titanium dioxide was separated by centrifugation. Evaporation of the solvent followed by chromatography on a silica-gel plate afforded the pure products. Ethyl 6-methyl-4-(4-chlorophenyl)pyrimidin-2(1H)-one-5-carboxylate (2). Pale yellow solid; mp 180–182 ºC. IR (KBr): 3255, 1705, 1642, 1410 and 1250 cm-1. 1H NMR (DMSO-d6): δ= 1.15 (t, 3H, J = 7.2 Hz), 2.52 (s, 3H), 4.03 (q, 2H, J = 7.2 Hz), 7.24-7.41 (m, 4H), 10.73 (s, 1H) ppm. Anal. Calcd. for C14H13ClN2O3: C 57.44, H 4.48, N 9.57% found: C 57.3, H 4.4, N 9.4% Ethyl 6-methyl-4-(2,6-dichlorophenyl)pyrimidin-2(1H)-one-5-carboxylate (4). Pale yellow solid; mp 185–186 ºC. IR (KBr): 3235, 2930, 1700, 1670, 1430 1200 cm-1. 1H NMR (DMSO- d6): δ= 0.85 (t, 3H, J = 7.3 Hz), 2.50 (s, 3H), 3.94 (q, 2H, J = 7.3 Hz), 7.17-7-29 (m, 3H), 12.73 (s, 1H) ppm. Anal. Calcd. for C14H12Cl2N2O3: C 51.40, H 3.70, N 8.56% found: C 51.5, H 3.8, N 8.7% Ethyl 6-methyl-4-(4-chlorophenyl)pyrimidin-2(1H)-one-5-carboxylate (2). Pale yellow solid; mp 180–182 ºC. IR (KBr): 3255, 1705, 1642, 1410 and 1250 cm-1. 1H NMR (DMSO-d6): δ= 1.15 (t, 3H, J = 7.2 Hz), 2.52 (s, 3H), 4.03 (q, 2H, J = 7.2 Hz), 7.24-7.41 (m, 4H), 10.73 (s, 1H) ppm. Anal. Calcd. for C14H13ClN2O3: C 57.44, H 4.48, N 9.57% found: C 57.3, H 4.4, N 9.4% Ethyl 6-methyl-4-(2,6-dichlorophenyl)pyrimidin-2(1H)-one-5-carboxylate (4). Pale yellow solid; mp 185–186 ºC. IR (KBr): 3235, 2930, 1700, 1670, 1430 1200 cm-1. Experimental Section 1H NMR (DMSO- d6): δ= 0.85 (t, 3H, J = 7.3 Hz), 2.50 (s, 3H), 3.94 (q, 2H, J = 7.3 Hz), 7.17-7-29 (m, 3H), 12.73 (s, 1H) ppm. Anal. Calcd. for C14H12Cl2N2O3: C 51.40, H 3.70, N 8.56% found: C 51.5, H 3.8, N 8.7% Ethyl 6-methyl-4-(3-bromophenyl)pyrimidin-2(1H)-one-5-carboxylate (9). Pale yellow solid; mp 167–168 ºC. IR (KBr): 3230, 2900, 1700, 1650, 1470 and 1226 cm-1. 1H NMR (DMSO-d6): δ= 0.92 (t, 3H, J = 7.2 Hz), 2.41 (s, 3H), 3.88 (q, 2H, J = 7.2 Hz), 7.20-7-45 (m, 3H), 9.25 (s, ©ARKAT USA, Inc. ISSN 1551-7012 ISSN 1551-7012 Page 262 Page 262 ARKIVOC 2009 (x) 255-264 ARKIVOC 2009 (x) 255-264 General Papers 1H) ppm. Anal. Calcd. for C14H13BrN2O3: C 49.87, H 3.89, N 8.31% found: C 50.0, H 3.8, N 8 4% 1H) ppm. Anal. Calcd. for C14H13BrN2O3: C 49.87, H 3.89, N 8.31% found: C 50.0, H 3.8, N 8.4% 1H) ppm. Anal. 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https://openalex.org/W3153322776	https://www.frontiersin.org/articles/10.3389/fmed.2021.639758/pdf	English	null	Behçet‘s Syndrome Apart From the Triple Symptom Complex: Vascular, Neurologic, Gastrointestinal, and Musculoskeletal Manifestations. A Mini Review	Frontiers in medicine	2,021	cc-by	8,235	MINI REVIEW MINI REVIEW published: 09 April 2021 doi: 10.3389/fmed.2021.639758 Edited by: Erkan Alpsoy, Akdeniz University, Turkey Reviewed by: Giacomo Emmi, Università degli Studi di Firenze, Italy Haner Direskeneli, Marmara University, Turkey Reviewed by: Giacomo Emmi, Università degli Studi di Firenze, Italy Haner Direskeneli, Marmara University, Turkey Reviewed by: Giacomo Emmi, Università degli Studi di Firenze, Italy Haner Direskeneli, Marmara University, Turkey Correspondence: Ina Kötter i.koetter@uke.de orcid.org/000000029262005X Correspondence: Ina Kötter i.koetter@uke.de orcid.org/000000029262005X Correspondence: Ina Kötter i.koetter@uke.de orcid.org/000000029262005X †These authors have contributed equally to this work and share ﬁrst authorship †These authors have contributed equally to this work and share ﬁrst authorship Keywords: BS, manifestations, vascular, neurologic, musculoskeletal, gastrointestinal, cluster Specialty section: This article was submitted to Rheumatology, a section of the journal Frontiers in Medicine Ina Kötter 1† and Fabian Lötscher 2† 1 Division of Rheumatology and Inﬂammatory Rheumatic Diseases, University Hospital Hamburg Eppendorf and Clinic for Rheumatology and Immunology Bad Bramstedt, Bad Bramstedt, Germany, 2 Department of Rheumatology and Immunology, Inselspital Bern, University of Bern, Bern, Switzerland Behçet‘s Syndrome (BS) is a variable vessel vasculitis according to the Chapel Hill Consensus Nomenclature (1) and may thus affect any organ, including major and minor arterial and venous vessels to a varying degree and with varying frequency. Although the main features of BS are recurrent oral and genital aphthous ulcers, cutaneous lesions, ocular inﬂammation and arthritis—major vessel and life—or organ threatening involvement of internal organs and the central and peripheral nervous system occur. In general, BS in Europe appears to form six phenotypes of clinical manifestations (2), which are (1) mucocutaneous only, (2) predominant arthritis/articular involvement, (3) vascular phenotype, (4) ocular manifestations, which are most likely associated with CNS manifestations and HLA-B51, (5) dominant parenchymal CNS manifestations (being associated with the ocular ones), and (6) gastrointestinal involvement. Mucocutaneous manifestations are present in almost all patients/all phenotypes. In the following review, we summarize the current knowledge concerning vascular, neurologic, gastrointestinal and musculoskeletal manifestations of the disease. INTRODUCTION Behçet‘s Syndrome (BS) is a variable vessel vasculitis according to the Chapel Hill Consensus Nomenclature (1) and may thus aﬀect any organ, including major and minor arterial and venous vessels to a varying degree and with varying frequency. Although the main features of BS are recurrent oral and genital aphthous ulcers, cutaneous lesions, ocular inﬂammation and arthritis—major vessel and life—or organ threatening involvement of internal organs and the central and peripheral nervous system occur. In general, BS in Europe appears to form six phenotypes of clinical manifestations (2), which are (1) mucocutaneous only, (2) predominant arthritis/articular involvement, (3) vascular phenotype, (4) ocular manifestations, which are most likely associated with CNS manifestations and HLA-B51, (5) dominant parenchymal CNS manifestations (being associated with the ocular ones), and (6) gastrointestinal involvement. Mucocutaneous manifestations are present in almost all patients/all phenotypes. A cluster analysis in China revealed 5 clusters, the most common being the mucocutaneous one, followed by Received: 10 December 2020 Accepted: 12 March 2021 Published: 09 April 2021 Keywords: BS, manifestations, vascular, neurologic, musculoskeletal, gastrointestinal, cluster Behçet‘s Syndrome Apart From the Triple Symptom Complex: Vascular, Neurologic, Gastrointestinal, and Musculoskeletal Manifestations. A Mini Review Ina Kötter 1† and Fabian Lötscher 2† Edited by: Erkan Alpsoy, Akdeniz University, Turkey Citation: Kötter I and Lötscher F (2021) Behçet‘s Syndrome Apart From the Triple Symptom Complex: Vascular, Neurologic, Gastrointestinal, and Musculoskeletal Manifestations. A Mini Review. Front. Med. 8:639758. doi: 10.3389/fmed.2021.639758 April 2021 \| Volume 8 \| Article 639758 Frontiers in Medicine \| www.frontiersin.org Vascular, Neurological, Gastrointestinal, Articular Manifestations Kötter and Lötscher may be involved (12–14). The occurrence of thrombosis at these unusual sites is relatively speciﬁc to BS (15). the articular one, the gastrointestinal, the ocular, and the cardiovascular cluster. These were distributed diﬀerentially among male and female patients, with cluster 1 occurring predominantly in female patients, whereas there was a strong male predominance in the uveitis and cardiovascular cluster (number 4 and 5). All other clusters were distributed almost equally among both genders (3). A considerable number of reviews and good prospective clinical trials exist for the typical mucocutaneous and ocular manifestations of BS. In the following review, we will provide an overview of the less frequently documented manifestations of the disease, which can nevertheless be decisive in terms of prognosis and diﬀerential diagnosis. Hence BCS, an occlusion of the intrahepatic veins is caused most frequently by BS in countries where BS is endemic. However, in BS itself it is rare with a frequency of <5% (16). In BS it is mostly accompanied by thrombosis of the vena cava inferior and often also by lower extremity DVT and has a high mortality rate as shown in Table 1 (4, 8, 13, 19–21). Interestingly, it could be shown that the thickness of the femoral vein wall measured with ultrasound is increased in patients with BS compared to healthy controls. This measurement may even facilitate the diﬀerentiation between inﬂammatory bowel disease and gastrointestinal involvement of BS. It may hint at a subclinical involvement of major vessels (veins) in all patients with BS independently of the clinical phenotype and may represent a new diagnostic tool (38, 39). Venous Manifestations Superﬁcial venous thrombosis (SVT) followed by deep vein thrombosis (DVT) are the most frequent vascular manifestations. There is a predominance of young males, and the DVT in contrast to those of other origin tend to relapse and are often bilateral. They aﬀect up to 13% of BS patients (8, 9) and may lead to post-thrombotic syndrome in the most severe cases (10, 11). Although thrombosis mainly occurs in the upper and lower limbs, also uncommon sites [such as superior or inferior vena cava, hepatic vein with Budd Chiari syndrome (BCS), portal vein, cerebral venous sinus, intracardiac thrombi in the right ventricle] VASCULAR INVOLVEMENT IN BS By deﬁnition, vessels of every size either arterial or venous can be aﬀected by the vasculitis in BS. Venous vessels are aﬀected much more frequently than arteries, which is unique among the vasculitides (4). Histopathologically, in the veins there is a scarce inﬂammatory inﬁltrate, ﬁbrous thickening of the vessel wall and focal aneurysmal dilatation, the vessel being occluded by an organized thrombus (5). Thrombembolism is not a feature of venous thrombosis in BS. Many common and well-known prothrombotic factors such as factor V Leiden mutation, protein C or S deﬁciency, prothrombin fragments, etc. have been discussed in the pathogenesis of thrombosis in BS, but the data are conﬂicting. A recent meta-analysis on antiphospholipid antibodies revealed a signiﬁcantly high prevalence of anticardiolipin (ACL) and anti-ß2-glycoprotein antibodies (6). Arterial Manifestations Peripheral arterial manifestations are much less common than the venous ones. Their frequency is estimated at <5% (8, 22). They occur late in the course of the disease, 5–10 years after the ﬁrst symptoms of BS (8). The majority present as aneurysms rather than with thrombi. Claudication of the aﬀected limb or digital ulcerations and necroses are the main symptoms. The thoracic and abdominal aorta may also be aﬀected, the abdominal aorta being the artery which is most commonly aneurysmatic (60%) (27, 28). Pulmonary artery involvement has a prevalence lower than 5%. However, it is the most common form of arterial involvement. It occurs early in the disease course and in ∼80% of the cases DVT of the extremities is present in parallel or has occurred 2–3 years before the involvement of the pulmonary artery. It manifests as pulmonary arterial aneurysms with hemoptysis and bilateral hilar opacities on imaging (Figure 1). Pulmonary artery thrombosis is present in one third of the patients with pulmonary artery aneurysms, and mild pulmonary artery hypertension may occur (23). In the large arteries, inﬂammatory inﬁltrates mainly consisting of neutrophils, lymphocytes, and plasma cells in the adventitia and media, sometimes resembling granulomas in Takayasu‘s arteritis, were found in 8 cases from Japan. In the media, loss of elastic ﬁbers and muscle ﬁbers and proliferation of ﬁbroblasts occurred. Intimal thickening of vasa vasorum was noted. Scarred arteritis of major aortic branches was noted in ﬁve of these patients, mainly aﬀecting aneurysms which had formed (7). Rarely, coronary arteries with the formation of giant or multiple aneurysms or pseudoaneurysms, thrombi and occlusions/stenosis are involved, causing the symptomatology of a myocardial ischemia or infarction in a young patient. Mostly, the coronary artery involvement is accompanied by other vascular manifestations such as aortic or other arterial aneurysms or DVT. Interestingly, catheterization leads to aneurysm formation in the punctured vessels comparable to a “vascular pathergy phenomenon” (40, 41). In addition to the above-mentioned intracardiac thrombi and coronary vessel aﬀections, further cardiac manifestations are pericarditis and endocardial involvement with valve insuﬃciencies (29). The frequency of vascular manifestations of BS is estimated at 5–40% (4), depending on the population in which the evaluation was performed, and males are aﬀected more often than females. The ﬁrst manifestation occurs a median time of 5 years after primary diagnosis of BS in 75% of the patients. Frontiers in Medicine \| www.frontiersin.org Coexistence of Venous and Arterial Involvement Mortality Morbidity References Mortality rate (%) Median follow up time (y) Behçet syndrome (all manifestations) 5–10 7.7–20 Increased mortality in major vessel and CNS disease young (15–24 year) and male patients, with high number of disease ﬂares with worse outcome and high unemployment and dependence rate (17, 18) Vascular 8 7.7 35.4% of patients with recurrent vascular events (8, 18) Venous 6–6.5 4.75–7.7 (18, 19) Deep venous thrombosis (DVT) 3 4.75 Severe post-thrombotic syndrome (in 50% of patients) venous claudication (in 30% of patients) most common type of recurrent vascular manifestation (8, 11, 19) Thrombosis of vena cava 12 4.75 (19) Budd Chiari syndrome (BCS) 18–47 4–9 Concomitant inferior vena cava thrombosis is common sequelae: portal hypertension, liver cirrhosis, hepatic failure, lower extremity edema poor prognosis in liver failure (13, 19–21) Arterial 13–14 7.7 45.6% undergo surgery of which 34% had surgical complications (mainly prosthetic thrombosis, less frequent if immunosuppressants are applied) (18, 22) Pulmonary artery aneurysm (PAA) 26–50 1–7 Anticoagulation can worsen hemoptysis frequent thrombus formation within PAA poor prognosis with highest mortality rate in BS (23–26) Pulmonary artery thrombosis (PAT) 23 7 PAT can transform/progress into PAA pulmonary artery hypertension (in up to 50% of patients) (23, 24) Extrapulmonary arteries 4–17 4 High frequency of new aneurysms frequent graft obstruction (well-tolerated due to collateral formation) (27, 28) Cardiac 15–28 3 Poor prognosis in coronary artery involvement (reduced cardiac function in 2/3 of patients) frequent relapse of pericarditis (12, 29) CNS 7–12 7.7–20 Worse prognosis in parenchymal involvement and abnormal CSF ﬁndings approximately 50% of patients with moderate to severe disability by 10 years (17, 18, 30– 32) Parenchymal 11–21 4–20 44% one attack and remission 28% attacks with secondary progression 10% primary progression 21% silent neurological involvement 25–33.9% disabled or dead factors associated with poor outcome (disability or death): baseline hemiparesis or paraparesis and brainstem involvement (17, 30–32) Vascular 7 4.75 Good short-term outcomes predominant sequel: optic nerve atrophy/reduced visual acuity (19, 33) Gastrointestinal 2–5 5–7.5 Cumulative operation rate (5 years) 32% remission or mild clinical activity 72% multiple relapses/chronic symptoms 28% (34, 35) Musculoskeletal 6 7.7 Mainly non-erosive, but impact on quality of life (18, 36, 37) Pericarditis, valve insufﬁciency, coronary artery involvement. TABLE 1 \| Overview of morbidity and mortality of different organ manifestations in BS. Pericarditis, valve insufﬁciency, coronary artery involvement. Coexistence of Venous and Arterial Involvement Within the vascular phenotype of BS, several associations of vascular manifestations clustering together have been described (4), encompassing diﬀerent combinations of vasculitic venous or arterial manifestations. April 2021 \| Volume 8 \| Article 639758 Frontiers in Medicine \| www.frontiersin.org 2 Vascular, Neurological, Gastrointestinal, Articular Manifestations Kötter and Lötscher TABLE 1 \| Overview of morbidity and mortality of different organ manifestations in BS. Coexistence of Venous and Arterial Involvement was also the case in the three of the four patients Hughes and Stovin (42). To date, it is hypothesized that all patients with HSS have BS and that isolated HSS without any other BS symptoms is incomplete BS (43, 44). Signiﬁcant correlations exist between cerebral venous thrombosis and pulmonary artery involvement (24), BCS and vena cava inferior syndrome. Lower (and, more rarely upper) extremity vein thrombosis is often present in all of these and may even precede them. Disease Course and Prognosis Frontiers in Medicine \| www.frontiersin.org Disease Course and Prognosis A special subtype of vascular involvement in BS is the Hughes-Stovin syndrome (HSS). This is the combination of deep vein thrombosis and pulmonary arterial aneurysms. Not uncommonly an intracardiac thrombus is also present, which Vascular involvement is often accompanied by systemic symptoms such as fever (45). Increased serological markers of inﬂammation such as ESR and CRP are present. It has a relapsing April 2021 \| Volume 8 \| Article 639758 Frontiers in Medicine \| www.frontiersin.org 3 Vascular, Neurological, Gastrointestinal, Articular Manifestations Kötter and Lötscher FIGURE 2 \| pNBS with a vasulitic lesion in the brainstem (MRI FLAIR sequence). Twenty eight years old male patient with aphasia, hemiparesis, who had deﬁnite BS according to the international study group criteria since the age of 21. FIGURE 1 \| Pulmonary arterial aneurysm (CT scan with contrast agent) of a 24 year old male patient with deﬁnite BS and hemoptysis. FIGURE 1 \| Pulmonary arterial aneurysm (CT scan with contrast agent) of a 24 year old male patient with deﬁnite BS and hemoptysis. FIGURE 2 \| pNBS with a vasulitic lesion in the brainstem (MRI FLAIR sequence). Twenty eight years old male patient with aphasia, hemiparesis, who had deﬁnite BS according to the international study group criteria since the age of 21. course, and the relapses may occur anywhere, although they tend to occur more often in the same segment or in close proximity to it (4, 22). Vascular involvement, especially of the arteries and large veins (BCS, vena cava), causes severe morbidity and increases mortality (17–19, 21, 25, 26) (Table 1). In 50% of the parenchymal CNS manifestations the brain stem is aﬀected, where inﬂammatory lesions can be found on MRI (Figure 2). Characteristic is a lesion in the midbrain or pons (30, 31). The clinical symptoms of these patients are primarily systemic such as fever and fatigue, followed by severe headache, and then after a few days focal neurological signs develop. These often consist of ophthalmoparesis in combination with ataxia and asymmetric long tract signs. Laboratory and Histopathological Findings in pNBS Laboratory and Histopathological Findings in pNBS Laboratory and Histopathological Findings in pNBS In the cerebrospinal ﬂuid (CSF), in two thirds of pNBS increased proteins and/or pleocytosis are found, in 54% with neutrophil predominance or a mixture of neutrophils and lymphocytes, 46% are purely lymphocytic. Oligoclonal bands are found in 16%. In one third the CSF may be completely normal in spite of typical pNBS (30). NEUROLOGIC INVOLVEMENT IN BS Neurologic involvement, also called Neuro-Behcet‘s syndrome (NBS) occurs with a frequency of 3–30% (46). Males are more frequently aﬀected than females, and it occurs a median time of 6 years after the ﬁrst manifestations of BS (47). However, it can also be the ﬁrst manifestation of BS, which in the cohort from Turkey was the case in 29% (47). Another pattern of pNBS on MRI is a more diﬀuse distribution of lesions in both hemispheres, clinically presenting with progressive encephalopathy consisting of headaches, focal signs such as dysphasia, hemiparesis, sensory loss, and cognitive impairment. NBS is divided into a parenchymal form (over 80% of all NBD cases) and a vascular form (∼20%). Both forms supposedly never occur simultaneously in one patient. Rarely, so-called “tumor-like lesions” are seen on MRI, a large mass develops in one hemisphere, mimicking glioma, or lymphoma. These lesions are frequently biopsied and resolve with treatment, but they often leave neurological sequelae (50). Peripheral nervous system (PNS) manifestations are very rare [3.9% of all NBS cases (47)], they include sensorimotor neuropathy, mononeuritis multiplex, and autonomic neuropathy, but also Guillain-Barré syndrome. Isolated cranial nerve neuropathies exist and are associated with signs of inﬂammation on lumbar puncture. Optic neuropathy is rare in the absence of panuveitis with retinal vasculitis (48). The peripheral nervous system manifestations can be associated with both parenchymal and vascular NBS manifestations. Parenchymal CNS Manifestations Histopathologically, either multifocal neutrophilic perivascular inﬂammation (51) or perivascular mononuclear cellular inﬁltrates were found, the latter consisting of CD45RO+ T-Lymphocytes and CD68+ monocytes with a few CD20+ B-lymphocytes. In long-term remission, gliosis, and atrophy of the aﬀected areas were shown, with some viable neurons. There were scattered inﬁltrates and foci of perivascular inﬂammation y Parenchymal NBS (pNBS) mainly occur in the brain, but the spinal cord may rarely (in up to 10% of NBS in large series) also be aﬀected in the form of a transverse myelitis (49). In the latter case, the leading clinical symptom is sensory disturbance, weakness, sphincter or sexual dysfunction, depending on the localization of the inﬂammatory lesions. April 2021 \| Volume 8 \| Article 639758 Frontiers in Medicine \| www.frontiersin.org 4 Vascular, Neurological, Gastrointestinal, Articular Manifestations Kötter and Lötscher GASTROINTESTINAL MANIFESTATIONS OF BS (GIBD) in a Japanese case series of 3 patients with NBD who were either diagnostically biopsied or histopathology was obtained upon autopsy (52). Symptoms (such as abdominal pain) indicating gastrointestinal manifestation are seen in up to 50% of patients with BS, especially in the Far East (62). However, the prevalence of endoscopically conﬁrmed cases is generally much lower, but still shows a clear geographic gradient: while the prevalence in Japan or Korea is reported in 3.2–37% of patients, the prevalence in Turkey is only just under 1–5% (34). Care should be taken when interpreting and comparing the diﬀerent data: on the one hand, the methods of diagnosis (symptom-based vs. endoscopy) diﬀered, on the other hand, various classiﬁcation criteria were applied. GIBD seems to occur some years after the ﬁrst manifestation of oral ulcers, there is no gender diﬀerence (63). Vascular Nbs (vNBS) VNBS almost exclusively consists of cerebral venous thrombosis of the superior sagittal or transverse sinus and accounts for up to 20% of all NBS cases (33). There is an association with systemic vascular manifestations of BS (53). Clinical symptoms are severe headache, evolving over a few weeks, and upon neurological examination papilledema, and occasionally sixth nerve palsy are revealed. MRI usually shows an occluded dural sinus, venous infarctions are rare. CSF ﬁndings are usually normal, except an increased pressure. Endoscopic Findings and Histology p g gy Colonoscopy remains the examination method of choice and in the majority of subjects with GIBD characteristic mucosal lesions can be found in the terminal ileum and the area of the coecum (64), but the disease can manifest along the entire gastrointestinal tract, including extra intestinal organs (pancreas, liver, and spleen). Typical ulcers are large (>1 cm) and deep with round or oval shape and appear localized, single, or in small groups (up to 5). Multi-segmental or diﬀuse distribution of the lesions are hardly seen (in 2 and 4%, respectively), as well as ano-rectal involvement (69). CD and GIBD share similar extraintestinal features and endoscopic diﬀerentiation is challenging (70). Lee et al. proposed a diagnostic algorithm to separate the two manifestations, shown in Figure 3 (71). The same group proposed diagnostic criteria for GIBD, validated in a Korean population (72). In many cases, histological processing of the samples taken does not provide any clariﬁcation: while a lymphoplasmocytic inﬁltration with sometimes vasculitic changes is usually observed in GIBS, granulomatous changes rather indicate the presence of a CD (73). The latter, however, are only described in 15–36% of patients with CD (66), and are occasionally also observed in GIBS (74). Other important diﬀerential diagnoses apart from CD are: intestinal tuberculosis Frontiers in Medicine \| www.frontiersin.org Clinical Manifestation and Laboratory Findings The diagnosis of NBS should be made according to the International Consensus Recommendation (ICR) criteria (54). They can be summarized as “the occurrence of neurological signs and symptoms in a patient that meets the ISG criteria for BS that are not otherwise explained by any other known systemic or neurological disease or treatment and in whom objective abnormalities consistent with NBS are detected either on neurological examination, neuroimaging studies, magnetic resonance imaging (MRI), or abnormal CSF examinations.” However, patients in whom NBS is the ﬁrst manifestation of BS, and who do not fulﬁll the diagnostic or classiﬁcation criteria for BS, diagnosis is very challenging. GIBD presents with a broad range of symptoms, dependent on the anatomic localization of involvement. Typically, aﬀected patients suﬀer from abdominal pain. More than 25% of patients complain about diarrhea and/or GI-bleeding, rarely vomiting, and weight loss are reported (64, 65). There are no speciﬁc laboratory tests reﬂecting GI- involvement in BS. Fecal calprotectin seems to be a promising tool for non-invasive detection of intestinal involvement and could also be used to monitor intestinal disease activity in the future (66, 67). A recent meta-analysis underscores the strong association of serum anti-Saccharomyces cervisiae antibodies (ASCA) with GIBD (68). But ASCA-positivity has only limited diagnostic value in GIBD, as the antibodies are frequently found in Crohn’s disease (CD), the main clinical diﬀerential diagnosis of GIBD. The diﬀerential diagnosis from multiple sclerosis (MS) may be diﬃcult, as clinical symptoms, as well as MRI lesions and even inﬂammatory markers in cerebrospinal ﬂuids may be very similar, even oligoclonal bands in CSF occur in MS as well as in NBS. Hence, there were attempts to ﬁnd either biomarkers in CSF (55–57) or even typical signs of vasculitis of BS in brain biopsies (58). One study suggests speciﬁc features of BS on MRI, such as a higher frequency of periventricular lesions in MS than in inﬂammatory vasculopathies, and the highest frequency of perivenular lesions (34%) in pNBS also in comparison to other CNS vasculitides (59). Disease Course and Prognosis For pNBS diﬀerent courses are described: Single attack, relapsing, secondary progressive, primary progressive (60). In a French cohort with 115 patients, 68% had acute forms of NBS, and 32% a progressive form. 40% of the patients had severe disability at baseline, the 5- and 7-year event-free survival rates were 65 and 53%. After a median follow-up of 73 months 25.2% of the patients became dependent, which means they were unable to perform activities of daily living or died. Factors independently associated with poor outcome were paresis at onset (OR 6.47), and location of inﬂammatory lesions at the brainstem on MRI (OR 8.41) (32) (Table 1). There appears to be an association with ocular involvement and with HLA-B51 positivity, the latter also indicating a worse prognosis (61) and also being associated with an increased relapse risk (OR 3.5) in the French cohort. April 2021 \| Volume 8 \| Article 639758 5 Vascular, Neurological, Gastrointestinal, Articular Manifestations Kötter and Lötscher TABLE 2 \| Characteristics of arthritis in BS. Localization* Knee 33% Ankle joint 24 Wrist 14 Hand 11 Foot 8 Elbow 5 Sacroiliac joint <5 Hip <5 Shoulder <5 Number of joints† Monoarticular 66 Oligoarticular 29 Polyarticular 4 Duration† Acute (<6–8 weeks) 89 Chronic (>8 weeks) 11 Synovial ﬂuid examination§ Cell count 11,000/ul (1,600–36,000/ul) Cell type Predominantly polymorphonuclear Radiography Typically non-erosive *Calculated with a total of 609 arthritis episodes extracted from the publications of Yurdakul et al. (79), Gur et al. (37), and Fatemi et al. (36). †Calculated with a total of 493 (duration) and 369 (number of Joints) arthritis episodes extracted from publications of Yurdakul et al. (79) and Fatemi et al. (36). §Calculated with extraction 38 synovial ﬂuid analyses from Yurdakul et al. (79) and Gibson et al. (81). TABLE 2 \| Characteristics of arthritis in BS. FIGURE 3 \| Simple decision tree of colonoscopic ﬁndings, distinguishing GIBD from CD Lee et al. (71). With permission of the author and the publisher. FIGURE 3 \| Simple decision tree of colonoscopic ﬁndings, distinguishing GIBD from CD Lee et al. (71). With permission of the author and the publisher. (especially in endemic areas), ulcerative colitis and NSAID induced colitis (34). Gastroduodenal or esophageal involvement have been reported to be rare, the latter being more common in men (75). However, due to the infrequency of gastroduodenoscopy in BS and a lack of large studies, underreporting of these manifestations might be possible (76). Disease Course Although more than 50% of Patients with GIBD have a rather mild disease course, intestinal manifestations bear the risk of life-threatening manifestations with complications such as intestinal perforation, perianal, or entero-enteric ﬁstulae as well as intestinal bleeding, requiring surgical intervention in up to 1/3 of all patients (35) (Table 1). Several studies report an association of arthritis with the extra- articular manifestation papulo-pustulosis, implying this form of disease-manifestation being a cluster of disease expression (36, 85–87). Furthermore, Hatemi et al. observed that in BS patients with arthritis and acne, enthesopathy (including enthesitis) is more commonly encountered on sonography than in patients without arthritis (88). The ﬁrst ﬁnding raises the question of a possible pathogenetic connection of BS to acne associated arthritis syndromes. There was a long debate about the inclusion of BS in the group of seronegative spondylarthritis (SpA) (89), as some investigators reported a high prevalence of sacroiliitis in small cohorts (90). Prospective studies have not yet been able to substantiate these observations (79, 91–93). Disease Course and Prognosis In cases of abdominal complaints, elevated inﬂammatory markers (as CRP or fecal calprotectin) with normal conventional endoscopic examinations, capsule endoscopy (CE) can be used to reveal pathologies of the small intestines (34, 77). oligoarticular, data concerning symmetry of joint distribution are conﬂicting: in some studies with a tendency to symmetric distribution if the manifestation is not monoarticular (79), favoring asymmetric involvement in others (37). Generally, polyarticular manifestation is rare in BS (36, 79). Some smaller studies observed a polyarticular course in the majority of cases (82). The arthritis in BS is recurrent in nature, with acute and self-limiting course, mostly of short duration (2 weeks to 2 months) but chronic courses were rarely observed (36, 78, 79). Radiographic evaluation is usually inconspicuous, although sporadic cases of joint erosion have been reported (83, 84). MUSCULOSKELETAL INVOLVEMENT IN BS Musculoskeletal involvement in BS is common, the frequency of joint involvement varies considerably depending on geographic location (36) and study design as well as the evaluating medical subspeciality and the deﬁnition of involvement (arthritis vs. arthropathy). Prevalence ranges from 39 to 70%, mostly around 50% (36, 78–80). Laboratory Findings, Synovial Fluid Characteristics, and Synovial Histology Vascular and neurological manifestations of BS are relatively rare but are associated with a high morbidity and mortality. This is especially true for the arterial vascular manifestations and parenchymal neurological manifestations involving the brainstem. Gastrointestinal involvement is very rare but is also associated with an increased morbidity. Even the much more common articular/locomotor involvement impairs quality of health and life of the patients with BS. A limitation of this mini review is, that there are only a few and mostly retrospective studies on the manifestations of BS covered here and the methods as well as evaluation criteria were not uniform. Furthermore, the cohorts published so far were quite small and mostly from a single specialized center. In the future, international registries may be helpful to collect data in larger, multicenter cohorts and the new OMERACT outcome measures will standardize and simplify the evaluation of the course of the diverse manifestations of BS (102). , y gy Acute phase reactants (ESR, CRP) are usually elevated in patients with arthritic episodes, antibody tests (rheumatoid factor, ACPA, and ANA) are negative (37, 79, 82). Synovial ﬂuid analysis typically reveals mild, unspeciﬁc inﬂammatory changes with predominantly polymorphonuclear leucocytes (79). Synovial ﬂuid cell counts range between 1,600 and 36,000/ul (Table 2) (79, 81), with normal glucose levels and elevated complement C3 and C4 levels (96). Pay et al. found synovial pro-inﬂammatory cytokines IL-18, TNF-alpha, and MMP-3 levels to be lower in BS than in RA patients but found IL-1 levels to be elevated (97). Synovial histopathology was only investigated in small studies, examinations mainly show non-speciﬁc inﬂammatory changes. In 1978 Vernon-Roberts et al. described replacement of the superﬁcial zone of the synovium by heavily inﬂamed granulation tissue, similar ﬁndings were conﬁrmed by Yurdakul et al. in (79) and Vernon Roberts et al. (84). Synovitis in BS is characterized by polymorphonuclear neutrophil and T-cell predominance, without signs of neutrophil vasculitis (98). Quality of Life and Fibromyalgia IK: wrote the part on vascular and neurologic manifestations. FL: wrote the part on articular and gastrointestinal manifestations. Both authors contributed to the article and approved the submitted version. A small study by Gur et al. in 2006 showed that arthritis in BS negatively aﬀects quality of life (in comparison to healthy subjects and BS patients without arthritis), with equal anxiety and depression levels (37). Limited evidence suggests that Further Locomotor Involvement Few cases of necrotizing myositis in BS have been reported (100), an both focal and generalized manifestations have been described. Osteonecrosis is rarely observed in patients with BS and could be corticosteroid-associated in a signiﬁcant proportion of cases described (101). Characteristics of Joint Involvement According to the largest prospective studies (36, 37, 78, 79) arthritis in BS usually aﬀects large peripheral joints, in decreasing frequency knees, ankle joints, wrists, hands, and rarely elbows, shoulders and feet, as shown in Table 2 (36, 78, 79, 82). The involvement is primarily mono- or Although SpA and BS share many clinical features (such as skin, joint, eye, and GI manifestations), the rarity of axial involvement in BS has led to the omission of BS from the SpA group. Nevertheless, SpA and BS (as well as psoriasis) share HLA class 1 association, which is a strong common April 2021 \| Volume 8 \| Article 639758 Frontiers in Medicine \| www.frontiersin.org 6 Vascular, Neurological, Gastrointestinal, Articular Manifestations Kötter and Lötscher there is increased prevalence (5.7–37%) of ﬁbromyalgia in BS patients compared with the general population (2.9–4.7%) (99). 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(2006) 26:608–13. doi: 10.1007/s00296-005-0040-0 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. 98. Cañete JD, Celis R, Noordenbos T, Moll C, Gómez-Puerta JA, Pizcueta P, et al. Distinct synovial immunopathology in Behçet disease and psoriatic arthritis. Arthritis Res Ther. (2009) 11:1–7. doi: 10.1186/ar2608 Copyright © 2021 Kötter and Lötscher. 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. 99. Jobanputra C, Richey RH, Nair J, Moots RJ, Goebel A. 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https://openalex.org/W4241646049	https://www.researchsquare.com/article/rs-26605/latest.pdf	English	null	Perceptions of patients with end-stage kidney disease (ESKD) and their informal caregivers on palliative care as a treatment option: a qualitative study	Research Square (Research Square)	2,020	cc-by	6,872	Perceptions of patients with end-stage kidney disease (ESKD) and their informal caregivers on palliative care as a treatment option: a qualitative study Catherine Sarfo-Walters Garden City University College Edward Appiah Boateng (  guardian2405@yahoo.com ) Kwame Nkrumah University of Science and Technology https://orcid.org/0000-0003-1000-8076 Research article Keywords: End-stage kidney disease, palliative care, conservative management, haemodialysis, Ghana Africa Posted Date: August 18th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-26605/v4 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on August 20th, 2020. See the published version at https://doi.org/10.1186/s12904-020-00640-y. Perceptions of patients with end-stage kidney disease (ESKD) and their informal caregivers on palliative care as a treatment option: a qualitative study Catherine Sarfo-Walters Garden City University College Edward Appiah Boateng (  guardian2405@yahoo.com ) Kwame Nkrumah University of Science and Technology https://orcid.org/0000-0003-1000-8076 Research article Keywords: End-stage kidney disease, palliative care, conservative management, haemodialysis, Ghana Africa Posted Date: August 18th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-26605/v4 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on August 20th, 2020. See the published version at https://doi.org/10.1186/s12904-020-00640-y. Research article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on August 20th, 2020. See the published version at https://doi.org/10.1186/s12904-020-00640-y. Page 1/15 Page 1/15 Abstract Background: Palliative care is increasingly becoming an accepted treatment choice for many individuals diagnosed with end-stage kidney disease (ESKD). Yet, its utilisation is non-existent in many lower- and middle-income countries (LMICs). This study explored the perceptions of individuals with ESKD and their informal caregivers on palliative care as a treatment option for the disease in Ghana. Methods: This was a phenomenological study, with an in-depth analysis of data collected from nine individuals with ESKD and six informal caregivers through individual, face-to-face semi-structured interviews. The study was conducted in two renal centres within the Kumasi metropolis, Ghana among individuals with ESKD seeking care from both renal centres and their informal caregivers. Results: Three main themes were derived from this study – motivation for initiating haemodialysis, facing realities of haemodialysis, and considering palliative care. Participants felt that haemodialysis (HD) was not meeting their health expectations and demonstrated a general willingness to utilise palliative care if it would reduce suffering. Conclusions: This study has shown that individuals with ESKD or their informal caregivers would consider palliative care services, if available. It paves the way for discussions about palliative care for ESKD to begin across renal centres within Ghana and other similar settings. Exploring perspectives of clinicians in such settings could inform strategies on how to implement palliative care for ESKD management in such settings. Background Palliative care for ESKD has been defined as “a transition from a conventional disease-oriented focus on dialysis as a rehabilitative treatment to an approach prioritizing comfort and alignment with preferences and goals of care to improve quality of life and reduce symptom burden” (1, 2). It comprises interventions that focus on slowing the deterioration of kidney function, minimising the risk of adverse events, shared decision-making, active symptom management, detailed communication, including advance care planning, psychological, social and family support, and cultural and spiritual care, but does not include RRT (3-5). The focus is on improving the quality of life of individuals with ESKD, in line with their values and goals, rather than merely extending their life (4, 6-8). This is increasingly becoming an accepted treatment choice and is highly recommended for individuals in whom renal replacement therapy (RRT) may offer little or no significant difference in terms of survival or quality of life (3, 4, 6, 9-14). Indeed, the World Health Organization (15) classifies ESKD among diseases that would benefit from palliative care. Individuals with ESKD, cared for in renal centres where palliative care services are well-established, choose palliative care more often than centres without palliative care services (2, 16). It has been reported that individuals with ESKD would prefer to be given adequate information about the condition and their treatment options, including palliative care, at an earlier stage of the disease (17). Yet, palliative care for managing ESKD is limited in several countries because of inadequate well-trained palliative care Page 2/15 Page 2/15 clinicians and poor access to palliative care services (4, 7). This is, especially, more profound in lower- and middle-income countries (LMICs) where financial, geographical and infrastructural challenges affect the provision of efficient renal services (18-22). Inadequate information provision and delayed initiation of discussions on palliative care also remain key barriers to its utilisation as a treatment option for ESKD (2, 17, 23, 24). Informal caregivers of individuals with ESKD, mainly relatives and friends, may also discourage clinicians’ efforts at providing open and honest information about palliative care services (25). Ghana is noted to have made improvements in the provision of palliative care services, being ranked 8th in Africa (26-28). However, like in other African countries, palliative care largely focuses on HIV and cancer-related diagnoses (20, 28). Background There are no established pathways to palliative care for ESKD management, and the subject of death is rarely broached in discussions between clinicians and patients (18). This study, therefore, explored the perceptions of individuals with ESKD and their informal caregivers on palliative care as a treatment option of the disease in Ghana. Ghana is noted to have made improvements in the provision of palliative care services, being ranked 8th in Africa (26-28). However, like in other African countries, palliative care largely focuses on HIV and cancer-related diagnoses (20, 28). There are no established pathways to palliative care for ESKD management, and the subject of death is rarely broached in discussions between clinicians and patients (18). This study, therefore, explored the perceptions of individuals with ESKD and their informal caregivers on palliative care as a treatment option of the disease in Ghana. Research setting Ghana has inadequate renal services, with fifteen renal centres all located in five out of the sixteen administrative regions of the country (29). These centres are managed by a few nephrologists, nephrology trainees, consultant physicians, specialist physicians, medical officers and, largely, by nurses who are trained-on-the-job (18, 29). Payment for the treatment of ESKD is mainly made out-of-pocket by patients or their significant others in Ghana as the National Health Insurance Scheme (NHIS) does not cover the treatment costs (18, 22, 29). This results in only a few patients with ESKD being able to initiate and sustain treatment, and the increased tendency of reducing the frequency of dialysis, withdrawing from treatment and/or pursuing other forms of treatment outside biomedicine (18, 30). This study was conducted in two renal centres that offer standard conservative care to individuals with ESKD who opt for it, including the treatment of anaemia, management of hypertension, pain as well as dietary and fluid management approaches, similar to other established renal centres (22, 31). Although there are a few specialist palliative care services in the country, these are largely utilised by patients with advanced types of cancers and largely inaccessible to a majority of the citizenry owing to costs (22, 28). Study design, sampling and data collection This was a qualitative study, utilizing the phenomenological approach as it focussed on the lived experiences of individuals with ESKD and their informal caregivers and their perceptions about palliative care (32, 33). The study targeted individuals with ESKD receiving haemodialysis at the two renal centres as well as their informal caregivers. A purposive sampling technique was employed for this study. After administrative approval was given, CS-W was introduced to eligible participants in both renal centres by the respective nurses-in charge to discuss the objectives of the study and their possible inclusion. Those Page 3/15 who agreed confirmed their participation by signing or thumb-printing the consent form before being interviewed by CS-W. All those who were approached agreed to be part of the study. Data for this study was collected through individual, face-to-face, semi-structured interviews. Each renal centre provided an office where the interviews were conducted, with only CS-W and the interviewee present in the office during each interview session. Two separate written interview guides were employed, one for the individual with ESKD and the other for informal caregivers to ensure comprehensive accounts from each interview session. Each interview guide was evaluated after the first session to ensure their accuracy, understandability, and appropriateness for this study, and to serve as a pilot test. There were no repeat interviews. Interview sessions lasted between 30 and 90 minutes and each was recorded using a digital audio-recording device. Field notes were written after each interview session to provide detailed observations and mannerisms that were not captured through the audio-recording. All audio-recordings were transcribed for data analysis. Two of the interview sessions were conducted in Twi, a popular Ghanaian dialect, and these were translated by CS-W. EAB and an independent researcher who are both fluent in the Twi and English languages verified the accuracy of the translation. Data processing and analysis All interviews were transcribed after each session. NVivo 10 software was used to manage and organize the data. Data collection and analysis occurred simultaneously, with the Colaizzi method (34, 35) guiding the analysis. All transcripts were proofread while listening to the audio recordings to ensure accuracy. Eleven participants agreed to look at their respective transcripts for comments and possible corrections and deletion of personal information – all were happy with the transcript and had no changes to be made. All transcripts were read multiple times and coded independently by CS-W and EAB. The field notes were also read to aid the contextualisation of various participant accounts during analysis. Significant statements about participants’ experiences with management of ESKD and their perceptions of palliative care were extracted from their respective transcripts and meanings formulated from these statements. Each participant’s experiences and perceptions were studied separately and then compared across transcripts. Both authors examined the data and used codes, patterns, and themes to classify them, meeting frequently to discuss emerging themes and agreeing on those that needed further exploration in subsequent interviews. These meetings continued until both authors agreed that themes had been adequately explored for the purpose of the study and ended data collection (36). Ethical considerations All participants gave their consent, either by signing or thumb-printing an informed consent form before participating in the study. Ethics approval (ID No: UCCIRB/CHAS/2015/105) was obtained from the Institutional Review Board of the University of Cape Coast, Ghana before data collection commenced. Participants were assured of confidentiality and anonymity, and their names and other personal identifiers have been replaced with pseudonyms in this paper to ensure this. Trustworthiness Page 4/15 Page 4/15 Data was collected from two renal centres as well as from two sources as a way of achieving triangulation and, in effect, ensuring credibility (37). Also, member checks allowed participants to correct errors to ensure credibility (38, 39). In this study, participants who were contacted did not find any errors or need for additional information. Both researchers coded and analysed the data independently, meeting to compare outcomes and resolve differences. Interpretation of data was supported by direct quotes from participants to enhance credibility and confirmability of the study findings (40-42). Transferability has been enhanced by providing an in-depth description of the population and the context of the study (41). This report has been guided by the Consolidated Criteria for Reporting Qualitative Research (43). Reflexivity In qualitative studies, the researcher becomes the instrument and is intimately involved in data collection and analysis. It was, therefore, important to reflect upon biases the researcher may have, a term called reflexivity (40). CS-W has over 10 years’ worth of experience in the management of ESKD. She is a nurse educator and served as a member of the palliative care team in her hospital. EAB has served as a nurse educator in Ghana for 9 years. He has a keen interest in enhancing the quality of life as well as decision- making experiences of individuals with ESKD. These potential biases were taken into consideration while collecting and analysing the data. Results Fifteen participants, comprising nine individuals with ESKD (patient-participants) receiving haemodialysis and six informal caregivers were included in this study. The sample size was determined after achieving data saturation (40). Seven of the patient-participants were male while the remaining two were female. Duration on haemodialysis ranged from 5 months to 5 years. The youngest patient-participant was 20 years old while the oldest was 65 years old. Four of the six informal caregivers were female, and all were closely related to the patients with ESKD. Details of all participants have been provided in Table 1. Three main themes were derived from the analysis of the data – motivation for initiating haemodialysis, facing realities of haemodialysis, and considering palliative care. Table 1 provides a summary of these themes and their sub-themes. Each of these three themes has been presented below, with relevant quotations from patient-participants, and from informal caregivers to corroborate statements from patient-participants. Motivation for initiating haemodialysis Analysis of data showed that two main factors served as motivation for haemodialysis initiation – facing life-threatening prognosis and mistaken expectations of a cure. The sub-theme ‘facing life-threatening prognosis’ described participants’ expression of a full realisation that ESKD is life-threatening, especially without any form of RRT. This was mainly as a result of the clinical manifestations experienced and information gained from the renal centre. Others were also as a result of the poor prognosis of other patients with ESKD that they had met at the renal centre. Page 5/15 Page 5/15 “It [ESKD] puts you in a state of fear because of the breathlessness, and blood issues [anaemia] – will I wake up the next day?” (Daniel, ESKD patient). “Yes, we think it can cause her death. Many times, she cannot breathe at night. All the time she needs to be transfused. Looking at how emaciated and the suffering she is going through I do not doubt that this will be her cause of death…I will not be surprised” (Gloria, caregiver). Some participants also started haemodialysis, hoping that a series of sessions would cure the disease. This mistaken expectation of a cure was the driving force for most of the initial decisions about treatment. “I thought it [haemodialysis] was only for a few sessions and I will no longer require the treatment” (Michael, ESKD patient). “Our understanding was that the haemodialysis is going to cure her of [the] symptoms she is experiencing” (Gloria, caregiver). Facing realities of haemodialysis This led to the next theme describing participants’ perceptions regarding death and their prevailing circumstances surrounding treatment with haemodialysis. Facing realities of haemodialysis Participants generally re-examined their health expectations and pondered the worth of haemodialysis, with its associated challenges, after receiving the treatment for some time. The theme ‘facing realities of haemodialysis’ describes this phase for participants. Expectations of haemodialysis were modified to the point where participants now hoped that their clinical manifestations would be as minimal as possible. Unfortunately, many participants felt that these modified expectations were also difficult to achieve. Some patient-participants reported that they had not noticed any improvements in their health status or quality of life even though they had been on haemodialysis for some time. “Sometimes I have swollen leg; you can’t know where it comes from… I vomit, I go to the toilet [diarrhoea], and these are abnormal things that will not happen to any normal human being. Even though I’m doing the haemodialysis, these continue to come” (Peter, ESKD patient). The burden of living with ESKD, including changes in health status and continuously raising large sums to finance their treatment but with little to no improvements led to feelings of despair among patient- participants. Indeed, they reported using all resources available to them to pursue treatment. Patient- participants also felt that they had become a burden to their significant others because they needed their assistance in performing activities of daily living, visiting the renal centre, or paying for the haemodialysis costs. “They are fed up with me, they come with me to haemodialysis but they complain all the time, you see they are tired of following me here and there” (David, ESKD patient). Informal caregivers reiterated the concerns about the realities of having their family member on haemodialysis and how onerous it is for the entire family. Page 6/15 Page 6/15 Page 6/15 “There are challenges because haemodialysis is costly. Everything is cash-and-carry and you are expected to pay without the National Health Insurance Scheme, not to talk about the cost of the [erythropoietin] injection, transportation and long hours on the machine, it is a worry” (Douglas, caregiver). The informal caregivers also corroborated the views of patient-participants that haemodialysis was not offering any improvements in their quality of life or health status. “She has never been well. Not a single day will this woman [patient] say ‘I feel well’. After six months of haemodialysis, there are no signs of her getting well. It is very frustrating” (Paulina, caregiver). Discussion This study aimed at exploring the perceptions of participants on palliative care as a treatment option for ESKD in Ghana. Many participants did not know much about the chronic and potential terminal nature of ESKD at the time of diagnosis and so were expectant of a cure when initiating treatment. Expectations of a cure around the time when treatment of ESKD is initiated has already been reported in Ghana (18) so this was, somewhat, not surprising. However, being on treatment for some time and experiencing symptoms associated with the disease made them perceive death as a real possibility. It is well-known that ESKD is a life-threatening condition and those living with it confront mortality as part of their daily experiences (44), and personal or vicarious experiences of clinical manifestations of ESKD led participants of this study to confront their mortality. Our study found that during such moments, deeper meanings about death were reflected upon. Comments that implied that death is part of life reflect their effort to ‘normalise’ death as a potential outcome of the disease. This did not always suggest acceptance of their prognosis though, especially as they had not had formal discussions with their clinicians about their prognosis and palliative care. Palliative care is recommended for patients with ESKD in whom RRT would not offer survival benefits or improved quality of life. These are usually the very elderly, those with comorbidities as well as those who initiate RRT within three months of diagnosis as a result of late presentation (3, 4, 6, 9-13). For such individuals, palliative care ensures that they receive quality care as they near the end of life. Although patients with ESKD are generally young in the Ghanaian setting, late presentation, poor functional status and high mortality rates associated with ESKD imply that many could benefit from palliative care services if this choice was to be available (9, 18, 19, 22, 45, 46). The treatment choices of the Ghanaian patient with ESKD are limited in so many ways when survival is the expected outcome, unlike in many high- income countries where there is a menu of life-saving treatment choices available to the patient (18, 47, 48). Despite the modality constraints, the initial drive to preserve life led participants to initiate haemodialysis until they realised that sustainability is nearly impossible or comes with its daunting adjustments. Considering palliative care If it is the will of God, little can we do but we wish we can support him…in that case if anything happens, you know it is the will of God” (Margaret, caregiver). Considering palliative care Participants perceived palliative care as a means to provide relief from pain and other symptoms, ensuring that death does not come through endless suffering. Participants generally perceived death as an event that befalls all mankind and preferred not to expend all their resources on haemodialysis, cognisant of the inevitability of death and the devastating effect that their continuous utilisation of haemodialysis could have on their family. They also believed that, through palliative care, they could avoid or control suffering before death. “I will opt for palliative care if that service is available for me so that I will not suffer but die peacefully” (Michael, ESKD patient). Generally, informal caregivers were also of the view that if haemodialysis was not meeting their expectations of improved quality of life for their relatives nor guaranteeing survival, then they would not be reluctant to choose palliative care if the service is available. “If we know the person will die eventually, I think she does not need to go through all these and lose [her] life. For me, I think that if the person cannot afford the haemodialysis, palliative care should be an option” (Gloria, caregiver). Some patient-participants advocated that palliative care should be implemented and awareness created to enhance choosing it as an alternative or alongside haemodialysis during decision-making on treatment. Some patient-participants advocated that palliative care should be implemented and awareness created to enhance choosing it as an alternative or alongside haemodialysis during decision-making on treatment. “Yes, in fact, please do that (implement palliative care). Life continues even after death, that’s our belief. So, if I’m not getting well via the haemodialysis and somebody has time to talk to me, discuss my problems and spiritual wellbeing, why not?” (Peter, ESKD patient). “Yes, in fact, please do that (implement palliative care). Life continues even after death, that’s our belief. So, if I’m not getting well via the haemodialysis and somebody has time to talk to me, discuss my problems and spiritual wellbeing, why not?” (Peter, ESKD patient). For relatively younger individuals with ESKD, however, caregivers felt uneasy about being introduced to palliative care. Yet, the belief that death is inevitable made them do all that they could in order not to appear non-supportive of their relative. Page 7/15 “It [palliative care] can help but can you look at a young man like this one to die? What can we say? Discussion The support of informal caregivers is instrumental in decisions regard ESKD (52, 53). Indeed, informal caregivers of patients with ESKD have many unexpected ways as a result of the disease, including having to centre and dealing with untoward psychological responses of patients such as depression (53). Yet, informal caregivers, sometimes, pressuri and sustain RRT, with the belief that patients could get better with time relatively young (25, 54). Our study found that, in some instances, info their power to ensure that the patient is sustained on treatment for as living with the disease, however, led to a change of perspective and a p services. The support of informal caregivers is instrumental in decisions regarding the treatment of patients with ESKD (52, 53). Indeed, informal caregivers of patients with ESKD have their usual routines disrupted in many unexpected ways as a result of the disease, including having to accompany them to the renal centre and dealing with untoward psychological responses of patients to the disease and its treatment such as depression (53). Yet, informal caregivers, sometimes, pressurise patients with ESKD to initiate and sustain RRT, with the belief that patients could get better with time, especially when patients are relatively young (25, 54). Our study found that, in some instances, informal caregivers did everything in their power to ensure that the patient is sustained on treatment for as long as possible. The realities of living with the disease, however, led to a change of perspective and a preference for palliative care services. Study strengths and limitation The phenomenological approach employed has given voice to those confronting mortality daily in an LMIC setting as a result of ESKD. Although the findings cannot be generalised, like all other qualitative study approaches, the study has raised insightful issues that could be explored further in other renal centres within the country and other similar settings. It paves the way for discussions about Palliative care for ESKD to begin across renal centres within Ghana and other similar settings. Discussion Studies on palliative care for ESKD report that this treatment choice is made because individuals do not want to burden their families, feel neglected, experience a deterioration of their quality of life, experience acute medical complications, or when they are unsure of good prognosis (7, 44, 49, 50). The desire not to burden others also seemed to play a role in making individuals living with ESKD in this study consider accepting palliative care. However, consideration for palliative care mainly stemmed from realising that there is no certainty of a good prognosis, coupled with a general deterioration of their quality of life. The impact of haemodialysis on individuals’ or their families’ finances also had a stronger role to play in their search for a different treatment option. Indeed, there are instances where comprehensive conservative Page 8/15 Page 8/15 care becomes the only option in the face of resource constraints or limited access to renal replacement therapy (5, 14). However, comprehensive conservative care of patients with ESKD must not be perceived as a low-cost option in place of well-developed RRT services in LMICs but as a component of integrated and patient-centred care for those with the disease (10, 51). This study noted that participants had not been extensively engaged in discussions regarding end-of-life care. An improvement in the provision of information on prognosis and available support is a key strategy in enhancing the utilisation of comprehensive conservative care, including palliative care (10). Indeed, the limitations around efficient renal services in LMICs cannot be understated, including unavailability of appropriately-trained clinicians to deliver specialist care as required. Unavailability of an established palliative care pathway for the management of ESKD means that death could not even be managed in a more ‘dignified’ way for those who could not benefit from RRT. Yet, this study brings to the fore the longing need for palliative care as a potential treatment choice that could be made by individuals with ESKD and their families in Ghana. It may well be that with improvements in access and dialysis outcomes, patients' views might be completely different. However, palliative care services should be developed for patients with multiple comorbidities or at the end of their lives in whom dialysis poses a risk of being a futile therapy (14). Timing for initiating a discussion on palliative care is essential as participants considered it only after being on haemodialysis for a while. ESKD – End-stage kidney disease LMICs – Lower- and middle-income countries RRT – Renal replacement therapy ESKD – End-stage kidney disease Consent for publication Not applicable Availability of data and materials Data can be made available upon reasonable request to the corresponding author so long as it complies with regulations of the institutional review board. Ethics approval and consent to participate Ethics approval (ID No: UCCIRB/CHAS/2015/105) was obtained from the Institutional Review Board of the University of Cape Coast, Ghana before data collection commenced. Eligible participants were given information about the study in either the English language or Twi, based on their preference, as a way of obtaining their informed consent. Participation was entirely voluntary and only individuals who gave their consent, either by signing or thumb-printing an informed consent form participated in the study. Thum- printing was in line with expectations of the institutional review board for participants who could not sign the consent form. Eligible participants were assured that non-participation will not affect the care they receive from the renal centres in any way. Participants’ names and other personal identifiers have been replaced with pseudonyms to maintain confidentiality and anonymity. Conclusions This study has shown that individuals with ESKD or their informal caregivers would consider palliative care services, if available. Indeed, as has always been advocated, primary prevention of ESKD, including Page 9/15 Page 9/15 meticulous management of ESKD precursor conditions is the way to go, especially in resource- constrained settings. Notwithstanding, a reasonable number of individuals who have already developed ESKD stand to benefit from integrated renal services, including palliative care. Seeking the opinions of clinicians in such settings would be a way to bring their perspectives about palliative care to the fore. meticulous management of ESKD precursor conditions is the way to go, especially in resource- constrained settings. Notwithstanding, a reasonable number of individuals who have already developed ESKD stand to benefit from integrated renal services, including palliative care. Seeking the opinions of clinicians in such settings would be a way to bring their perspectives about palliative care to the fore. Competing interest The authors declare that there is no conflict of interest. Authors’ contributions Both authors conceptualised the study. CS-W collected data for this study. Both CS-W and EAB were involved in the analysis and interpretation of the data. Both authors drafted the manuscript and approved the final draft of this paper. Funding This research received no grant from any funding agency in the public, commercial, or not-for-profit sectors. Page 10/15 Authors’ information – ORCID iDs Catherine Sarfo-Walters https://orcid.org/0000-0002-3389-7171 Edward Appiah Boateng https://orcid.org/0000-0003-1000-8076 Acknowledgements The authors would like to express their gratitude to all participants who willingly shared their experiences in this study. The authors also acknowledge the support of Prof. Funmilayo Okalanwon and Dr Jerry Ninnoni all of the School of Nursing and Midwifery, University of Cape Coast, Ghana during the conduct of this study. References 1. Grubbs V, Moss AH, Cohen LM, Fischer MJ, Germain MJ, Jassal SV, et al. A Palliative Approach to Dialysis Care: A Patient-Centered Transition to the End of Life. Clinical Journal of the American Society of Nephrology. 2014;9(12):2203. 2. Axelsson L, Benzein E, Lindberg J, Persson C. End-of-life and palliative care of patients on maintenance hemodialysis treatment: a focus group study. BMC Palliative Care. 2019;18(1):89. 2. Axelsson L, Benzein E, Lindberg J, Persson C. End-of-life and palliative care of patients on maintenance hemodialysis treatment: a focus group study. BMC Palliative Care. 2019;18(1):89. 3. Johnston S, Noble H. Factors influencing patients with stage 5 chronic kidney disease to opt for conservative management: A practitioner research study. Journal of Clinical Nursing. 2012;21(9- 10):1215-22. 4. Morton RL, Webster AC, McGeechan K, Howard K, Murtagh FE, Gray NA, et al. Conservative management and end-of-life care in an Australian cohort with ESRD. Clinical Journal of the American Society of Nephrology. 2016;11(12):2195-203. 5. Murtagh FEM, Burns A, Moranne O, Morton RL, Naicker S. Supportive Care: Comprehensive Conservative Care in End-Stage Kidney Disease. Clinical Journal of the American Society of Nephrology. 2016;11(10):1909. 6. O'Connor NR, Kumar P. Conservative management of end-stage renal disease without dialysis: a systematic review. Journal of Palliative Medicine. 2012;15(2):228-35. 7. Chen JC-Y, Thorsteinsdottir B, Vaughan LE, Feely MA, Albright RC, Onuigbo M, et al. End of life, withdrawal, and palliative care utilization among patients receiving maintenance hemodialysis therapy. Clinical Journal of the American Society of Nephrology. 2018;13(8):1172-9. Page 11/15 Page 11/15 8. Scherer JS, Wright R, Blaum CS, Wall SP. Building an Outpatient Kidney Palliative Care Clinical Program. Journal of Pain and Symptom Management. 2018;55(1):108-16.e2. 9. Davison R, Sheerin NS. Prognosis and management of chronic kidney disease (CKD) at the end of life. Postgraduate Medical Journal. 2014;90(1060):98-105. 10. Harris DC, Davies SJ, Finkelstein FO, Jha V, Donner J-A, Abraham G, et al. Increasing access to integrated ESKD care as part of universal health coverage. Kidney international. 2019;95(4):S1-S33. 11. Dionne JM, d’Agincourt-Canning L. Sustaining life or prolonging dying? Appropriate choice of conservative care for children in end-stage renal disease: an ethical framework. Pediatric Nephrology. 2015;30(10):1761-9. 12. Noble H. An aging renal population–is dialysis always the answer? British Journal of Nursing. 2011;20(9):545-7. 13. Douglas A. Palliative care for patients with advanced chronic kidney disease. The journal of the Royal College of Physicians of Edinburgh. 2014;44(3):224-31. 14. References Davison SN, Levin A, Moss AH, Jha V, Brown EA, Brennan F, et al. Executive summary of the KDIGO Controversies Conference on Supportive Care in Chronic Kidney Disease: developing a roadmap to improving quality care. Kidney International. 2015;88(3):447-59. 15. World Health Organization. Palliative Care, Factsheet No 402 2015 [Available from: http://www.who.int/mediacentre/factsheets/fs402/en/. 16. Tonkin-Crine S, Okamoto I, Leydon GM, Murtagh FE, Farrington K, Caskey F, et al. Understanding by older patients of dialysis and conservative management for chronic kidney failure. American Journal of Kidney Diseases. 2015;65(3):443-50. 16. Tonkin-Crine S, Okamoto I, Leydon GM, Murtagh FE, Farrington K, Caskey F, et al. Understanding by older patients of dialysis and conservative management for chronic kidney failure. American Journal of Kidney Diseases. 2015;65(3):443-50. 17. Lazenby S, Edwards A, Samuriwo R, Riley S, Murray MA, Carson-Stevens A. End-of-life care decisions for haemodialysis patients – ‘We only tend to have that discussion with them when they start deteriorating’. Health Expectations. 2017;20(2):260-73. 17. Lazenby S, Edwards A, Samuriwo R, Riley S, Murray MA, Carson-Stevens A. End-of-life care decisions for haemodialysis patients – ‘We only tend to have that discussion with them when they start deteriorating’. Health Expectations. 2017;20(2):260-73. 18. Boateng EA, East L, Evans C. Decision-making experiences of patients with end-stage kidney disease (ESKD) regarding treatment in Ghana: a qualitative study. BMC nephrology. 2018;19(1):1-12. 18. Boateng EA, East L, Evans C. Decision-making experiences of patients with end-stage kidney disease (ESKD) regarding treatment in Ghana: a qualitative study. BMC nephrology. 2018;19(1):1-12. 19. Ashuntantang G, Osafo C, Olowu WA, Arogundade F, Niang A, Porter J, et al. Outcomes in adults and children with end-stage kidney disease requiring dialysis in sub-Saharan Africa: a systematic review. The Lancet Global Health. 2017;5(4):e408-e17. 20. Bates MJ, Chitani A, Dreyer G. Palliative care needs of patients living with end-stage kidney disease not treated with renal replacement therapy: An exploratory qualitative study from Blantyre, Malawi. African Journal of Primary Health Care & Family Medicine. 2017;9:1-6. 20. Bates MJ, Chitani A, Dreyer G. Palliative care needs of patients living with end-stage kidney disease not treated with renal replacement therapy: An exploratory qualitative study from Blantyre, Malawi. African Journal of Primary Health Care & Family Medicine. 2017;9:1-6. 21. Ddungu H. Palliative care: what approaches are suitable in developing countries? British Journal of Haematology. 2011;154(6):728-35. 21. Ddungu H. Palliative care: what approaches are suitable in developing countries? British Journal of Haematology. 2011;154(6):728-35. 22. References Tannor EK, Norman BR, Adusei KK, Sarfo FS, Davids MR, Bedu-Addo G. Quality of life among patients with moderate to advanced chronic kidney disease in Ghana - a single centre study. BMC Nephrology. 2019;20(1):122. 22. Tannor EK, Norman BR, Adusei KK, Sarfo FS, Davids MR, Bedu-Addo G. Quality of life among patients with moderate to advanced chronic kidney disease in Ghana - a single centre study. BMC Nephrology. 2019;20(1):122. Page 12/15 Page 12/15 23. McIlfatrick S, Hasson F, McLaughlin D, Johnston G, Roulston A, Rutherford L, et al. Public awareness and attitudes toward palliative care in Northern Ireland. BMC palliative care. 2013;12(1):34. 24. McIlfatrick S, Noble H, McCorry NK, Roulston A, Hasson F, McLaughlin D, et al. Exploring public awareness and perceptions of palliative care: A qualitative study. Palliative medicine. 2014;28(3):273-80. 25. Noble H, Price JE, Porter S. The challenge to health professionals when carers resist truth telling at the end of life: a qualitative secondary analysis. Journal of clinical nursing. 2014;24(7-8):927-36. 26. Connor S, Bermedo MS. Global atlas of palliative care at the end of life Geneva, Switzerland/London, UK: Worldwide Palliative Care Alliance; World Health Organization; 2014 [Available from: 26. Connor S, Bermedo MS. Global atlas of palliative care at the end of life Geneva, Switzerland/London, UK: Worldwide Palliative Care Alliance; World Health Organization; 2014 [Available from: https://www.who.int/nmh/Global_Atlas_of_Palliative_Care.pdf. UK: Worldwide Palliative Care Alliance; World Health Organization; 2014 [Available from: https://www.who.int/nmh/Global_Atlas_of_Palliative_Care.pdf. 27. Rhee JY, Garralda E, Namisango E, Luyirika E, De Lima L, Powell RA, et al. An analysis of palliative care development in Africa: A ranking based on region-specific macroindicators. Journal of pain and symptom management. 2018;56(2):230-8. 28. Ofosu-Poku R, Owusu-Ansah M, Antwi J. Referral of Patients with Nonmalignant Chronic Diseases to Specialist Palliative Care: A Study in a Teaching Hospital in Ghana. International Journal of Chronic Diseases. 2020;2020. 29. Tannor E, Awuku Y, Boima V, Antwi S. The geographical distribution of dialysis services in Ghana. Renal Replacement Therapy. 2018;4(1):3. 29. Tannor E, Awuku Y, Boima V, Antwi S. The geographical distribution of dialysis services in Ghana. Renal Replacement Therapy. 2018;4(1):3. 30. Jha V, Arici M, Collins AJ, Garcia-Garcia G, Hemmelgarn BR, Jafar TH, et al. Understanding kidney care needs and implementation strategies in low- and middle-income countries: conclusions from a “Kidney Disease: Improving Global Outcomes” (KDIGO) Controversies Conference. Kidney International. 2016;90(6):1164-74. 31. Pommer W, Wagner S, Thumfart J. References Conservative Care, Dialysis Withdrawal, and Palliative Care: Results from a Survey of a Non-Profit Dialysis Provider in Germany. Kidney and Blood Pressure Research. 2019;44(2):158-69. 32. Starks H, Trinidad SB. Choose your method: A comparison of phenomenology, discourse analysis, and grounded theory. Qualitative health research. 2007;17(10):1372-80. 32. Starks H, Trinidad SB. Choose your method: A comparison of phenomenology, discourse analysis, and grounded theory. Qualitative health research. 2007;17(10):1372-80. 33. Pringle J, Drummond J, McLafferty E, Hendry C. Interpretative phenomenological analysis: a discussion and critique. Nurse researcher. 2011;18(3):20-4. 33. Pringle J, Drummond J, McLafferty E, Hendry C. Interpretative phenomenological analysis: a discussion and critique. Nurse researcher. 2011;18(3):20-4. 34. Colaizzi PF. Psychological research as the phenomenologist views it. In: Vale R, King M, editors. Existential-Phenomenological Alternatives for Psychology. New York: Oxford University Press; 1978. 34. Colaizzi PF. Psychological research as the phenomenologist views it. In: Vale R, King M, editors. Existential-Phenomenological Alternatives for Psychology. New York: Oxford University Press; 1978. 35. Wirihana L, Welch A, Williamson M, Christensen M, Bakon S, Craft J. Using Colaizzi’s method of data analysis to explore the experiences of nurse academics teaching on satellite campuses. Nurse Researcher. 2018;25(4):30. 36. O’reilly M, Parker N. ‘Unsatisfactory Saturation’: a critical exploration of the notion of saturated sample sizes in qualitative research. Qualitative Research. 2013;13(2):190-7. 36. O’reilly M, Parker N. ‘Unsatisfactory Saturation’: a critical exploration of the notion of saturated sample sizes in qualitative research. Qualitative Research. 2013;13(2):190-7. 37. Connelly LM. Trustworthiness in qualitative research. Medsurg Nursing. 2016;25(6):435-7. 37. Connelly LM. Trustworthiness in qualitative research. Medsurg Nursing. 2016;25(6):435-7. Page 13/15 Page 13/15 38. Guba E, Lincoln Y. Competing paradigms in qualitative research. In: Denzin N, Lincoln Y, editors. Handbook of qualitative research. California: Sage; 1994. p. 105-17. 39. Thomas DR. Feedback from research participants: are member checks useful in qualitative research? Qualitative Research in Psychology. 2017;14(1):23-41. 40. Polit DF, Beck CT. Nursing research: Generating and assessing evidence for nursing practice. 9 ed. Philadelphia: Lippincott Williams & Wilkins; 2012. 41. Noble H, Smith J. Issues of validity and reliability in qualitative research. Evidence Based Nursing. 2015;18(2):34-5. 42. Korstjens I, Moser A. Series: practical guidance to qualitative research. Part 4: trustworthiness and publishing. European Journal of General Practice. 2018;24(1):120-4. 43. Tong A, Sainsbury P, Craig J. Consolidated criteria for reporting qualitative research (COREQ): a 32- item checklist for interviews and focus groups. International journal for quality in health care. 2007;19(6):349-57. 44. Tables Due to technical limitations Table 1 and 2 are available as a download in the Supplementary Files. Due to technical limitations Table 1 and 2 are available as a download in t References Morton R, Tong A, Howard K, Snelling P, Webster A. The views of patients and carers in treatment decision making for chronic kidney disease: Systematic review and thematic synthesis of qualitative studies. BMJ: British Medical Journal. 2010;340(7742):1-11. 45. Luyckx VA, Miljeteig I, Ejigu AM, Moosa MR. Ethical Challenges in the Provision of Dialysis in Resource-Constrained Environments. Seminars in Nephrology. 2017;37(3):273-86. 46. Eghan BA, Amoako-Atta K, Kankam CA, Nsiah-Asare A. Survival pattern of hemodialysis patients in Kumasi, Ghana: a summary of forty patients initiated on hemodialysis at a new hemodialysis unit. Hemodialysis International. 2009;13(4):467-71. 47. Harwood L, Clark AM. Understanding pre-dialysis modality decision-making: A meta-synthesis of qualitative studies. International journal of nursing studies. 2013;50(1):109-20. 48. Murray MA, Brunier G, Chung JO, Craig LA, Mills C, Thomas A, et al. A systematic review of factors influencing decision-making in adults living with chronic kidney disease. Patient Education and Counseling. 2009;76(2):149-58. 49. Ashby M, op’t Hoog C, Kellehear A, Kerr PG, Brooks D, Nicholls K, et al. Renal dialysis abatement: lessons from a social study. Palliative medicine. 2005;19(5):389-96. 50. Tong A, Cheung KL, Nair SS, Kurella Tamura M, Craig JC, Winkelmayer WC. Thematic Synthesis of Qualitative Studies on Patient and Caregiver Perspectives on End-of-Life Care in CKD. American Journal of Kidney Diseases. 2014;63(6):913-27. 51. Hole B, Hemmelgarn B, Brown E, Brown M, McCulloch MI, Zuniga C, et al. Supportive care for end- stage kidney disease: an integral part of kidney services across a range of income settings around the world. Kidney international supplements. 2020;10(1):e86-e94. 52. Grubbs V, Tuot DS, Powe NR, O’Donoghue D, Chesla CA. Family Involvement in Decisions to Forego or Withdraw Dialysis: A Qualitative Study of Nephrologists in the United States and England. Kidney Medicine. 2019;1(2):57-64. Page 14/15 Page 14/15 53. DePasquale N, Cabacungan A, Ephraim PL, Lewis-Boyér L, Powe NR, Boulware LE. Family members’ experiences with dialysis and kidney transplantation. Kidney Medicine. 2019;1(4):171-9. 54. Sellars M, Clayton JM, Morton RL, Luckett T, Silvester W, Spencer L, et al. An interview study of patient and caregiver perspectives on advance care planning in ESRD. American Journal of Kidney Diseases. 2018;71(2):216-24. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. COREQChecklistSarfoWaltersandBoatengBMCPallCare.DOC InterviewGuidesSarfoWaltersandBoateng.docx Tables.pdf Page 15/15
https://openalex.org/W4386587583	https://www.qeios.com/read/FCCW0Q/pdf	English	null	Review of: "Culture Lockdown, Nature Freedom: Respite for Biodiversity during the COVID Pandemic – A Limited Case Study in La Union, Philippines"	null	2,023	cc-by	385	Qeios, CC-BY 4.0 · Review, September 10, 2023 Review of: "Culture Lockdown, Nature Freedom: Respite for Biodiversity during the COVID Pandemic – A Limited Case Study in La Union, Philippines" Debal Deb1 1 Center for Interdisciplinary Studies Potential competing interests: No potential competing interests to declare. Qeios ID: FCCW0Q · https://doi.org/10.32388/FCCW0Q The article is interesting. I have a few suggestions to improve upon this work. The article is interesting. I have a few suggestions to improve upon this work. 1. The title is misleading, as it deals with a single species of bird, rather than a range of biodiversity. There is no evidence that Brahminy kite is a keystone species, which may represent the biodiversity status in an ecosystem. The title and introduction should clearly state that this is a study of BK sighting frequency in association with COVID-19 restriction phases, and nothing more. 2. The data is scanty, with df = 12, with a signficant kurtosis. Also, the figure shows that the mean of the mean of sighting records is less than the variance. In this case, a general linear model (GLM) with negative binomial link function is more appropriate than a linear model of correlation. Kendall's tau alone in this small sample size is not enough to detect significance at a high confidence level. 3. Given that human excursions and depredation of wild habitats led to a higher frequency of sighting of BK, an ecological explanation is warranted. The author describes the bird as the top predator in the food web, so one must show that the availability and abundance of prey at lower trophic levels increased during the lock down periods. Alternatively, more nesting/ breeding habitat for the bird became available during the lock down. In either case, some data about the availability of more resources (food or habitat) would give the paper more ecological meaning. 4. The usage of the term “dialectical” is somewhat obfuscating and unnecessary, because it is not clearly defined in the context, and because it does not reveal any new insight. The “Dialectics of Nature" sensu Friedrich Engels is evident in the process of advancement of science building on novel discoveries, which change our understanding of the workings of nature. In this paper's context, the “dialectical process” is at best unclear. Qeios ID: FCCW0Q · https://doi.org/10.32388/FCCW0Q 1/1
https://openalex.org/W3093342870	https://www.nature.com/articles/s41597-020-00675-z.pdf	English	null	Harmonised LUCAS in-situ land cover and use database for field surveys from 2006 to 2018 in the European Union	Scientific data	2,020	cc-by	11,869	www.nature.com/scientificdata www.nature.com/scientificdata 1European Commission Joint Research Centre (JRC), Ispra, Italy. 2European Commission, Eurostat (ESTAT), Luxembourg, Luxembourg. 3GOPA Luxembourg, Luxembourg, Luxembourg. ✉e-mail: raphael.dandrimont@ ec.europa.eu; marijn.van-der-velde@ec.europa.eu Data Descriptor Raphaël d’Andrimont 1 ✉, Momchil Yordanov1, Laura Martinez-Sanchez 1, Beatrice Eiselt2, Alessandra Palmieri2, Paolo Dominici2, Javier Gallego1, Hannes Isaak Reuter 2, Christian Joebges3, Guido Lemoine 1 & Marijn van der Velde 1 ✉ Accurately characterizing land surface changes with Earth Observation requires geo-located ground truth. In the European Union (EU), a tri-annual surveyed sample of land cover and land use has been collected since 2006 under the Land Use/Cover Area frame Survey (LUCAS). A total of 1351293 observations at 651780 unique locations for 106 variables along with 5.4 million photos were collected during five LUCAS surveys. Until now, these data have never been harmonised into one database, limiting full exploitation of the information. This paper describes the LUCAS point sampling/surveying methodology, including collection of standard variables such as land cover, environmental parameters, and full resolution landscape and point photos, and then describes the harmonisation process. The resulting harmonised database is the most comprehensive in-situ dataset on land cover and use in the EU. The database is valuable for geo-spatial and statistical analysis of land use and land cover change. Furthermore, its potential to provide multi-temporal in-situ data will be enhanced by recent computational advances such as deep learning. Background & Summary specific variables on different aspects of land cover, land use, and land and water management6), has remained comparable for all five surveys. Survey design summary. LUCAS collects information on land cover and land use, agro-environmental variables, soil, and grassland. The surveys also provide spatial information to analyse the mutual influences between agriculture, environment, and countryside, such as irrigation and land management. The in-situ point data is collected according to a harmonised classification with separate land cover and land use codes. Data qual- ity is assured by a regular two-level quality control (i.e. internal and external), in which all points are evaluated by quality controllers (see7 for more details). At each LUCAS point, standard variables are collected including land cover, land use, environmental parameters (the so called micro data), as well as one downward facing photo of the point (P) and four landscape photos in the cardinal compass directions (N, E, S, W). Additionally to the core variables collected, other specific modules were carried out on demand such as (i) the transect of 250 m to assess transitions of land cover and existing linear features (2009, 2012, 2015), (ii) the topsoil module (2009, 2012 (partly), 2015 and 2018), (iii) the grassland module (2018), and (iv) the Copernicus module collecting the homo- geneous and continuous extent of land cover on a 50-m radius (2018)8. Due to the specificity of these modules, their corresponding collected data are not included in the data harmonisation presented in this paper. The topsoil module datasets for 2009, 2012 and 2015 were harmonised and documented separately as an open-access dataset of topsoil properties in the EU9.hi LUCAS is a two phase sample survey. The LUCAS first phase sample is a systematic selection of points on a grid with a 2 km spacing in Eastings and Northings covering the whole of the EU’s territory10. Currently, it includes around 1.1 million points (Fig. 1) and is referred to as the master sample. Each point of the first phase sample is classified in one of ten land-cover classes via visual interpretation of ortho-photos or satellite images11. The core sampling and survey methods have remained the same throughout the five surveys. Nevertheless evolv- ing goals of the surveys have led to slightly different sample point allocations for different land covers. Background & Summary Background & Summary g y Accurate, timely, and representative in-situ observations across large areas have always been needed to report statistics on land use, land cover, and the environment. Precise geo-located in-situ information is also indispen- sable to train and validate algorithms that characterize the Earth’s surface based on remotely sensed observations. Comprehensive and thematically rich in-situ data can lead to better classifiers and more accurate multi-temporal land surface mapping. This is especially true since increasingly frequent and detailed Earth Observations are being made, for instance by the fleet of Sentinel satellites of the EU’s Copernicus program. These developments are opening avenues to better combine classical statistical surveying and Earth Observation (EO) derived prod- ucts in the domains of land use and land cover change and environmental monitoring (e.g1.). Historical background. The Land Use/Cover Area frame Survey (LUCAS) in the European Union (EU) was set-up exactly to provide such statistical information2. It represents a triennial in-situ land cover and land use data collection exercise that extends over the whole of the EU’s territory (https://ec.europa.eu/eurostat/web/ lucas). The LUCAS project was implemented following Decision 1445/2000/EC of the European Parliament and of the Council of 22 May 2000 “On the application of area-frame survey and remote-sensing techniques to the agricultural statistics for 1999 to 2003” and has continued since. While the LUCAS survey concept was initiated and tested in 2001 and 20033, it has been restructured in 20064 and then slightly modified to result in the actual survey design5. In 2006, Eurostat, the statistical office of the EU, launched a pilot survey project in 11 countries to test the stratified sampling design. The primary focus was on agricultural areas with emphasis given to easily accessible points. Since then Eurostat has carried out LUCAS surveys every three years with the survey design ever evolving, however the LUCAS core component (i.e. the identification of the point, and the surveying of 1European Commission Joint Research Centre (JRC), Ispra, Italy. 2European Commission, Eurostat (ESTAT), Luxembourg, Luxembourg. 3GOPA Luxembourg, Luxembourg, Luxembourg. ✉e-mail: raphael.dandrimont@ ec.europa.eu; marijn.van-der-velde@ec.europa.eu Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z www.nature.com/scientificdata/ Fig. 1 Schematic overview of the LUCAS and harmonisation methodologies. The left side illustrates the sampling at the basis of the production of the LUCAS primary data. The top right side shows the raw base data (micro data). Background & Summary The process of harmonising is contained within the multi-year harmonised aggregation block and is the subject of the following two sections. The bottom right presents the four main outputs associated with this manuscript (more in section Data Records) - a harmonised, legend-explicit, multi-year, ready-to-use, version of the LUCAS micro data (section Overview of multi-year harmonised LUCAS survey database49,), a database with all cardinal-direction landscape and point photos collected during the surveys, including their respective EXIF attributes (section Overview of EXIF photos database, EXIF table49, photos on https://gisco-services.ec.europa. eu/lucas/photos/, the survey geometries49 and a R package to generate the data51. Fig. 1 Schematic overview of the LUCAS and harmonisation methodologies. The left side illustrates the h Fig. 1 Schematic overview of the LUCAS and harmonisation methodologies. The left side illustrates the sampling at the basis of the production of the LUCAS primary data. The top right side shows the raw base data (micro data). The process of harmonising is contained within the multi-year harmonised aggregation block and is the subject of the following two sections. The bottom right presents the four main outputs associated with this manuscript (more in section Data Records) - a harmonised, legend-explicit, multi-year, ready-to-use, version of the LUCAS micro data (section Overview of multi-year harmonised LUCAS survey database49,), a database with all cardinal-direction landscape and point photos collected during the surveys, including their respective EXIF attributes (section Overview of EXIF photos database, EXIF table49, photos on https://gisco-services.ec.europa. eu/lucas/photos/, the survey geometries49 and a R package to generate the data51. Fig. 1 Schematic overview of the LUCAS and harmonisation methodologies. The left side illustrates the sampling at the basis of the production of the LUCAS primary data. The top right side shows the raw base data (micro data). The process of harmonising is contained within the multi-year harmonised aggregation block and is the subject of the following two sections. The bottom right presents the four main outputs associated with this manuscript (more in section Data Records) - a harmonised, legend-explicit, multi-year, ready-to-use, version of the LUCAS micro data (section Overview of multi-year harmonised LUCAS survey database49,), a database with all cardinal-direction landscape and point photos collected during the surveys, including their respective EXIF attributes (section Overview of EXIF photos database, EXIF table49, photos on https://gisco-services.ec.europa. eu/lucas/photos/, the survey geometries49 and a R package to generate the data51. Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z Methods Having contextualized the LUCAS survey, we proceed with describing the full methodological workflow to harmonise the data, as schematically shown in Fig. 1. The Sampling and Survey sub-figures provide an over- view of the methodological framework of the LUCAS data collection itself (see previous section Background & Summary). The Data aggregation and Results sub-figures illustrate the work carried out in this study. The datasets collected during the five surveys (in 2006, 2009, 2012, 2015, 2018) are the main LUCAS products available (more in section Micro data collection and documentation (Protocol 1)). These datasets and their respective data docu- mentation were used to create the multi-year harmonised database. The harmonisation process is described below and in Table 1. Associated with the summary Table 1, the Table 2 provides name changes, the Table 3 provides the new columns added, the Online-only Table 1 provides the missing column adding and the Online-only Table 2 provides the variable re-coding. The results are consolidated in one single consistent and legend-explicit table along with hard-coded links to the full resolution photos (stored on the GISCO, https://gisco-services.ec.europa. eu/lucas/photos/). The LUCAS primary data includes alpha-numerical variables and field photographs linked to the geo-referenced points. Micro data collection and documentation (Protocol 1). The first step is to collect the data from the source for each survey year (see Table 1 Source). The raw micro data for the harmonised database presented here are the five LUCAS campaigns, which can be downloaded from the official Eurostat website (https://ec.europa.eu/ eurostat/web/lucas). The LUCAS micro data for 200621 is divided into a separate file for each of the 11 surveyed countries (Belgium22, Czechia23, Germany24, Spain25, France26, Italy27, Luxembourg28, Hungary29, Netherlands30, Poland31, and Slovakia32). The LUCAS micro data is provided aggregated for all countries for every other survey years, whereby the data can also be downloaded separately by country (200933, 201234, 201535) in CSV format and 201836 in 7z zipped format. The second step is to collect the documentation that facilitates translating the alpha-numerical class-description in the original datasets into explicit information. For 2006, 2009 and 2012, the survey data comes with a content descriptor (200637, 200938, 201239), though the content descriptor doesn’t neces- sarily have the same number of variables as the data; and the variables themselves sometimes have a slightly differ- ent name. Background & Summary In 2006, the main objective was to “make early estimates of the main crop areas”, along with the ability to collect information on agri-environmental indicators in the context of the monitoring of the Common Agriculture Policy (CAP)3. In 2009, the main objective was to estimate areas, especially in conjunction with other data sources such as Corine Land Cover (CLC)10. In 2018, the main objective was to “monitor social and economic use of land as well as ecosystems and biodiversity”5. Additionally, in 2018, a linear logistic regression model based on LUCAS 2015 and 16 additional variables were used as co-variates to forecast the most probable land cover for each of these points5. From this stratified first phase sample, the second phase sample of points is selected to obtain the desired statistically representative spatial distribution of sampled land cover classes according to the first phase visual interpretation. With LUCAS 2018 this amounts to 337845 points, out of which approximately 240000 points are visited in the field by surveyors to collect additional information that cannot be assessed remotely.h i y y y The in-situ nature of the survey implies that the majority of the data are gathered through direct observations made by surveyors on the ground. Those points which are unlikely to change and points which are too difficult Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z 2 www.nature.com/scientificdata/ to access are classified by photo-interpretation in the office, using the latest available ortho-photos or Very High Resolution (VHR) images. Although most of the points a-priori assigned for in-situ assessment can effectively be visited in the field, those that cannot be reached, because of lack of access to the point or the point location being at more than 30 minutes walking distance from the closest point reachable by car. Those points are thus photo-interpreted on ortho-photos or Very High Resolution (VHR) images in the field by the field surveyor. Furthermore, sometimes a significant difference exists between the theoretical LUCAS point and the actual GPS location reached by the surveyor. Observations are collected for the LUCAS point, while the photos are taken at the actual GPS location. Both locations and the distance between them is noted down. Previous LUCAS use cases and shortcomings. Background & Summary In the scientific literature, LUCAS land cover and land use survey data have been used to derive statistical estimates2, to describe land cover/use diversity at regional level12, and its sampling frame was used as a basis for various applications including assessing the availability of crowd-sourced photos potentially relevant for crop monitoring across the EU13. LUCAS was designed to derive statistics for area estimation (e.g3. and10). Recently, several researchers have started to use LUCAS data in large scale land cover mapping processes, especially as a source of training and/or validation data for supervised clas- sification approaches at regional/national scale14–20. i pp g Several drawbacks become apparent when working with the original LUCAS datasets. While the inconsisten- cies could be due to the enumerators’ subjectivity in interpretation of the legends and the legend itself, it is also related to the complexity of the field survey: large number of surveyors (>700), complex documentation for the enumerators (>400 pages combining all the documents), translated to 20 languages. These drawbacks hinder the further use of the LUCAS data by the scientific community as a whole and in particular by users who are active in emerging fields of big data analytics, data fusion, and computer vision. Such drawbacks include: • Inconsistencies and errors between legends and labels from one LUCAS survey to the next which is hamper- ing temporal analysis. g p y Missing internal cross-references in the datasets that would facilitate computation and linking observed var- iables, photos, etc.h • The original full resolution photos taken at each surveyed point are not available for download. Th l k f i l i lid d d b h i d i d bi d h The lack of a single-entry point or consolidated database hampering automated processing and big data analysis. Therefore, we have gone through an extensive process of cleaning by semantic and topological harmonisation, along with connecting the originally disjoint LUCAS datasets in one consolidated database with hard-coded links to the full-resolution photos, openly accessible along with this paper. www.nature.com/scientificdata/ www.nature.com/scientificdata/ Source Year Points (n) Protocol 1 Protocol 2 21 2006 168401 Data download, Data documentation37,40, Preparation (year aggregation), Generate mapping files Column renaming (Table 2), Missing column adding (Online-only Table 1), New column adding (Table 3), Character case uniformity, Variable re-coding (Online-only Table 2), Column order 33 2009 234623 Data download, Data documentation38,41, Preparation, Generate mapping files Column renaming (Table 2), Missing column adding (Online-only Table 1), New column adding (Table 3), Character case uniformity, Variable re-coding (Online-only Table 2), Column order 34 2012 270272 Data download, Data documentation39,42, Preparation, Generate mapping files Column renaming (Table 2), Missing column adding (Online-only Table 1), New column adding (Table 3), Character case uniformity, Variable re-coding (Online-only Table 2), Column order 35 2015 340143 Data download, Data documentation46, Preparation, Generate mapping files Column renaming (Table 2), Missing column adding (Online-only Table 1), New column adding (Table 3), Character case uniformity, Variable re-coding (Online-only Table 2), Column order 36 2018 337854 Data download, Data documentation47, Preparation, Generate mapping files Missing column adding (Online-only Table 1), New column adding (Table 3), Character case uniformity Table 1. Aggregation of micro data - summarizing the different steps applied to harmonise the survey data. Table 1. Aggregation of micro data - summarizing the different steps applied to harmonise the survey data. is changed permanently. All transformations are done by recoding ordinal variables to be compliant with the encoding of variables used in the last survey (2018). These mappings serve as a blueprint for the transformation and data integration described in Protocol 2. is changed permanently. All transformations are done by recoding ordinal variables to be compliant with the encoding of variables used in the last survey (2018). These mappings serve as a blueprint for the transformation and data integration described in Protocol 2. Micro data harmonisation (Protocol 2). The harmonisation workflow, alongside the performed database consistency checks, is shown in Fig. 2 and the code is described in code section (section Code availability). The general principle of the harmonisation workflow was to convert all the field legends to fit with the latest i.e. the 2018 database layout (the next LUCAS is planned for 2022). y Some notable changes in the source tables had to be made in order to make the harmonisation and subsequent merger into one complete table possible. This was accomplished with the above-mentioned instance-mapping files (Section Micro data collection and documentation (Protocol 1)). www.nature.com/scientificdata/ All manipulations executed over the sepa- rate tables prior to the merger are listed in Table 1 under heading ‘Protocol 2’: 1. Rename columns - iteratively renaming columns to align them with the last (in this case 2018) survey. 1. Rename columns - iteratively renaming columns to align them with the last (in this case 2018) surv 1. Rename columns - iteratively renaming columns to align them with the last (in this case 2018) Performed on all tables but 2018 by using the Rename_cols() function from the package. y g g ( Performed on all tables but 2018 by using the Rename_cols() function from the packag 2. Add photo column 2006 - adds columns photo_north, photo_south, photo_east, photo_west, and photo_ point on account of them missing from the 2006 base data. Adding is done by cross-referencing the EXIF picture database (see section Overview of EXIF photos database). Performed solely on table for 2006 by using the Add photo field 2006() function. g _p _i _ () 3. Add Missing columns - iteratively adding all columns that are present in one table and not present in the others. Performed on all tables by using the Add missing cols() function.h 4. Add new columns - iteratively adding all newly created columns. These include the variables ‘letter group’, ‘year’, and ‘file_path_gisco_n/s/e/w/p’ (for more information check Online-only Table 3). Performed on all tables using the Add new cols() function. g _ _ () 5. Upper case - iteratively converting all characters of selected fields to upper case. Performed on all tables using the Upper case() function.i g pp _ () 6. Re-code variable - iteratively re-coding selected variables according to created mapping CSV files, designed referring back to the reference documents. Performed on all tables but 2018 by using the Recode_ vars() function. g pp _ () 6. Re-code variable - iteratively re-coding selected variables according to created mapping CSV files, designed referring back to the reference documents. Performed on all tables but 2018 by using the Recode_ vars() function. () 7. Order columns - iteratively ordering all columns according to the template from the 2018 survey. Per- formed on all tables but 2018 by using the Order_cols() function. () 7. Order columns - iteratively ordering all columns according to the template from the 2018 survey. Per- formed on all tables but 2018 by using the Order_cols() function. Merge and post-processing (Protocol 3). Methods These inconsistencies were resolved with assistance from the technical documents (LC1 (Instructions, 200640, 200941, 201242) and LC3 (Classification, 200643, 200944, 201245). From 2015 and 2018, the data is provided with a record descriptor (201546, 201847), which contains information on variable name, data type and description in a more consolidated fashion, making it easier to find information about the relevant variable.hiih gi The third and final step in Protocol 1 is the generation of the mapping files used for value recoding. The work- flow maps the ascertained relationship between those variables that are the same but have changed in name or alpha-coding between surveys. To recode all variables coherently from one survey to the next, the original data Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z 3 www.nature.com/scientificdata/ www.nature.com/scientificdata/ Year added to Old name New name 2006 surv_date surveydate 2006 x_laea th_lat 2006 y_laea th_long 2009, 2012, 2015 area_size parcel_area_ha 2009, 2012, 2015 date surveydate 2009, 2012, 2015 lc1_pct lc1_perc 2009, 2012, 2015 lc2_pct lc2_perc 2009, 2012, 2015 lc1_species lc1_spec 2009, 2012, 2015 lc2_species lc2_spec 2009, 2012, 2015 land_mngt grazing 2009, 2012, 2015 obs_dir obs_direct 2009, 2012, 2015 photo_e photo_east 2009, 2012, 2015 photo_w photo_west 2009, 20122009, 2012, 2015 photo_n photo_north 2009, 2012, 2015 photo_s photo_south 2009, 2012, 2015 photo_p photo_point 2009, 2012, 2015 tree_height_surv tree_height_survey 2009, 2012, 2015 soil_stones soil_stones_perc 2012, 2015 tree_height_mat tree_height_maturity 2015 lu1_pct lu1_perc 2015 lu2_pct lu2_perc 2015 protected_area special_status 2015 pi_extension office_pi Table 2. Table of renamed variables. Column name Description letter_group First level of LUCAS LC1/2 classification year Year of the survey file_path_gisco_n/s/e/w/p Path to cardinal or point photo on GISCO th_geom Geometry of theoretical LUCAS point according to grid gps_geom Geomtery at the point the surveyor reached th_gps_dist Calculated distance between the two points visit Numbers of years of visit for the LUCAS point Table 3. Table of newly added columns. Table 3. Table of newly added columns. Table 3. Table of newly added columns. 7. Convert encoding to label - Create columns with labels for coded variables and decodes all variables where possible to explicit labels. Performed with User_friendly() function. Consistency check performed after this successful execution.i 7. Convert encoding to label - Create columns with labels for coded variables and decodes all variables where possible to explicit labels. Performed with User_friendly() function. Consistency check performed after this successful execution.i 7. Convert encoding to label - Create columns with labels for coded variables and decodes all variables where possible to explicit labels. Performed with User_friendly() function. Consistency check performed after this successful execution.i t 8. Final column order - Re-order columns of final tables with the Final_order_cols() function. 9. Remove variables - optional function to remove variables which the technician deems not necessary for the new harmonised product. Done with the Remove vars() function.i 8. Final column order - Re-order columns of final tables with the Final_order_cols() function. 9. Remove variables - optional function to remove variables which the technician deems not necessary for the new harmonised product. Done with the Remove_vars() function.i p _ 10. www.nature.com/scientificdata/ The third part of the harmonisation process includes the merg- ing of the harmonised tables of each survey year plus additional steps listed below before exporting the final data outputs. Merge and post-processing (Protocol 3). The third part of the harmonisation process includes the merg- ing of the harmonised tables of each survey year plus additional steps listed below before exporting the final data outputs. Merge and post-processing (Protocol 3). The third part of the harmonisation process includes the merg- ing of the harmonised tables of each survey year plus additional steps listed below before exporting the final data outputs. 1. Merge into single table - Merge the five harmonised tables to one unique table via Merge_harmo() function. Consistency check performed after this successful execution on newly generated Table. y pt y g 2. Correct theoretical location - Applying a correction of the values of columns th_long and th_lat for mer harmonised table according to the latest LUCAS grid via the Correct th loc() function. g g _ _ 3. Add geometry columns - Location of theoretical point(th_geom), location of lucas survey (gps_geom), lucas transect geometries (trans_geom) and distance between theoretical and survey point (th_gps_dist Done by the Add geom() function. y _g () 4. Create database tags - Primary key, index, and spatial index via the Create_tags() function. 5. Add number of visits column - column to show the number of times between the years when the point was visited thanks to the Add num visits() function.f _ _ 6. Align mapping CSVs - Corrects any typo, spelling mistake, or spelling difference in the user-created map- ping CSVs, used to generate labels in subsequent function that converts encoding to label by aligning them to the mapping CSV of the latest survey. Done by the Align_map_CSVs() function. Consistency check performed after this successful execution on newly generated mapping CSVs. 6. Align mapping CSVs - Corrects any typo, spelling mistake, or spelling difference in the user-created map- ping CSVs, used to generate labels in subsequent function that converts encoding to label by aligning them to the mapping CSV of the latest survey. Done by the Align_map_CSVs() function. Consistency check performed after this successful execution on newly generated mapping CSVs. Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z Storage. g 1. Multi-year harmonised LUCAS survey data. The harmonised database (available for download here49 and also archived as compressed folder here50) contains 106 variables and 1351293 records corresponding to a unique combination of survey year and field location. The same dataset is also available for each year with a different file for users interested only in one specific survey. The database is provided with a Record de- scriptor (Online-only Table 3 presents a summary, the complete table is available here in CSV format49 in the supporting files). This record descriptor specifies variable name, data type, range of possible values and meanings. In the documentation one can find more information about the variable and a short description, along with comments concerning the variable that the authors have deemed important. Additionally, the tables in LUCAS-Variable and Classification Changes, in the supporting files, contain documentation for users to quickly identify the differences between LUCAS campaigns and the harmonised database. The file contains four tables: 1. “References”: Description and a legend of the used colors of the different tables; p gf 2. “Harmonised DB”: a comparison of all the collected variables of the 2018 survey with the variables of the harmonised database and an overview of the actions to harmonise the data; 3. “Variable changes”: an overview/ comparison of all collected variables between all campaigns from 2009 to 2018 highlighting the changes; g g g g 4. “LC (LU) changes”: an overview of the possible LC and LU codes of each campaign highlighting the changes. 2. LUCAS survey geometries/point locations. To facilitate spatial analysis and usability, three types of geometries are provided as distinct shapefiles (see the geometries folder downloadable on49): 1. LUCAS theoretical points (th_long, th_lat), p g 2. LUCAS observed points (gps_lon, gps_lat) and p g 2. LUCAS observed points (gps_lon, gps_lat) and p gp gp 3. LUCAS transect lines (250-m east looking lines). 3. High resolution LUCAS photo archive. The 5.4 millions of photos collected during the five surveys are available at https://gisco-services.ec.europa.eu/lucas/photos/. For each in-situ point, landscape (N, E, S, W), and point (P) photos are available. The EXIF information of all the photos were extracted and are provided as an additional table (lucas_harmo_exif.csv49).h p f 4. R package. The scripts to harmonise the LUCAS data is provided as an open source R package along with the documentation51. Overview of multi-year harmonised LUCAS survey database. www.nature.com/scientificdata/ Update record descriptor - Updates Record descriptor by adding a field (year) showing the year for which the variable exists and removing variables listed in the optional function for removing variables from record descriptor. Done with the Update_RD() function. _ 10. Update record descriptor - Updates Record descriptor by adding a field (year) showing the year for which the variable exists and removing variables listed in the optional function for removing variables from record descriptor. Done with the Update_RD() function. The workflow ends with the output exports. The table is exported as CSV and the geometries as shapefiles. The full workflow is dependent on two software prerequisites. Firstly, one must have a running PostgreSQL server, and secondly, an installation of R (more about the versions used in section Code availability). The pipeline is provided as a R package for ease of reproducibility and transparency (section Code availability). Full resolution LUCAS photos. In addition to the alphanumerical and geometry information of the sur- vey, a complete database with full-resolution point and landscape photos was set up with photos retrieved from Eurostat. This archive was organised as a table with all the exchangeable image file (EXIF) variables for each of the images, among which a unique file path, as stored on the Eurostat GISCO server for easy retrieval by other researchers. Besides the EXIF attributes, each photo is also hard-coded with the respective point ID of the LUCAS point and the year of survey. The photos’ metadata were extracted with ExifTool (v 10.8)48 resulting in a database of photos that was compared for completeness with the survey data records. The hard-coded HTTPS links to each photo in the consolidated database allow for large data volume queries and selection tasks. Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z 5 www.nature.com/scientificdata/ Fig. 2 Processing workflow to harmonise the survey data. Asterix used to indicate steps after which there is a performed consistency check (Merge into single table, Align mapping CSVs, and Convert encoding to label). Fig. 2 Processing workflow to harmonise the survey data. Asterix used to indicate steps after which there is performed consistency check (Merge into single table, Align mapping CSVs, and Convert encoding to label). Fig. 2 Processing workflow to harmonise the survey data. Asterix used to indicate steps after which there is a performed consistency check (Merge into single table, Align mapping CSVs, and Convert encoding to label). Data Recordshi The first section Storage describes each data-set provided along with this manuscript including the table, photo, and geometry databases along with the R package created to compile and construct all the data. The second sec- tion Overview of multi-year harmonised LUCAS survey database provides an outline of the resulting harmonised database and the last section Overview of EXIF photos database provides an overview of the photo database. www.nature.com/scientificdata/ www.nature.com/scientificdata/ (Table 4). The total number of surveyed points has increased significantly from the 2006 pilot study (168401) to 2015 (340143) (Table 4). This rise is mainly due to the increase in terms of thematic richness, scope, and scale of the study from what was primarily an evaluation of agricultural areas (2006) to a more holistic and exhaustive inspection of the EU territory. Further, the total number of surveyed countries increased from 11 in 2006 to 28 in 2018 (Table 4). Over the five surveys, 1 031 813 observations (76.36%) were done in-situ. Out of these in-situ observations, 94% have been surveyed within 100 m distance of the theoretical LUCAS point and 6% were more than 100 m away from the point. The proportion of points where actual in-situ data was collected has decreased from 92.18% in 2006 to 63.67% in 2018. Furthermore, 10.92% of the points (i.e. 147574) that were visited in-situ turned out not be accessible in practice and are photo-interpreted in the field. The number of points surveyed per country and per year ranged between 79 (Malta) to 48215 (France). Finally, over the five surveys, 1677 points were out of national territory, i.e. “NOT EU” corresponding to water outside national borders or countries includ- ing Russia, Turkey, Albania and Switzerland). g y Figure 3 provides the accumulative frequency of assigned level-3 classes (out of 77 classes in total) to the sur- veyed points, sorted by reference year. Land Cover/Land Use (LC/LU) classification specifications can be found in the new reference document, containing the harmonised C3 legend (see Harmonized C3 legend in49).hi g g g The classification system follows rules on spatial and temporal consistency - it can be applied and compared both between locations in the EU and by survey years. Additionally, excluding 2006, it is ‘as much as possible’ compatible with other existing LC/LU systems (e.g. Food and Agriculture Organization (FAO), statistical classifi- cation of economic activities in the European Community (NACE) (2009–2018) and fulfills the specifications of the European Infrastructure for Spatial Information in Europe (INSPIRE) (2015-2018)). To inform about changes in two consecutive surveys, the data providers describe the adjustments to the terminology in the documentation. The 3-level legend system is arranged hierarchically, whereby the first level (letter group) corresponds to the eight main classes obtained by ortho-photo-interpretation during the second level stratification phase (Fig. www.nature.com/scientificdata/ 1); the sec- ond and third level, representing subcategories of these main classes are indicated by a combination of the letter group and further digits. g p g The number of point visits is shown in Table 5. Some LUCAS points were visited once in 15 years (n = 332605) while others were visited each time, thus totaling five visits (n = 35204). This means that 651780 locations were at least visited once. Figure 4 shows a map with the visit frequency for each point over Europe. Overview of EXIF photos database. The available photos (N, E, S, W, P, i.e. North, East, South, West, and Point) were catalogued totaling 5440459 photos for the 5 surveys (see Table 6 for detailed distribution). The lucas_harmo_exif.csv table contains the essential and available LUCAS EXIF information (27 variables) for all the photos 2006, 2009, 2012, 2015, 2018. While the observation location is recorded by the surveyor during the LUCAS field survey (gps_lon, gps_lat), the digital cameras with GPS could also capture the location where the photos were taken as well as the orientation, i.e. the azimuth angle. In the first surveys, the digital camera and the GPS were separate devices. The orientation was determined with a traditional compass. The data were used to cross-validate the geo-location reported during the survey. To assess the availability of this information, the EXIF information of the 5440459 photos was retrieved. As summarised in the two last columns of Table 6, the photos with geo-location information have increased considerably through time, i.e. 0% in 2006, 5.4% in 2009, 34.2% in 2012, 68.5% in 2015 and finally 72.9% in 2018. For azimuth angle, there is no information on orientation for the photos taken in 2006 and 2009. However, respectively 15.3%, 22%, and 6.7% of the photos have EXIF orientation information for 2012, 2015, and 2018.ih Each point surveyed has potentially five photos (N, E, S, W, P) per surveyed year (Fig. 5(a)). The EXIF table database is a table of records, corresponding to the photos taken in the cardinal orientations plus the point for each one of the points for the five surveys. The table holds information on the point ID, year of survey, path to the full resolution image and an wide variety of EXIF attributes, including coordinates, orientation, camera model, exact time and date, Eurostat metadata, etc. www.nature.com/scientificdata/ It was decided that having this information in a separate table is more sensible in terms of storage size and accessibility, whereby cross-table checks can easily be performed by executing joins between the tables based on point ID and year of survey. By combining this information from the two tables (i.e. the multi-year harmonised LUCAS survey database and the EXIF table database) one arrives at a significantly large set of labeled examples, corresponding to images of the 77 different types of recorded land cover.The background RGB imagery for (c) and (d) is obtained from “Map data ©2019 Google”. Storage. Among the data provided with the current study described in the previous section, the multi-year harmonised LUCAS survey database contains the five LUCAS surveys, i.e. a total of 1351293 observations that have been made at 651676 unique locations Overview of multi-year harmonised LUCAS survey database. Among the data provided with the current study described in the previous section, the multi-year harmonised LUCAS survey database contains the five LUCAS surveys, i.e. a total of 1351293 observations that have been made at 651676 unique locations Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z 6 Table 4. Number of LUCAS points per country and per year. The total number of records is provided by year and also split according to the type of observation: In-situ (direct observation), In-situ PI (In-situ Photo- Interpreted if point is not accessible) or Office PI (Photo-Interpreted in the office and thus not in-situ). Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z Technical Validationhi The first part of this section briefly summarises the LUCAS field surveys quality check. The section then focuses on analyses carried out specifically to support the technical quality of the multi-year harmonised LUCAS data- base process. p The LUCAS surveyed observations are subject to detailed quality checks (see LUCAS metadata52 and the data quality control documents available for 200953, 201254, 201555). First, an automated quality check verifies the completeness and consistency after field collection. Second, all surveyed points are checked visually at the offices responsible for collection. Third, an independent quality controller interactively checks 33% of the points for accuracy and compliance against pre-defined quality requirements, including the first 20% observations for each surveyor, to prevent systematic errors during the early collection phase.hf y p y g y p The presented data consolidation effort seeks to enhance the quality of an existing product. Ensuring data quality by harmonisation throughout the years is thus essential. Data quality was ensured by taking into account validity, accuracy, completeness, consistency, and uniformity throughout data processing (Fig. 2): 7 Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z www.nature.com/scientificdata/ www.nature.com/scientificdata/ 2006 2009 2012 2015 2018 Total # AT — 4961 6469 8839 8840 29109 BE 2370 1804 2446 2899 3659 13178 BG — — 6641 7677 7678 21996 CY — — 1442 1726 2313 5481 CZ 5626 4662 5514 5712 5713 27227 DE 27507 21113 24939 26598 26777 126934 DK — 2540 3442 3665 3703 13350 EE — 2663 2200 2637 2665 10165 EL — 7758 7821 12521 12622 40722 ES 34489 29912 35377 50281 45314 195373 FI — 19895 13476 16116 16182 65669 FR 39070 32318 38324 48188 48215 206115 HR — — — 3532 4239 7771 HU 8422 5513 4637 5169 5514 29255 IE — 4164 3484 4907 4975 17530 IT 20291 17790 20985 28693 28294 116053 LT — 3860 3889 4505 4584 16838 LU 197 152 213 251 340 1153 LV — 3825 4420 5374 5376 18995 MT — — 79 79 79 237 NL 2916 2449 2237 2521 5011 15134 PL 24128 18487 21797 22980 23086 110478 PT — 5423 7332 9006 7168 28929 RO — — 14278 16720 16725 47723 SE — 26656 22420 26648 26709 102433 SI — 1203 1621 1923 1922 6669 SK 3385 2898 2455 2755 2898 14391 UK — 14438 12214 16803 17253 60708 NOT EU — 139 120 1418 — 1677 Total # records 168401 234623 270272 340143 337854 1351293 Total # countries 11 23 27 28 28 — In-situ # 155238 175029 243603 242823 215120 1031813 In-situ [%] 92.18 74.6 90.13 71.39 63.67 76.36 In-situ PI # 13163 59594 26669 25254 22894 147574 In-situ PI [%] 7.82 25.4 9.87 7.42 6.78 10.92 Office PI # — — — 71970 99803 171773 Office PI [%] — — — 21.16 29.54 12.71 Other # — — — 96 37 133 Other [%] — — — 0.03 0.01 0.01 Table 4 Number of LUCAS points per country and per year The total number of reco Table 4. Number of LUCAS points per country and per year. The total number of records is provided by year and also split according to the type of observation: In-situ (direct observation), In-situ PI (In-situ Photo- Interpreted if point is not accessible) or Office PI (Photo-Interpreted in the office and thus not in-situ). www.nature.com/scientificdata/ This is important for several reasons - firstly, it allows to ascertain the real distance between the point actu- ally surveyed and the point supposed to be surveyed, which is, in a sense, a proxy for the quality of the surveyed observation itself; secondly, it is an accuracy check of the surveyed distance between the theoretical point and the survey observation point, as collected by the surveyor, “as provided by the GPS (in m)” (column obs_dist), and the distance between the same points as calculated from the data (column th_gps_dist). It must be noted that for Frequency of point visits 1 2 3 4 5 LUCAS points (n) 332605 101052 91112 91807 35204 Table 5. Number of LUCAS points and visits. Frequency of point visits 1 2 3 4 5 LUCAS points (n) 332605 101052 91112 91807 35204 Table 5. Number of LUCAS points and visits. Frequency of point visits 1 2 3 4 5 LUCAS points (n) 332605 101052 91112 91807 35204 Table 5. Number of LUCAS points and visits. To further asses spatial accuracy of the data, the distance between the theoretical point from the LUCAS grid (th_long, th_lat), and the actual GPS measurement of the survey observation point (gps_lon, gps_lat) were com- pared. This is important for several reasons - firstly, it allows to ascertain the real distance between the point actu- ally surveyed and the point supposed to be surveyed, which is, in a sense, a proxy for the quality of the surveyed observation itself; secondly, it is an accuracy check of the surveyed distance between the theoretical point and the survey observation point, as collected by the surveyor, “as provided by the GPS (in m)” (column obs_dist), and the distance between the same points as calculated from the data (column th_gps_dist). It must be noted that for To further asses spatial accuracy of the data, the distance between the theoretical point from the LUCAS grid (th_long, th_lat), and the actual GPS measurement of the survey observation point (gps_lon, gps_lat) were com- pared. www.nature.com/scientificdata/ 9 Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z To further asses spatial accuracy of the data, the distance between the theoretical point from the LUCAS grid (th_long, th_lat), and the actual GPS measurement of the survey observation point (gps_lon, gps_lat) were com- pared. This is important for several reasons - firstly, it allows to ascertain the real distance between the point actu- ally surveyed and the point supposed to be surveyed, which is, in a sense, a proxy for the quality of the surveyed observation itself; secondly, it is an accuracy check of the surveyed distance between the theoretical point and the survey observation point, as collected by the surveyor, “as provided by the GPS (in m)” (column obs_dist), and the distance between the same points as calculated from the data (column th_gps_dist). It must be noted that for Fig. 3 Distribution of land cover classes in the multi-year harmonised LUCAS database. In cases where survey years are not present please orientate oneself with reference to adjacent classes of the same color. Counting for the distribution of each class begins at 2018 and ends with 2006 due to the relative abundance of 2018 in terms of classes compared to other years. Frequency of point visits 1 2 3 4 5 LUCAS points (n) 332605 101052 91112 91807 35204 Table 5. Number of LUCAS points and visits. Fig. 3 Distribution of land cover classes in the multi-year harmonised LUCAS database. In cases where survey years are not present please orientate oneself with reference to adjacent classes of the same color. Counting for the distribution of each class begins at 2018 and ends with 2006 due to the relative abundance of 2018 in terms of classes compared to other years. To further asses spatial accuracy of the data, the distance between the theoretical point from the LUCAS grid (th_long, th_lat), and the actual GPS measurement of the survey observation point (gps_lon, gps_lat) were com- pared. www.nature.com/scientificdata/ • Validity of the harmonised database was ensured via data type (for which information can be found in the record descriptor) and a unique constraint of a composite key (consisting of the point ID and year of survey). • Accuracy of the data relies on the source data for which the quality was assessed as described in the previous paragraphs. • Validity of the harmonised database was ensured via data type (for which information can be found in the record descriptor) and a unique constraint of a composite key (consisting of the point ID and year of survey). • Accuracy of the data relies on the source data for which the quality was assessed as described in the previous paragraphs. p g p • Completeness checking shows that since several variables have been added over the years, many missing val- ues exist. In such cases, fields were populated with null values. Consistency across surveys has been enhanced. All surveys were harmonised towards the 2018 survey. y y • Consistency of the presented dataset was internally ensured through running checks at various stage processing.f • Uniformity checks revealed that the geographical coordinates in columns th_long and th_lat show different locations between some survey years. In the interest of complete uniformity, it was decided to have the values of these variables hard coded from the LUCAS grid. Because the LUCAS grid is a non-changing feature of all LUCAS surveys, the location of each point remains the same throughout the years. Thus any discrepancy between the recorded theoretical location of a LUCAS point in the micro data and the grid must be corrected. This was done for all but 64 points from 2006 which where recorded on an inaccurate location and were thus removed from the grid. Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z 8 www.nature.com/scientificdata/ Fig. 3 Distribution of land cover classes in the multi-year harmonised LUCAS database. In cases where survey years are not present please orientate oneself with reference to adjacent classes of the same color. Counting for the distribution of each class begins at 2018 and ends with 2006 due to the relative abundance of 2018 in terms of classes compared to other years. www.nature.com/scientificdata/ This is important for several reasons - firstly, it allows to ascertain the real distance between the point actu- ally surveyed and the point supposed to be surveyed, which is, in a sense, a proxy for the quality of the surveyed observation itself; secondly, it is an accuracy check of the surveyed distance between the theoretical point and the survey observation point, as collected by the surveyor, “as provided by the GPS (in m)” (column obs_dist), and the distance between the same points as calculated from the data (column th_gps_dist). It must be noted that for Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z 9 www.nature.com/scientificdata/ Fig. 4 Number of visits to each LUCAS survey point over the five surveys between 2006 and points were at least surveyed once. Visit ranges from one to five. Fig. 4 Number of visits to each LUCAS survey point over the five surveys between 2006 and 2018, 651780 points were at least surveyed once. Visit ranges from one to five. Year East North Point South West TOTAL Location [%] Orientation [%] 2006 137461 137426 134538 137368 137179 683972 0 0 2009 199208 199264 171165 199129 199117 967883 5.4 0 2012 269329 269286 243074 269277 269205 1320171 34.2 15.3 2015 265421 265392 242772 265368 265285 1304238 68.5 22 2018 237259 237529 215190 237262 236955 1164195 72.9 6.7 Total 1108678 1108897 1006739 1108404 1107741 5440459 Table 6. Number of LUCAS photos per year, per type (N, E, S, W, P) with proportions that have EXIF geo- location (Location [%]) and orientation information (Orientation [%]). Table 6. Number of LUCAS photos per year, per type (N, E, S, W, P) with proportions that have EXIF geo- location (Location [%]) and orientation information (Orientation [%]). Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z 10 www.nature.com/scientificdata/ Fig. 5 Overview of the data available for a LUCAS point that was visited five times: (a) Point, North, East, South and West photos for 2006, 2009, 2012, 2015 and 2018, (b) Location of the point in the EU, (c) Zoom showing the point (3-m diameter in green, 50-m diameter in dashed red), (d) Visit frequency on a 20 by 20 km square centered on the point, and (e) In-situ land cover observation of the point for the different years. Fig. www.nature.com/scientificdata/ 5 Overview of the data available for a LUCAS point that was visited five times: (a) Point, North, East, South and West photos for 2006, 2009, 2012, 2015 and 2018, (b) Location of the point in the EU, (c) Zoom showing the point (3-m diameter in green, 50-m diameter in dashed red), (d) Visit frequency on a 20 by 20 km square centered on the point, and (e) In-situ land cover observation of the point for the different years. the 2006 survey the variable obs_dist was collected as a range, whereas for the other years it represents the actual value of the distance. Because of this lack of uniformity, it was decided to hard code the values for 2006 to match exactly with the calculated distance. In this way we ensure consistency in the data type of the column, yet sacrifice the nuances from changing the original data. The procedure explains that, in 2006, we see a 100% match between recorded and calculated distance (Table 7), whereby for 2009 a match of 96.3%, meaning that for only 3.7% of the cases did the value not match. In carrying out this comparison it became apparent that the percentage of match- ing distances has increased throughout years probably due to better precision of positioning sensors. Thus the total amount of error in 2018 is reduced to a negligible 0.31%. Furthermore, the comparison was instrumental in the flagging and removing of a number of records that have inaccurate GPS coordinates most probably due to sensor malfunction. Cross-checking with the source data, we found that the error is indeed present in the source data, rather than introduced during processing - something which would have been hard to spot otherwise. The distribution of these calculated distances, alongside an equivalent distribution of the surveyed distances, can be found in Fig. 6. The distance between 75% of the points (1–3 quantile) is between 1.1 and 21.2 meters, meaning that only a fourth of the points have a distance greater than this. For the surveyed distances the ranges are similar the 2006 survey the variable obs_dist was collected as a range, whereas for the other years it represents the actual value of the distance. Because of this lack of uniformity, it was decided to hard code the values for 2006 to match exactly with the calculated distance. www.nature.com/scientificdata/ In this way we ensure consistency in the data type of the column, yet sacrifice the nuances from changing the original data. The procedure explains that, in 2006, we see a 100% match between recorded and calculated distance (Table 7), whereby for 2009 a match of 96.3%, meaning that for only 3.7% of the cases did the value not match. In carrying out this comparison it became apparent that the percentage of match- ing distances has increased throughout years probably due to better precision of positioning sensors. Thus the total amount of error in 2018 is reduced to a negligible 0.31%. Furthermore, the comparison was instrumental in the flagging and removing of a number of records that have inaccurate GPS coordinates most probably due to sensor malfunction. Cross-checking with the source data, we found that the error is indeed present in the source data, rather than introduced during processing - something which would have been hard to spot otherwise. The distribution of these calculated distances, alongside an equivalent distribution of the surveyed distances, can be found in Fig. 6. The distance between 75% of the points (1–3 quantile) is between 1.1 and 21.2 meters, meaning that only a fourth of the points have a distance greater than this. For the surveyed distances the ranges are similar Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z 11 www.nature.com/scientificdata/ 2006 2009 2012 2015 2018 Match 100.00 96.32 97.92 99.08 99.77 No match 0.00 3.68 2.08 0.92 0.23 Table 7. Percentage (%) of points for which the distances between the theoretical point from the LUCAS grid (th_long, th_lat) and the actual GPS measurement (gps_lon,gps_lat) taken during surveying and calculated post factum match or not. Table 7. Percentage (%) of points for which the distances between the theoretical point from the LUCAS grid (th_long, th_lat) and the actual GPS measurement (gps_lon,gps_lat) taken during surveying and calculated post factum match or not. Table 7. Percentage (%) of points for which the distances between the theoretical point from the LUCAS grid (th_long, th_lat) and the actual GPS measurement (gps_lon,gps_lat) taken during surveying and calculated post factum match or not. Fig. 6 Comparison of distributions between (a) calculated distances and (b) surveyed distances between LUCAS theoretical points and actual GPS position of surveyor. The red-colored part of the distribution in subfigure (b) represents the data from 2006, which is copied from the calculated distances (th_gps_dist). Fig. www.nature.com/scientificdata/ 6 Comparison of distributions between (a) calculated distances and (b) surveyed distances between LUCAS theoretical points and actual GPS position of surveyor. The red-colored part of the distribution in subfigure (b) represents the data from 2006, which is copied from the calculated distances (th_gps_dist). Fig. 7 Stability of points and location change over time as illustrated: (a) Example of a surveyed point (id 40402278) at close distance (<2 m) and (b) Example of a surveyed point (id 63861648) at large distance (1938m). Location change can be either because of survey conditions, the accessibility of the terrain, and/or accuracy of GPS positioning. The background RGB imagery is obtained from “Map data ©2019 Google”. Fig. 7 Stability of points and location change over time as illustrated: (a) Example of a surveyed point (id 40402278) at close distance (<2 m) and (b) Example of a surveyed point (id 63861648) at large distance (1938m). Location change can be either because of survey conditions, the accessibility of the terrain, and/or accuracy of GPS positioning. The background RGB imagery is obtained from “Map data ©2019 Google”. - 75% of the values fall between 1.0 and 30.0 meters. From the distributions we see that there is a lot more nuance in the values of the calculated distances, which makes sense as they are represented by numbers with decimals, which have a lower frequency than the integers, representing the surveyed distances. The values shown in the red part of the histogram of surveyed distances represent the values from 2006, which are copied from the calculated distance in order to hard code a numerical in the place of the categorical value of the variable in the source data. The theoretical grid of LUCAS point location is stable over time. However, according to the survey conditions and the terrain and accuracy of the GPS positioning, the surveyor may not be able to reach the point. This results in effective variations of the position of the observer through time (Fig. 7). f g g In addition to the theoretical grid and survey point location, this data descriptor provides the East-facing transect geo-location data. No additional geo-located spatial information is collected in the transect module and this is probably a shortcoming in the survey design resulting from trade-offs between the cost of the survey and its objectives. www.nature.com/scientificdata/ The theoretical transect line (with the same geometry as the one provided with this data descriptor) is displayed on the ground document of the surveyor. The surveyor has then to walk on the line and to record the successive land cover and landscape elements as described in the survey methodology. The only geo-location accuracy information relevant for the transect module is thus the same as presented previously, i.e. distance between the theoretical point and the GPS measured surveyed point. Then the successive land covers and land- scapes surveyed along the 250-m line are collected as a sequence without distance or geo-located information. Usage Notes g To summarize, the work documented in this data descriptor consists of49: (1) Multi-year harmonised LUCAS table, (2) Archive with high resolution LUCAS photos, (3) LUCAS survey geometries and point locations, (4) R package51, (5) Data descriptor of resulting database and (6) a Documentation table for users to quickly identify the differences of collected data between LUCAS campaigns micro-data and harmonised database. Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z 12 www.nature.com/scientificdata/ The harmonised LUCAS product reduces the complexity and layered nature of the original LUCAS datasets. In doing so, it valorizes the effort of many surveyors, data cleaners, statisticians, and database maintainers. The database’s novelty lies in the fact that for the first time, users can query the whole LUCAS archive concurrently, allowing for comparisons and combinations between all variables collected during the relevant reference years. The homogeneity of the product facilitates the unearthing of temporal and spatial relations that were otherwise jeopardized by the physical separation between survey results. Moreover, by avoiding the burden of combing through the cumbersome documentation, the user is now free to concentrate on the research, thereby facilitating scientific discovery and analysis. Naturally, the product suffers from the shortcomings inherent in the source data, such as any inadequate surveying, surveyor or technology-related errors of precision while taking coordinates or measurements, etc. The harmonisation process itself also reveals some inconsistencies in the source data. For instance, certain variables could not be harmonised between survey years. These are mostly related to measure- ments of percentage or extent of coverage. Where in the early stages of LUCAS surveyors were asked to fill in a multiple choice questionnaire, listing a range of values, in subsequent surveys the surveyor was asked to fill in the actual value in quantified units. www.nature.com/scientificdata/ This situation applies mostly, though not exclusively, to the 2006 survey, which makes it impossible for these variables to be translated into the user friendly version; therefore in these cases the variables of 2006 must remain in their original coding. Additional information can be found in the comments section of the record descriptor. p Another shortcoming is the change of hierarchy of the LUCAS classification system between the different sur- veys, mainly concerning LC/LU, as well as LC and LU types. A table is provided to document this shortcoming (see special remarks in the Table (“LC (LU) changes” in the file LUCAS-Variable_and_Classification _Changes.xlsx49). Code availability T y To guarantee transparency and reproducibility, the harmonisation workflow was carried out with open-source tools, namely PostgreSQL (9.5.17)/PostGIS (2.1.8 r13775)) and R (3.4.3)56). The code is provided as a R package containing 17 functions along with the documentation on51. The LUCAS package includes all the scripts and documentation (also provided in pdf). Additionally, along with the package, a script (main.R) builds the harmonised database step by step. The workflow is schematically shown in Fig. 2. All the processing is done with SQL with only column reordering and consistency checks being done in R. The code is freely available under GPL (> = 3) license. Received: 13 May 2020; Accepted: 9 September 2020; Received: 13 May 2020; Accepted: 9 September 2020; Published: xx xx xxxx References 1. Johnson, D. M. Using the Landsat archive to map crop cover history across the United States. 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Harmonised LUCAS in-situ land cover and use database for field surveys from 2006 to 2018 in the European Union. figshare https://doi.org/10.6084/m9.figshare.9962765.v2 (2020). 50. d’Andrimont, R. et al. Harmonised LUCAS in-situ land cover and use database for field surveys from 2006 to 2018 in the European Union. figshare https://doi.org/10.6084/m9.figshare.9962765.v2 (2020). Union. figshare https://doi.org/10.6084/m9.figshare.9962765.v2 ( fig p gi g ( ) 51. Yordanov, M., Martinez, L. & d’Andrimont, R. LUCAS R PAckage. CRAN repository, https://cran.r-project.org/web/packages/lucas/ index.html (2020). fig p gi g ( ) 51. Yordanov, M., Martinez, L. & d’Andrimont, R. LUCAS R PAckage. CRAN repository, https://cran.r-project.org/web/packages/lucas/ index.html (2020). fig p gi g 51. Yordanov, M., Martinez, L. & d’Andrimont, R. LUCAS R PAckage. CRAN repository, https://cran.r-project.org/web/packages/l index.html (2020). ( ) 52. ESTAT. 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Competing interestsh p g The authors declare no competing interests. www.nature.com/scientificdata/ www.nature.com/scientificdata/ Acknowledgementsh The authors would like to acknowledge the many surveyors, quality controllers, and support personal who have been carrying out the LUCAS survey. Additionally, we would like to thank members of the Eurostat LUCAS team (M. Kasanko, M. Fritz, H. Ramos, P. Jaques, C. Wirtz, L. Martino,…) who have been instrumental over the years in implementing LUCAS at various stages. The authors would also like to thank N. Elvekjaer for her precious comments on the manuscript. The authors would also like to thank the Google Earth Engine team for their support in the harmonisation process, in particular, D. Sherwood, S. Ilyushchenko and T. Erickson. The authors would also like to thank the JRC Big Data Platform (JEODPP) for the support provided. Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z 14 Author contributions Author contributions B.E., A.P., P.D. are responsible of the LUCAS data collection. M.Y., R.D., G.L., L.M.-S. and M.v.d.V. processed and analyzed the data. H.I.R. provides a storage solution to distribute the photos. C. J. reviewed the DB and made the documentation table. R.D., M.Y., G.L., B.E., A.P., P.D., J.G., H.I.R., L.M.-S., M.v.d.V. wrote the paper, provided comments and suggestions on the manuscript. B.E., A.P., P.D. are responsible of the LUCAS data collection. M.Y., R.D., G.L., L.M.-S. and M.v.d.V. processed and analyzed the data. H.I.R. provides a storage solution to distribute the photos. C. J. reviewed the DB and made the documentation table. R.D., M.Y., G.L., B.E., A.P., P.D., J.G., H.I.R., L.M.-S., M.v.d.V. wrote the paper, provided comments and suggestions on the manuscript. Additional information d d f Correspondence and requests for materials should be addressed to R.d. or M.v. 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 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 Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted 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 license, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the metadata files associated with this article. © The Author(s) 2020 Scientific Data \| (2020) 7:352 \| https://doi.org/10.1038/s41597-020-00675-z 15
https://openalex.org/W2972622391	http://uu.diva-portal.org/smash/get/diva2:1373199/FULLTEXT01	English	null	Celiac disease and complement activation in response to Streptococcus pneumoniae	European journal of pediatrics	2,019	cc-by	5,628	Celiac disease and complement activation in response to Streptococcus pneumoniae Anna Röckert Tjernberg1,2 & Hanna Woksepp3 & Kerstin Sandholm4 & Marcus Johansson5,6 & Charlotte Dahle6,7 & Jonas F Ludvigsson8,9 & Jonas Bonnedahl6 & Per Nilsson4,10 & Kristina Nilsson Ekdahl4,11 Received: 11 June 2019 /Revised: 16 September 2019 /Accepted: 26 September 2019 # The Author(s) 2019 European Journal of Pediatrics https://doi.org/10.1007/s00431-019-03490-w European Journal of Pediatrics https://doi.org/10.1007/s00431-019-03490-w ORIGINAL ARTICLE Abstract Individuals with celiac disease (CD) are at increased risk of invasive pneumococcal disease (IPD). The aim of this study was to explore whether the complement response to Streptococcus pneumoniae differed according to CD status, and could serve as an explanation for the excess risk of IPD in CD. Twenty-two children with CD and 18 controls, born 1999–2008, were included at Kalmar County Hospital, Sweden. The degree of complement activation was evaluated by comparing levels of activation products C3a and sC5b-9 in plasma incubated for 30 min with Streptococcus pneumoniae and in non-incubated plasma. Complement analyses were performed with enzyme-linked immunosorbent assay (ELISA). Pneumococcal stimulation caused a statistically significant increase in C3a as well as sC5b-9 in both children with CD and controls but there was no difference in response between the groups. After incubation, C3a increased on average 4.6 times and sC5b-9 22 times in both the CD and the control group (p = 0.497 and p = 0.724 respectively). Conclusion: Complement response to Streptococcus pneumoniae seems to be similar in children with and without CD and is thus unlikely to contribute to the increased susceptibility to invasive pneumococcal disease in CD. What is Known: • An excess risk of pneumococcal infections has been demonstrated in individuals with celiac disease. • I f ti li ti d d h l i b t lt ti h i l i • An excess risk of pneumococcal infections has been demonstrated in individuals with celiac disease. • Infectious complications can depend on hyposplenism but alternative mechanisms are sparsely examine • Infectious complications can depend on hyposplenism but alternative mechanisms are sparsely examined. What is New: • Complement activation in response to Streptococcus pneumoniae was examined in children with and without celiac disease but no differences could be demonstrated. Keywords Coeliac . Pneumococcal . Infection . Innate immunity . MBL Per Nilsson and Kristina Nilsson Ekdahl contributed equally to this work Communicated by Peter de Winter * Anna Röckert Tjernberg anna.rockert@regionkalmar.se Hanna Woksepp hanna.woksepp@regionkalmar.se Kerstin Sandholm kerstin.sandholm@lnu.se Marcus Johansson marcus.johansson@regionkalmar.se Charlotte Dahle charlotte.dahle@regionostergotland.se Per Nilsson and Kristina Nilsson Ekdahl contributed equally to this work. Abstract Communicated by Peter de Winter Jonas F Ludvigsson jonasludvigsson@yahoo.com Jonas Bonnedahl jonas.bonnedahl@regionkalmar.se Per Nilsson per.h.nilsson@lnu.se Kristina Nilsson Ekdahl kristina.nilsson_ekdahl@igp.uu.se Extended author information available on the last page of the article Extended author information available on the last page of the article Extended author information available on the last page of the article Eur J Pediatr Abbreviations CD Celiac disease ELISA Enzyme-linked immunosorbent assay GFD Gluten-free diet HR Hazard ratio IPD Invasive pneumococcal disease MAC Membrane attack complex MBL Mannan-binding lectin tTG Antibodies against tissue transglutaminase lectin (MBL) and ficolins to microbes [1, 23, 24]. Complement deficiencies, including MBL deficiency, lead to an increased risk of pneumococcal infections or adverse out- come in IPD [24]. The knowledge of the complement sys- tem’s role in CD is limited. Some older studies have shown reduced levels of C3 and C4 [17, 27]; however, none of them has measured the levels of complement activation markers in response to a pathogen. Abbreviations CD Celiac disease ELISA Enzyme-linked immunosorbent assay GFD Gluten-free diet HR Hazard ratio IPD Invasive pneumococcal disease MAC Membrane attack complex MBL Mannan-binding lectin tTG Antibodies against tissue transglutaminase The aim of this study was to investigate whether the com- plement response to Streptococcus pneumoniae differed be- tween young individuals with and without CD. Material and methods Celiac disease (CD) is a chronic enteropathy affecting about 1% of the population worldwide [15]. The last decade, severe infections as complications of CD have received increased attention [14, 29]. In particular, pneumococcal infections have attracted interest and several studies, including our own [25], have shown an increased risk of invasive pneumococcal dis- ease (IPD) in CD (HRs = 1.4–2.5) [13, 25, 28]. Since IPD is a potentially life-threatening condition, these findings have re- sulted in changed guidelines for CD management [2]. In the UK, pneumococcal immunization is now recommended to individuals with CD [16]. CD is an autoimmune disease and is considered to be one of the most common disorders associ- ated with splenic hypofunction and atrophy [8], even though the most recent estimates are slightly lower than the first re- ports [6]. The spleen is crucial for the defense against encap- sulated bacteria and the elevated risk of IPD in CD has been attributed to this possible hyposplenism [7]. However, an im- paired splenic function is rarely seen in children [3] and more- over, it is believed to improve after introduction of a gluten- free diet (GFD) [4] whereas the excess risk of IPD seems to persist beyond one year of follow-up [13, 25, 28]. In the light of this, we deemed that alternative explanations for this in- creased susceptibility deserved further attention. CD Individuals with CD, born 1999–2008, were identified through computerized medical records and invited to partici- pate in the study through a study-specific letter. Patients with an upcoming visit in near-time were invited first. Patients with additional autoimmune diseases or ongoing infection were excluded. Controls The innate immunity is the first line of defense against pathogens, in which the complement system plays an impor- tant role [24]. The complement system is a complex network of proteins acting in a cascade-like manner [24]. Its key func- tions are the opsonization of the surface of the pathogen, ac- tivation and recruitment of circulating and tissue-resident in- flammatory cells, and the direct killing of the bacteria via formation of the membrane attack complex (MAC) [23, 24]. The complement system can be activated through different pathways (classical, lectin, or alternative) [23, 24]. Data from mice models have shown that complement protection against pneumococci is mainly mediated through the classical path- way which is activated by antigen-bound antibodies and by acute phase pentraxins. The alternative pathway is in general also activated but the response seems, in comparison, weaker. The lectin pathway, also important in the defense against pneumococci, is activated by binding of mannan-binding Individuals, born 1999–2008, visiting the Pediatric Clinic for other reasons than CD were invited to participate as controls. Individuals with autoimmune diseases or ongoing infection were excluded. Study participants All study participants were included at the Pediatric Clinic at Kalmar County Hospital, Sweden. The participants were born between 1999 and 2008. Since plasma levels of complement components as well as complement activation are independent of age and sex in this age category [5, 19], no matching was performed. We chose to include individuals born 2008 and earlier since pneumococcal immunization was included in the Swedish national vaccine program for children in 2009 [26]. Blood sampling and preparation of plasma Blood samples for analyses of complement activation prod- ucts (C3a and sC5b-9), C3, MBL, pneumococcal serology, and IgA antibodies against tissue transglutaminase (tTG) were collected from all study participants. Plasma-EDTA for com- plement analyses was centrifuged at 2500×g for 20 min and frozen at −70 °C, within 4 h from sampling. MBL was measured by sandwich ELISA using mouse mono- clonal antibody (clone HYB 131-01) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Analyses were per- formed at the Department of Clinical Immunology and Transfusion Medicine, Uppsala, Sweden. IgA antibodies against tissue transglutaminase IgA antibodies against tissue transglutaminase were analyzed with Thermo Fisher Scientific Phadia 250 (Thermo Fisher Scientific, Uppsala, Sweden) at the accredited hospital labo- ratory at Kalmar County Hospital. The method includes a screening for detecting IgA deficiency (EliA™Celikey® IgA on Phadia 250, Thermo Fisher Scientific, Uppsala, Sweden). In case of a low response, samples are further ana- lyzed on BN ProSpec (Siemens, Erlangen, Germany) to get an actual IgA level [12]. If IgA is ≤0.07 g/L, IgG antibodies against transglutaminase and IgG antibodies against deamidated gliadin peptide are measured. Statistics Variables were examined with normality tests (Kolmogorov- Smirnov, Shapiro-Wilk, and plots). Non-parametric tests (Mann-Whitney U test) were used for group comparisons. In addition, parametric tests (independent sample t test) were used when variables were normally distributed. To statistically verify that pneumococcal stimulation caused complement ac- tivation, the Wilcoxon signed-rank test was used. Spearman’s rho was used for analyzing correlations. Pneumococcal incubations in lepirudin plasma IgG antibodies against pneumococcal serotypes 19F, 23F, and 6B were quantified using ELISA meeting World Health Organization standard [21]. The analyses were performed at the Department of Clinical Immunology and Transfusion Medicine, Lund, Sweden. Prior to pneumococcal stimulation, the plasma anticoagulant EDTA was removed to allow for complement activation. Samples were spinned through Bio-Spin P-6 gel columns (Bio-Rad Laboratories AB, Solna, Sweden), saturated with veronal-buffered saline and lepirudin 50 μg/mL (Refludan®, Celgene, Windsor, UK) as previously described [9]. For pneu- mococcal stimulation, Streptococcus pneumoniae (serogroup 23F) isolated from a patient suffering from invasive infection was chosen. The isolate was retrieved from the Department of Clinical Microbiology, Kalmar County Hospital, Sweden. Pneumococcal stimulation was carried out by mixing 20 μL of S. pneumoniae, 108 CFU/mL in NaCl with 180 μL plasma. As control 20 μL 0.9% NaCl without bacteria was added. Incubation was done in polypropylene microtubes, 30 min in 37 water-bath. An additional control was included: 20 μL NaCl mixed with 180 μL plasma, without incubation. The reaction was stopped by adding 0.2 M EDTA (10 mM final concentration). Incubations were carried out as dupli- cates or triplicates, depending on sample volume. Samples were centrifuged at 4500×g for 5 min and 150 μL was frozen at −80 °C prior to complement analysis. Analysis of complement activation (C3a and sC5b-9) Complement activation was monitored as the generation of activation products C3a and sC5b-9 complexes measured in the plasma by employing enzyme-linked immunosorbent as- say (ELISA) with antibodies specific for neo-epitopes in C3a and C9 respectively, as previously described [18, 20]. For statistical analyses, we used the median of the triplicates and duplicates. The concentration was presented in μg/mL. Analyses and figures were performed using SPSS 24 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA). p values < 0.05 were considered statistically significant. C3 Clinical data All study participants filled out a questionnaire about diet, medication, autoimmune diseases, previous pneumonias/men- ingitis, pneumococcal vaccine, and known splenic affection. In addition, medical records were reviewed when there were uncertainties. Eur J Pediatr Total C3 was measured by nephelometry (Beckman Coulter Immage 800, Bromma, Sweden) using Immunochemistry Diagnostic C3 (Beckman). Analyses were performed at the Department of Clinical Immunology and Transfusion Medicine, Uppsala, Sweden. Ethics Total C3 was measured by nephelometry (Beckman Coulter Immage 800, Bromma, Sweden) using Immunochemistry Diagnostic C3 (Beckman). Analyses were performed at the Department of Clinical Immunology and Transfusion Medicine, Uppsala, Sweden. The study conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethical Review Board in Linköping, Sweden (2016/366/31). Written Eur J Pediatr Fig. 1 Distribution of C3a in individuals with CD and controls before and after pneumococcal stimulation. Bars representing mean and 95%CI. (−) Non-stimulated samples. (+) Stimulated samples informed consent was obtained from all participants and their guardians. Characteristics of study participants A total of 25 individuals with CD and 23 controls were in- cluded. However, during the process, 8 study participants had to be excluded: two individuals with CD and two controls due to bacterial contamination, one CD and one control due to too low bacterial concentration, and one control did, for unknown reason, not activate C3a and sC5b-9 (despite C3 level within normal range), in addition, plasma from one control was miss- ing. Accordingly, the final study population consisted of 22 individuals with CD and 18 controls. The median age was 15.4 (10.2–17.1) years in the CD group and 11.5 (9.3–17.4) years in the control group. The majority of the study partici- pants were female (CD 16/22 and controls 10/18). None of the participants had received pneumococcal vaccination. One CD patient claimed to have had more than one pneumonia but the diagnoses could not be verified. No recent (last month) pneu- monias or previous meningitis were reported. No participant had a known affection of the spleen or reported use of medi- cation that potentially could affect pneumococcal stimulation or complement activity. One of the CD patients had a suspected asthma diagnosis; otherwise, no comorbidities known to influence complement levels were reported or found when reviewing medical records. All but one of the CD pa- tients received their diagnosis after small intestinal biopsy showing findings compatible with CD. The remaining patient was diagnosed without biopsy as proposed by the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidelines from 2012 [10]. The mean duration of CD was 7.6 years (1.3–14.9). Out of the control individuals, 15/18 attended the hospital for undergoing mag- netic resonance imaging. Four of the controls reported having a relative with CD (responding rate 17/18). Fig. 1 Distribution of C3a in individuals with CD and controls before and after pneumococcal stimulation. Bars representing mean and 95%CI. (−) Non-stimulated samples. (+) Stimulated samples 4.7; range 2.3–6.7). Additional statistical analyses comparing C3a concentrations in non-stimulated and stimulated samples showed no significant differences between the groups (p = 0.415 and p = 0.786 respectively). C3 and C3a/C3 ratio Since C3a is dependent on the total C3 level, we also mea- sured C3 concentrations. The C3 values were normally dis- tributed and statistical testing showed no significant difference between the groups (p = 0.124) (Fig. 2). In addition, we ex- amined the ratio between C3a and C3 since this is reported to be a more sensitive indicator than C3a alone [30] (Fig. 3). No statistically significant differences between the study groups could be found. Three CD patients had C3 levels below the reference. Excluding these from the statistical analyses did not affect the results. Fig. 2 Distribution of C3 in individuals with CD and controls. Bars representing mean and 95%CI C3a Pneumococcal stimulation did cause a statistically significant increase in C3a levels in both the CD (p < 0.001) and the control group (p < 0.001) but the levels did not differ between the groups (p = 0.497) (Fig. 1). After incubation, C3a in- creased on average 4.6 times in both the CD and the control group (CD: median 4.4; range 1.6–11.4 and controls: median Pneumococcal stimulation did cause a statistically significant increase in C3a levels in both the CD (p < 0.001) and the control group (p < 0.001) but the levels did not differ between the groups (p = 0.497) (Fig. 1). After incubation, C3a in- creased on average 4.6 times in both the CD and the control group (CD: median 4.4; range 1.6–11.4 and controls: median Eur J Pediatr Fig. 3 Distribution of C3a/C3 ratio in individuals with CD and controls before and after pneumococcal stimulation. Bars representing mean and 95%CI. (−) Non-stimulated samples. (+) Stimulated samples sC5b-9 The majority of the study participants showed signs of previ- ous pneumococcal infection (IgG antibody level > 1 mg/L). In the CD group, the median IgG antibody concentration for pneumococcal serotype 19F was 3.10 (0.10 to 38.00) mg/L, for serotype 23F 2.00 (0.22 to > 26) mg/L and for serotype 6B 5.25 (0.29 to > 50) mg/L. The corresponding numbers for controls (n = 16, samples missing from two controls) were 4.25 (0.53 to 23) mg/L, 1.5 (0.34 to > 26) mg/L, and 3.50 (0.22 to > 50) mg/L respectively. Mann-Whitney U tests showed no statistically significant differences in distribution between the study groups in any of the serotypes (p = 0.535, p = 0.431, and p = 0.460 respectively). Antibody levels did not correlate to the degree of complement response in any of the serotypes. Pneumococcal stimulation was statistically significantly asso- ciated with an increase in sC5b-9 activity in both the CD (p < 0.001) and the control group (p < 0.001) but no significant difference was seen between the groups (p = 0.724) (Fig. 4). In the CD group, sC5b-9 increased on average 22 times (median 19; range 8–47), equal to the also 22-fold increase observed in the control group (median 20; range 5–39). Comparing the study groups with regard to sC5b-9 concentrations in non- stimulated and stimulated samples separately did not reveal any statistically significant differences (p = 0.881 and p = 0– 664 respectively). Fig. 4 Distribution of sC5b-9 in individuals with CD and controls before and after pneumococcal stimulation. Bars representing mean and 95%CI. (−) Non-stimulated samples. (+) Stimulated samples Concentration of Streptococcus pneumoniae The bacterial load was evaluated after each pneumococcal stimulation. CFU varied between 6.8 × 106 and > 5 × 108 (median 5 × 108). Statistical testing showed no difference in distribution of bacterial concentration between the CD group and controls (p = 0.757). IgA antibodies against tissue transglutaminase Two individuals in the CD group had a slightly elevated tTG (8 and 13 kIE/L respectively [ref < 7 kIE/L]). Excluding these individuals from statistical analyses did not change the results. None of the study participants had p-IgA < 0.07 g/L wherefore there was no need for assessment of alternative serological CD markers. Mannan-binding lectin The median concentration of MBL was 481.5 kU/L (7 to > 2000 kU/L) in the CD group and 492.5 kU/L in controls (102 to > 2000 kU/L). There was no statistically significant differ- ence between the study groups (p = 0.508). One study partic- ipant (CD individual) had an MBL concentration below the reference level (7 kU/L [ref > 40 kU/L]). The results from the statistical analyses did not change when this participant was removed from the data set. Fig. 3 Distribution of C3a/C3 ratio in individuals with CD and controls before and after pneumococcal stimulation. Bars representing mean and 95%CI. (−) Non-stimulated samples. (+) Stimulated samples Discussion The first reports of severe pneumococcal infections in individ- uals with CD were published in the 1980s [22]. The last de- cade, the association between CD and IPD has been examined more thoroughly [13, 25, 28] and recent international CD guidelines recommend pneumococcal vaccine in CD [2, 16]. Fig. 4 Distribution of sC5b-9 in individuals with CD and controls before and after pneumococcal stimulation. Bars representing mean and 95%CI. (−) Non-stimulated samples. (+) Stimulated samples Eur J Pediatr The complement system plays an important role in the im- mune system’s first line of defense against invading patho- gens, including Streptococcus pneumoniae [23] but also against other encapsulated bacteria (e.g., meningococci and Haemophilus Influenzae). These latter pathogens have been very sparsely examined in CD although some experts argue that vaccine against these bacteria should be offered in addi- tion to pneumococcal immunization. Even though comple- ment deficiency is not uncommon in children with recurrent IPD [11], to our knowledge, ours is the first study to investi- gate whether alterations in the complement system are linked to the increased risk of IPD in CD. We used plasma samples from children (9–18 years old) with and without CD to mea- sure complement activation, i.e., levels of complement activa- tion markers C3a and sC5b-9, after in vitro stimulation with pneumococci. Even though pneumococci might resist the MAC (C5b-9) [23], we chose to measure the soluble form of this complex (sC5b-9) in addition to C3a since they are the complement cascade’s terminal products [24] and accordingly reflect the function of the entire complement system. A com- plement response was evident in all individuals after incuba- tion with S. pneumoniae, but no differences in activation be- tween the CD group and controls could be demonstrated. The laboratory work was performed in several steps. Prior to stimulation, pilot tests were performed to estimate bacterial load and incubation time needed to induce com- plement activation in vitro. Initially lower concentrations of pneumococci (100, 1000, and 10,000 CFU/mL) were evaluated but these failed to activate the complement and a concentration of 108 CFU/mL was required. High bacte- rial loads are commonly required to activate complement in vitro. Even though this could be considered a limit, it is unlikely to affect group comparisons. Discussion Incubation times of 15, 30, and 60 min were evaluated and since there was no difference in complement response between 30 and 60 min, 30 min was chosen. Another limitation of the study is that not all samples could be analyzed simultaneously. However, samples from both study groups were analyzed at each occasion and there was no statistically significant difference in bacterial load between groups. To further confirm that the methodological concept worked, we veri- fied that the increase in complement activity that we no- ticed in both study groups after pneumococcal stimulation was statistically significant. We cannot entirely exclude that the study was underpow- ered. The lack of previous research was of course one of the rationales for performing the study but naturally it hampered sample size calculations. What size a difference in comple- ment activation should have to be clinically significant is not known. However, considering the strikingly similar response between the groups, it is doubtful whether a larger study would have resulted in a different outcome. Besides assessing the increased susceptibility to infec- tions in CD from a totally new angle, one of the major strengths of the study was the availability of clinical data. All CD diagnoses were confirmed by reviewing relevant medical records. All CD cases but one were biopsy-verified. Theabsenceof concomitantautoimmunediseasescouldalso be confirmed. We also had access to vaccine data. The Swedish national vaccine register (Swevac) has a good cov- erage in the county where the study was performed. Likewise, medical records of control children were reviewed. This was done in addition to the questionnaire to confirm that the controls did not have any autoimmune dis- easesother thanCDorotherconditionsthatpotentiallycould affect complement activity, thereby avoiding selection bias. Sincethe studydidnot includeinfants,wedidnot requireage and sex matching between the study groups. The youngest patient in the study was 9 years old and by that age comple- mentlevelsandresponsehavereachedadultlevels[5,19].To further confirm this (and by that excluding selection bias in regardtosexandage),weanalyzedtotalC3andnodifference was found according to CD status. The mean duration of CD was 7.6 years and all patients claimed to adhere to a GFD which makes ongoing intestinal inflammation unlikely. However, two CD patients had a slightly elevated IgA tTG. They had only been diagnosed with CD 1.3 and 1.8 years ago respectively and it is therefore possible that they had not yet acquired complete mucosal recovery. Authors’ contributions ICMJE criteria for authorship read and met: ART, HW, KS, MJ, CD, JFL, JB, PN, KNE Discussion To examine if these patients somehow influenced our results, we also performed all statistical tests after excluding them from the analyses but this did not affect the results. As a secondary finding, we noticed that the majority of the study participants, despite being unvaccinated, showed immu- nity to all the three investigated pneumococcal serotypes. This most likely supports studies showing that most individuals are exposed to and infected by pneumococci during childhood [23]. The antibody levels were equally distributed between individuals with and without CD so we found nothing that indicated differences in antibody response to pneumococci with regard to CD status. In conclusion, this study tested a novel idea and even though no differences in complement response to pneumococ- ci were found between CD patients and controls, this is an important pilot study which hopefully can be followed by further studies investigating involvement of complement as well as other immune mechanisms (e.g., IgG2 levels) that in addition to hyposplenism could contribute to the increased susceptibility to infectious complications in CD. Acknowledgments The authors thank Ivar Tjernberg for valuable ideas prior to study start. The authors would also like to thank the staff at the Pediatric Clinic at Kalmar County Hospital for including study participants. Eur J Pediatr Agree with the manuscript’s results and conclusions: ART, HW, KS, MJ, CD, JFL, JB, PN, KNE 6. Di Sabatino A, Rosado MM, Cazzola P, Riboni R, Biagi F, Carsetti R, Corazza GR (2006) Splenic hypofunction and the spectrum of autoimmune and malignant complications in celiac disease. Clin Gastroenterol Hepatol 4(2):179–186 6. Di Sabatino A, Rosado MM, Cazzola P, Riboni R, Biagi F, Carsetti R, Corazza GR (2006) Splenic hypofunction and the spectrum of autoimmune and malignant complications in celiac disease. Clin Gastroenterol Hepatol 4(2):179–186 MJ, CD, JFL, JB, PN, KNE Designed the experiments/the study: ART, JB, KNE, HW, KS, PN, MJ Collected data: ART Analyzed the data: ART Wrote the first draft of the paper: ART Contributed to study design, interpretation of data, and writing: ART, Analyzed the data: ART 7. Di Sabatino A, Carsetti R, Corazza GR (2011) Post-splenectomy and hyposplenic states. Lancet 378(9785):86–97. https://doi.org/ 10.1016/S0140-6736(10)61493-6 Wrote the first draft of the paper: ART Contributed to study design, interpretation of data, and writing: ART, 8. Di Sabatino A, Brunetti L, Carnevale Maffe G, Giuffrida P, Corazza GR (2013) Is it worth investigating splenic function in patients with celiac disease? Discussion World J Gastroenterol 19(15):2313–2318. https:// doi.org/10.3748/wjg.v19.i15.2313 HW, KS, MJ, CD, JFL, JB, PN, KNE Interpretation of data; approved the final version of the manuscript: ART, HW, KS, MJ, CD, JFL, JB, PN, KNE Responsible for data integrity: ART Obtained funding: ART, JB, KNE 9. Ekdahl KN, Norberg D, Bengtsson AA, Sturfelt G, Nilsson UR, Nilsson B (2007) Use of serum or buffer-changed EDTA-plasma in a rapid, inexpensive, and easy-to-perform hemolytic complement assay for differential diagnosis of systemic lupus erythematosus and monitoring of patients with the disease. Clin Vaccine Immunol 14(5):549–555. https://doi.org/10.1128/CVI.00486-06 Funding information Open access funding provided by Örebro University. The study was mainly funded by the Medical Research Council of Southeast Sweden (658741). ART, HW, MJ, and JB were supported by Region Kalmar County. JFL, KNE, and PN were supported by the Swedish Research Council (522-2A09-195), (2016-2075-5.1), and (2018-04087). JFL was supported by the Swedish Celiac Society and Fulbright Commission. KS, KNE, and PN were supported by faculty grants from Linnaeus University. PN was supported by the Norwegian Research Council (274332). 10. Husby S, Koletzko S, Korponay-Szabo IR, Mearin ML, Phillips A, Shamir R, Troncone R, Giersiepen K, Branski D, Catassi C, Lelgeman M, Maki M, Ribes-Koninckx C, Ventura A, Zimmer KP, EWGoCD D, Committee EG, European Society for Pediatric Gastroenterology H, Nutrition (2012) European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr 54(1):136–160. https://doi.org/10.1097/MPG.0b013e31821a23d0 Compliance with ethical standards 11. Ingels H, Schejbel L, Lundstedt AC, Jensen L, Laursen IA, Ryder LP, Heegaard NH, Konradsen H, Christensen JJ, Heilmann C, Marquart HV (2015) Immunodeficiency among children with re- current invasive pneumococcal disease. Pediatr Infect Dis J 34(6): 644–651. https://doi.org/10.1097/INF.0000000000000701 Conflict of interest The authors declare that they have no conflicts of interest. Ethical approval All procedures performed in studies involving hu- man participants were in accordance with the ethical standards of the Ethical Review Board in Linköping, Sweden (2016/366/31), and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. 12. Lowbeer C, Wallinder H (2010) Undetectable anti-tissue transglutaminase IgA antibody measured with EliA Celikey indi- cates selective IgA deficiency. Clin Chim Acta 411(7-8):612. https://doi.org/10.1016/j.cca.2010.01.020 13. 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Indian J Pediatr 72(9):745– 749 Anna Röckert Tjernberg1,2 & Hanna Woksepp3 & Kerstin Sandholm4 & Marcus Johansson5,6 & Charlotte Dahle6,7 & Jonas F Ludvigsson8,9 & Jonas Bonnedahl6 & Per Nilsson4,10 & Kristina Nilsson Ekdahl4,11 Anna Röckert Tjernberg1,2 & Hanna Woksepp3 & Kerstin Sandholm4 & Marcus Johansson5,6 & Charlotte Dahle6,7 & Jonas F Ludvigsson8,9 & Jonas Bonnedahl6 & Per Nilsson4,10 & Kristina Nilsson Ekdahl4,11 1 Department of Pediatrics, Kalmar County Hospital, SE-391 85 Kalmar, Sweden 7 Department of Clinical Immunology and Transfusion Medicine, Linköping University Hospital, SE-581 85 Linköping, Sweden 2 School of Medical Sciences, Örebro University, SE-701 82 Örebro, Sweden 8 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, SE-171 77 Stockholm, Sweden 8 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, SE-171 77 Stockholm, Sweden 3 Research section, Department of Development and Public Health, Kalmar County Hospital, SE-391 85 Kalmar, Sweden 9 Departme 85 Örebro 3 Research section, Department of Development and Public Health, Kalmar County Hospital, SE-391 85 Kalmar, Sweden 9 Department of Pediatrics, Örebro University Hospital, SE-701 85 Örebro, Sweden 9 Department of Pediatrics, Örebro University Hospital, SE-701 85 Örebro, Sweden 4 Linnaeus Center for Biomaterials Chemistry, Linnaeus University, SE-391 82 Kalmar, Sweden 10 Department of Immunology, Oslo University Hospital, University of Oslo, 0424 Oslo, Norway 10 Department of Immunology, Oslo University Hospital, University of Oslo, 0424 Oslo, Norway 5 Department of Clinical Microbiology, Kalmar County Hospital, SE- 391 85 Kalmar, Sweden 11 Department of Immunology, Genetics and Pathology, Uppsala University, SE-751 85 Uppsala, Sweden 6 Department of Clinical and Experimental Medicine, Linköping University, SE-581 85 Linköping, Sweden 6 Department of Clinical and Experimental Medicine, Linköping University, SE-581 85 Linköping, Sweden
W2884863995.txt	https://journals.univie.ac.at/index.php/voebm/article/download/2113/1691	de		Neue Anforderungen – viele offene Fragen. Zu den vielfältigen Rollen von Repositorien am Beispiel der UB Wien	Mitteilungen der Vereinigung Österreichischer Bibliothekarinnen & Bibliothekare	2,018	cc-by	3,977	 NEUE ANFORDERUNGEN – VIELE OFFENE FRAGEN. ZU DEN VIELFÄLTIGEN ROLLEN VON REPOSITORIEN AM BEISPIEL DER UB WIEN von Susanne Blumesberger Zusammenfassnung: Der Aufbau und Betrieb von Repositorien stellt wissenschaftliche Bibliotheken vor komplexe Herausforderungen. Zahlreiche technische, juristische und ethische Fragen sind ungelöst und können nur in einer engen Zusammenarbeit mit einer Vielzahl an ExpertInnen aus unterschiedlichen Fachrichtungen beantwortet werden. Die größte Aufgabe und zugleich eine wichtige Zukunftsperspektive für Bibliotheken besteht darin, sich rasch auf ändernde technische Gegebenheiten, Publikationskulturen und wissenschaftliche Arbeitsabläufe einzustellen und den Forschenden Tools anzubieten, die nicht nur das bloße Ablegen von Daten ermöglichen sondern weit darüber hinaus den gesamten Forschungslebenszyklus optimal unterstützen. Schlüsselwörter: Repositorien; Forschungsdaten; wissenschaftliche Bibliotheken; Forschungsdatenlebenszyklus; Datenmanagement NEW REQUIREMENTS – MANY OPEN QUESTIONS. ON THE DIVERSE ROLES OF REPOSITORIES USING THE EXAMPLE OF THE VIENNA UNIVERSITY LIBRARY Abstract: The construction and operation of repositories presents academic libraries with complex challenges. Numerous technical, legal and ethical questions remain unresolved and can only be answered in close cooperation with a large number of experts from different disciplines. The biggest challenge, and at the same time an important future for libraries, is to be able to adapt quickly to changing technical conditions, publication cultures and scientific workflows, and to offer researchers tools that not only allow data to be stored, but also optimally cover the entire research life cycle support. Keywords: Repositories; research data; academic libraries; research data life cycle; data management DOI: http://doi.org/10.31263/voebm.v71i1.2003 Dieses Werk ist lizenziert unter einer Creative-Commons-Lizenz Namensnennung 4.0 International Mitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich 149 Inhalt 1. Einleitung 2. Repositorien – von der Datenarchivierung zum vielfältigen Instrumentarium 3. Das Chamäleon Repositorium? 4. Forschungsunterstützung morgen? 5. Die Archivierung von Forschungsdaten in der Zukunft Bibliotheken müssen ihr Geschäftsmodell radikal ändern. Wer das nicht tut, der wird in den nächsten 20 Jahren verschwinden.1 Rafael Ball 1. Einleitung In den letzten Jahren gab es einen unübersehbaren Wandel in den Bibliotheken, vor allem forschungsunterstützende Services und hier meist die langfristige Sicherung der Daten betreffend. Die Erwartungen und Anforderungen an Repositorien passten sich gemäß der veränderten Arbeitsgewohnheiten der WissenschaftlerInnen ebenfalls an. Eine Überlegung, die hier gestellt werden soll ist, inwieweit Repositorienbetreibende die sich ändernden Erwartungen und Forderungen berücksichtigen können und sollen. Einige Fragen, die sich in diesem Zusammenhang stellen werden: Wo braucht es technisch und nichttechnisch gesehen Kontinuität und Stabilität, wo sind ständige Anpassungen angebracht? Welche Infrastrukturen werden in Zukunft nötig sein um möglichst auf alle Bedürfnisse der Forschenden eingehen zu können? Auch auf inhaltlicher Ebene ist ein ständiger Diskurs wichtig. Die Frage des Zugangs zum Repositorium, beispielsweise die Entscheidung ob alle MitarbeiterInnen mit dem eigenen Account archivieren dürfen oder ob das Archivieren nur einer eingeschränkten Benutzergruppe möglich sein soll, muss ebenso hinterfragt und entschieden werden, wie die grundsätzliche Festlegung, wer bestimmen darf, was langfristig verfügbar gehalten werden soll? Denn davon leitet sich im Grunde genommen ab, wer heute das kulturelle Erbe von morgen definiert. Abgesehen von diesen Überlegungen stellt sich auf Datenebene stets die Frage, wie man einerseits den geforderten FAIR-Principles2 entsprechen kann, die Daten also auffindbar, zugänglich, austauchbar und wiederverwendbar hält und andererseits bewusst mit jenen Daten umgeht, die aus rechtlichen und/oder ethischen Gründen eines sehr reflektierten und vorsichtigen Umgangs bedürfen. Von diesem Spannungsfeld zwischen den Forderungen nach Open Science und Open Data auf der einen Seite und einer hohen 150 Mitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich Sensibilität gegenüber Daten auf der anderen Seite sind nicht nur WissenschaftlerInnen der unterschiedlichen Fachdisziplinen betroffen, sondern auch BibliothekarInnen, ArchivarInnen und Sammlungsbeauftragte, denn auch für diese Personengruppen sind institutionelle Repositorien wichtig. Bei sensiblen Daten, die evtl. eines eingeschränkten Zugangs, einer Pseudonymisierung, Anonymisierung und besonderen technischen Sicherheitsvorkehrungen bedürfen, tragen Repositorien eine hohe Verantwortung im Bereich Datenschutz und sind nicht nur in der Forschung unverzichtbar geworden, sondern übernehmen auch gesellschaftspolitische Aufgaben. 2. Repositorien – von der Datenarchivierung zum vielfältigen Instrumentarium Zehn Jahre bedeuten in der digitalen Welt eine lange Zeitspanne. Entsprechend den technischen Gegebenheiten – Handys waren noch nicht so smart wie heute und Tablets kaum verbreitet – gestalteten sich die Arbeits- und Publikationsabläufe von ForscherInnen noch etwas anders als im Jahr 2018. Digitalisierung und Retrodigitalisierung befanden sich im Aufschwung, man war als Forschender, vor allem aus den Geisteswissenschaften kommend, froh, schwer erreichbare Quellen plötzlich im Netz per Link rund um die Uhr auffinden und nutzen zu können. Ein Repositorium aufzubauen um als wissenschaftliche oder kulturelle Einrichtung selbst wertvolle Materialien weltweit digital anbieten zu können war mit einer gewissen Euphorie verbunden. Endlich hatte man die Infrastruktur zur Verfügung um bisher schwer zugängliche und deshalb kaum genutzte Schätze aus Bibliotheken und Archiven für alle Welt sichtbar und einfach nutzbar machen zu können. Zehn Jahre später gehen die meisten Forschenden selbstverständlich davon aus, dass sämtliche benötigte Quellen rund um die Uhr frei und ohne rechtliche Einschränkungen verfügbar im Netz sind und rasch kommentiert und mit anderen geteilt werden können. Mit der Etablierung von Repositorien erwartete man sich auch einen tiefgreifenden Wandel im Bereich des Publikationswesens. Hat man als WissenschaftlerIn Zugang zu einem Repositorium, kann man prinzipiell ohne Verlag rasch und sicher eigene Texte samt Begleitmaterialien aller Art weltweit verbreiten – ohne Zeitverlust durch Peer-Review-Prozesse und Drucklegung. Der Wegfall qualitätssichernder Maßnahmen wird heute jedoch sehr kritisch gesehen. Den Open Access Policies folgend wird empfohlen entweder die Publikation im Rahmen des goldenen Wegs, also über Mitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich 151 Open Access-Zeitschriften oder im Rahmen des grünen Wegs, in Form von Zweitveröffentlichungen bereits peer-reviewter und publizierter Artikel vorzunehmen. Während in den Natur- und Sozialwissenschaften schon seit längerer Zeit auf diese Weise publiziert wird, herrschen in den Geisteswissenschaften oft noch Printmedien, vor allem Monografien und Sammelbände, vor. Erst langsam wird auch hier ein Umdenken sichtbar. Einig sind sich die Forschenden bzgl. Repositorien auf jeden Fall in ihren Forderungen, dass das Hochladen eigener Publikationen und Daten sowie deren Beschreibung kaum Zeit in Anspruch nehmen darf und von allen Endgeräten aus jederzeit möglich sein soll. Archivierte Objekte müssen rasch und ohne weiteren Aufwand in unterschiedliche Systeme weiterverbreitet werden können. NutzerInnenzahlen, Downloadstatistiken für bibliometrische Auswertungen und diverse Evaluierungen müssen ebenfalls jederzeit problemlos abrufbar sein. Ausfälle des Systems – wenn auch noch so kurz – werden nicht mehr toleriert. Abgesehen von den WissenschaftlerInnen, sind auch jene Personengruppen zu berücksichtigen, die das Repositorium dazu verwenden, Sammlungsbestände, Archivbestände, Nachlässe, historische Bücher und Karten digital zur Verfügung zu stellen und damit wieder die Datengrundlage für weitere Forschungen nutzbar machen. Auch für sie muss das Repositorium, wenn es wie PHAIDRA3 universell für eine Institution eingesetzt wird, Anwendungsmöglichkeiten bieten, die mit dem ressourcenaufwändigen Prozess der Digitalisierung und Beschreibung der Daten, in Einklang stehen und gerechtfertigt sind. Dazu zählen geeignete Uploadtools, ein effizientes Metadatenmanagement und diverse Visualisierungsmöglichkeiten. Repositorien werden oft auch für die Archivierung und Publikation von Hochschulschriften verwendet. Daraus entsteht ein ganzes Bündel an Handlungsfeldern rund um den Betrieb eines Repositoriums. Kenntnisse des Forschungsdatenzyklus, im Bereich Open Science, Open Data, Open Access, über Lizenzen, ethische und rechtliche Gegebenheiten, über technische Schnittstellen, Fragen der Langzeitarchivierung, Metadaten, Sammlungsrichtlinien, diverse Policies usw. werden erwartet. Darüber hinaus spielt auch das Wissen um das institutionsinterne Zusammenspiel von Forschungsservices, Drittmittelstellen und die enge Zusammenarbeit mit IT-Services eine große Rolle. Auch die von den Fördergebern teilweise bereits eingeforderten Datenmanagementpläne verlangen Beratungen hinsichtlich der optimalen Vorbereitung der Daten für die Langzeitverfügbarkeit in Repositorien. All diese Aspekte zusammengenommen erfordern heute zugleich einen breiten Überblick über diverse Tätigkeitsfelder aber auch Spezialwissen in 152 Mitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich verschiedenen Bereichen, die sich zum Teil erst in den letzten Jahren entwickelt haben. Um diese Fragen gemeinsam diskutieren zu können, wurde unter anderem das Netzwerk RepositorienmanagerInnen4 gegründet, dem sich inzwischen zahlreiche VertreterInnen von Institutionen in ganz Österreich angeschlossen haben. 3. Das Chamäleon Repositorium? Als 2006 an der UB Wien eine Arbeitsgruppe gegründet wurde um die Implementierung eines digitalen Repositoriums vorzubereiten, gab es kaum Erfahrungswerte, die man hreanziehen konnte. Die Repositorienlandschaft war noch sehr dünn besiedelt, das Bewusstsein für digitale Langzeitarchivierung in den meisten Wissenschaften noch kaum vorhanden. Auch die technischen Möglichkeiten waren nicht sehr breit gefächert, kein fertiges Produkt schien richtig zu passen. Die Lösung, eine Open Source Software wie Fedora, heranzuziehen und nach den Bedürfnissen einer sehr großen und heterogenen Universität zu adaptieren, lag nahe. Als im April 2007 das zunächst für drei Jahre geplante Projekt „Digital Asset Management System“ startete, lag die Hauptaufgabe beim nichttechnischen Team an der Universitätsbibliothek Wien spätere NutzerInnen zu befragen, welche Aspekte dieses neuen Tools für sie wichtig seien. Gemeinsam mit VertreterInnen unterschiedlicher Fachrichtungen wurde ein Anforderungskatalog erstellt, der in enger Zusammenarbeit mit dem Zentralen Informatikdienst der Universität Wien abgestimmt und verfeinert wurde. Genau ein Jahr nach Projektstart ging PHAIDRA5, wie das System genannt wurde, in Betrieb. Damit war die Möglichkeit für sämtliche MitarbeiterInnen und Studierende gegeben, wertvolle digitale Inhalte weltweit und permanent zitierbar zur Verfügung zu stellen. Neben der ständigen Optimierung der Software und der Überarbeitung der Oberfläche, stand vor allem die Bewerbung des neuen Systems im Vordergrund. Eine Serviceseite wurde aufgebaut, die Vorteile einer langfristigen Sicherung von wertvollen Objekten angepriesen und Hilfestellung bei sämtlichen Archivierungsschritten angeboten, denn der Upload und die Beschreibung der Daten obliegen laut der Policy von PHAIDRA den NutzerInnen. Damit hatte die UB Wien im Bereich Repositorien eine Vorreiterrolle inne. Recht bald meldeten sich auch andere Universitäten, die auf der Suche nach Langzeitarchivierungssystemen waren und wurden Partner. 2010 schloss beispielsweise die Universität Padua einen Vertrag mit der UniMitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich 153 versität Wien. Das dortige System wird vor allem für die Präsentation von Sammlungen verwendet.6 Inzwischen betreut die Università di Padova auch die Università Ca‘ Foscari und die Università Iuav di Venezia mit. In den letzten Jahren sind zahlreiche Universitäten und Institutionen der PHAIDRA-Community beigetreten, unter anderem die FH St. Pölten, die Kunstuniversität Linz, die Universität für angewandte Kunst in Wien, Universitäten in Serbien, Bosnien und Herzegowina und Montenegro, die vor allem Abschlussarbeiten und wissenschaftliche Beiträge archivieren. Die Landesbibliothek Vorarlberg verwendet VOLARE7 vor allem um die Kultur Vorarlbergs digital zugänglich zu machen. Fotosammlungen, Ansichtskarten, Karten und vieles mehr werden übersichtlich und in hoher Auflösung für alle zugänglich angeboten. Der FWF betreibt seine E-Book Library8, die die geförderten Open Access Publikationen enthält, auf der Basis von PHAIDRA. Jüngste PHAIDRA-Partner sind die Donau-Universität Krems, die Anton Bruckner-Privatuniversität und die Veterinärmedizinische Universität Wien.9 In den letzten Jahren konnten an zahlreichen Universitäten und anderen Forschungseinrichtungen Repositorien und diverse Infrastrukturen aufgebaut werden. Dazu hat wesentlich das dreijährige Projekt „e-Infrastructures Austria“10, koordiniert von der UB Wien, beigetragen. 2014 vom Bundesministerium für Wissenschaft, Forschung und Wirtschaft (BMWFW) initiiert, sollte es österreichweit den koordinierten Aufbau sowie die Weiterentwicklung von Repositorieninfrastrukturen fördern. In den drei Jahren konnte viel Know-how in den Bereichen Open Access, Technik, rechtliche Fragen, Metadaten usw. aufgebaut werden. Es entstand ein umfassendes Netzwerk mit BibliothekarInnen, TechnikerInnen, ForscherInnen, Forschungsservices und anderen ExpertInnen sowie ein Wissenspool an dem alle partizipieren können. Das Nachfolgeprojekt „e-Infrastructures Austria Plus“ wird – koordiniert von der Universität Innsbruck – von 2017–2019 durchgeführt. Ziel ist es, eine Infrastruktur für eScience in Österreich aufzubauen. Es werden so unterschiedliche Themen wie RDM-Policies, Datenmanagementpläne, der Aufbau von institutionellen Repositorien für Forschungsdaten, Standards für Metadaten nach den FAIR Principles, Aufbau einer DOI-Infrastruktur und erste Schritte zur Implementierung von Electronic Lab Notebooks behandelt. Durch die Einbettung der Repositorien in die bereits bestehenden Infrastrukturen der jeweiligen Institutionen und durch die Ausweitung der Aufgaben von RepositorienbetreiberInnen entstanden neue Services, die sowohl institutionell verankert werden müssen, als auch einer großen Expertise der MitarbeiterInnen bedürfen. 154 Mitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich 4. Forschungsunterstützung morgen? Die beiden HRSM-Projekte bzw. die Erfahrungen der RepositorienmanagerInnen haben gezeigt, dass das bloße Bereitstellen von Repositorien nicht ausreicht, um WissenschaftlerInnen bei ihren Forschungen bzw. BibliothekarInnen, ArchivarInnen und Sammlungsbeauftragte zu unterstützen. Neben Schulungen und Workshops mit den unterschiedlichen Stakeholdern müssen zusätzlich auch weitere Dienste angeboten werden. Diese können individuell sehr unterschiedlich sein. Für Forschende zählen unter anderem auch sämtliche Fragen dazu, die auch in einem Datenmanagementplan zu finden sind, z.B. nach der Möglichkeit der Zwischenspeicherung von Materialien, nach den günstigsten Formaten, nach ethischen und rechtlichen Fragen, nach Kostenmodellen, nach der dauerhaften Abrufbarkeit von komplexen Objekten und beispielsweise Datenbanken. Aber auch technische Fragen, wie zum Beispiel nach Schnittstellen zu anderen Systemen werden immer wieder gestellt. Vor allem bei Forschungsprojekten sind inhaltliche Überlegungen rund um Metadaten und Metadatenschemata wichtig. Die Usability der Systeme hat ebenfalls einen großen Stelenwert. Dazu zählen der Prozess des raschen und sicheren Uploadens von Publikationen und Objekten und deren Verknüpfung. Auf strategischer Ebene ist es wichtig langfristig zu planen wie sich Repositorien in schon an den Institutionen bestehenden anderen Systemen eingliedern und optimal vernetzen lassen. Dazu kommen noch Fragen nach dem Marketing, nach der Positionierung des Repositoriums in den Institutionen bis zu Überlegungen, welche Ausbildung MitarbeiterInnen benötigen, wenn sie ein Repositorium mit all den oben beschriebenen Handlungsfeldern betreuen sollen. Diese Fragen variieren von Institution zu Institution, wobei alle RepositorienmanagerInnen immer wieder mit juristischen und ethischen Fragen konfrontiert sind. Eine der zentralen Fragen wird sein, welchen Stellenwert Repositorien in Zukunft für die Forschung haben werden, bzw. welche Funktionalitäten gebraucht werden. 5. Die Archivierung von Forschungsdaten in der Zukunft Forschungsdaten haben in der wissenschaftlichen Reputationsökonomie nur einen indirekten Wert. (Fecher 2018, 213) Im Sinne der European Open Science Cloud (EOSC) Declaration11 heißt es: Mitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich 155 „European science must be grounded in a common culture of data stewardship, so that research data is recognised as a significant output of research and is appropriately curated throughout and after the period conducting the research. Only a considerable cultural change will enable long-term reuse for science and for innovation of data created by research activities: no disciplines, institutions or countries must be left behind.“ (European Open Science Cloud Declaration, 1). Demnach müssen Daten per default Open Access sein. Großen Wert wird auf Research Datamanagement, Data Stewardship, Datenmanagementpläne und die Einhaltung der FAIR-Principles gelegt. Wie müssen Repositorien der Zukunft beschaffen sein um auf diese Anforderungen reagieren zu können? Aber auch weitere zukunftsweisende Überlegungen sind wichtig: Für welche Zwecke werden Repositorien zukünftig eingesetzt werden? Werden eher Fachrepositorien wichtig sein oder institutionelle Repositorien, die, wie PHAIDRA heute, offen für sämtliche Daten sind? Welche Rolle spielen Forschungsdaten in Zukunft. Werden diese wirklich wiederverwendet? Inwieweit lässt sich die Forderung nach Daten gemäß den FAIR-Principles realisieren? In vielen Forschungsbereichen, wie etwa der Medizin oder in der Ethnologie unterliegen die Forschungen juristischen und/oder ethischen Einschränkungen. Mit dem Appell zur Offenheit wird von Seiten der Förderer und politischen Entscheidungsträger an Digitalisierung ein großes Fortschrittsversprechen geknüpft, das bislang noch nicht eingelöst wird. Am Beispiel von Forschungsdaten wird dies überraschend deutlich. (Fecher 2018, 15) BefürworterInnen des Data Sharing begründen dies wie folgt: 1. Synergien und Spill-Over-Effekte, also Faktoren, die Forschung effizienter, effektiver und innovativer machen. 2. Überprüfbarkeit von Ergebnissen, also deren Replizierbarkeit und Nachvollziehbarkeit. 3. Austausch und Feedback, also die Vernetzung und Kollaboration von Wissenschaftlern“ (Fecher 2018, 118) Derzeit werden nur wenige Forschungsdaten so aufbereitet und archiviert, dass sie für die Nachnutzung in Frage kommen, wie die 2015 in Österreich durchgeführte Studie Forschende und ihre Daten gezeigt hat: Zugriff auf selbst generierte Forschungsdaten für Dritte ermöglichen Forschende in der Regel nur eingeschränkt. Während etwas mehr als die Hälfte der Befragten den Zugang nur auf Anfrage ermöglicht, stellt nur jede/jeder Zehnte seine Forschungsdaten als Open Data für die Öffentlichkeit zur Verfügung; ebenso viele verweigern den Zugriff ganz. (siehe Forschende und ihre Daten, 7) 156 Mitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich Jene, die ihre Daten zur Verfügung stellten, nutzten dafür kaum Repositorien: Zugriff auf Forschungsdaten wird von der Mehrheit der Forschenden entweder über physische Datenträger oder per E-Mail ermöglicht. Mehr als zwei Drittel der Forschenden setzen hierfür Cloud- oder Website-Anwendungen ein; Datenarchive/Repositorien werden nur von jeder/ jedem siebenten Forschenden genutzt (siehe Forschende und ihre Daten, 7) Der Umgang mit Forschungsdaten ist von Fach zu Fach sehr unterschiedlich wie auch die Definition, die die WissenschaftlerInnen selbst für ihre Forschungsdaten wählen. In der Studie ging man von folgender Definition aus: Unter Forschungsdaten sind alle Daten zu verstehen, die im Zuge wissenschaftlicher Forschungs- und künstlerischer Schaffensprozesse entstehen (z.B. Text, Tabellen, Video, Audio, Grafik etc.) und auf deren Grundlage ihre Forschungsergebnisse und/oder Kunstwerke basieren – z.B. durch Experimente, Quellenforschungen, Messungen, Erhebungen, Digitalisate oder Entwürfe. (siehe Forschende und ihre Daten, 15) Die Wiederverwendung der Daten variiert ebenfalls von Fach zu Fach: Ungefähr ein Drittel der Befragten ermöglicht die Nachnutzung ihrer eigenen Forschungsdaten; tendenziell häufiger geschieht dies in der Geographie, Biologie und Chemie, verhältnismäßig seltener in der Medizin, den Sozialwissenschaften und Geisteswissenschaften. (siehe Forschende und ihre Daten, 7) Auf die Frage, warum Forschungsdaten nicht mit anderen geteilt werden, wurden unter anderem „der erhöhte Zeit- und Kostenaufwand, ein möglicher Datenmissbrauch, rechtliche Unsicherheiten, eine potenzielle Datenverfälschung, unerwünschte Kommerzialisierung sowie Erhöhung des Konkurrenzdrucks“ genannt. (Forschende und ihre Daten, 7) Vor allem in der Medizin, den Sozial- und Verhaltenswissenschaften sowie den Ingenieurwissenschaften nannten die Befragten rechtliche Einschränkungen als größte Hindernisse. (siehe Forschende und ihre Daten, 7f.) Fecher verweist in seiner Studie jedoch auch auf ein anderes Hemmnis: Im Mai 2016 veröffentlichten die Autoren Dan L. Longo und Jeffrey M. Drazen den Leitartikel „Data Sharing“ in der renommierten Fachzeitschrift New England Journal of Medicine (NEJM). In dem Artikel bezeichneten sie WissenschaftlerInnen, die für ihre Forschung Daten anderer Wissenschaftler verwenden, als „research parasites“. (Longo und Drazen 2016, 276) Häufig werden auch rechtliche und ethische Bedenken genannt, wenn Daten nicht geteilt werden: Rechtliche Unsicherheiten entstehen weiterhin aufgrund der fehlenden Verständigung auf einheitliche Lizenzmodelle zur Nachnutzung von Daten. Im Bereich der Artikelpublikationen hat sich mit den Creative Commons-Lizenzen ein Quasi-Standard zur Beschreibung der Open Access-Bedingungen durchgesetzt. Vor dem Hintergrund des AusMitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich 157 baus der europäischen Dateninfrastruktur, insbesondere mit Hinblick auf die geplante European Open Science Cloud, sind verstärkte Harmonisierungsbemühungen und die Verständigung auf Standard-Lizenzmodelle dem Austausch zuträglich. (Fecher 2018, 132) Aber auch folgende Gründe können gegen eine Nachnutzung von Daten sprechen: 1. die Falsifizierung, also der Nachweis der Ungültigkeit einer Aussage des Primärforschers durch den Nachnutzer, 2. die nachteilige kommerzielle Nutzung, als die nachteilige oder unerwünschte Nutzung der bereitgestellten Daten durch kommerzielle Unternehmen, 3. die kompetitive Nutzung, also die Publikation von Ergebnissen auf Grundlage von bereitgestellten Daten durch konkurrierende Forscher und 4. die fehlerhafte Interpretation der bereitgestellten Daten durch andere Wissenschaftler. (Fecher 2018, 139) Da die Publikationen für Forschende im Vordergrund stehen, weil derzeit ihre Forschungsleistung daran gemessen wird, wäre es evtl. denkbar, Forschungsdaten erst zeitverzögert einzufordern, erst nach der Veröffentlichung der Daten in Form einer Publikation. Datenzeitschriften oder Anerkennungen für viel genutzte Daten könnten weitere Anreize schaffen, wie es auch die EOSC empfiehlt: „Researchers who make research data open and FAIR for reuse and/or reuse and reproduce data should be rewarded, both in their career assessment and in the evaluation of projects (initial funding, review of performance and impact). This should go hand in hand with other career policies in universities and research institutions (appointments, promotions etc.).“12 Bzgl. der Datenarchive stellten die Befragten keine großen Ansprüche, aber „[m]ehr als die Hälfte der Forschenden erwartet die Bereitstellung von zusätzlichem qualifiziertem Personal sowie die Verabschiedung von Policies zum Umgang mit Forschungsdaten [...] Die Mehrzahl der Forschenden wünscht sich technische Infrastrukturen sowie projektspezifische Unterstützung für das Forschungsdatenmanagement. Darüber hinaus zeigt mehr als ein Drittel Interesse an Rechtsberatung, einem allgemeinen Helpdesk sowie an Schulungsangeboten.“ (Forschende und ihre Daten, 8) Die Zertifizierung von Repositorien, etwa das „Data Seal of Approval“, wird ebenfalls an Bedeutung gewinnen: „Trusted research data repositories play a fundamental role in modern science. Scientist must be able to 158 Mitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich find, re-use, deposit and share data via trusted data repositories that implement FAIR data principles and that ensure long-term sustainability of research data across all disciplines. Data repositories must be easy to find and identify, and provide to users full transparency about their services.“13 Konzepte für eine gesicherte Langzeitarchivierung der Daten, auch jener, die sehr komplex sind, sind bisher kaum umgesetzt. Ebenso gibt es in den Bereichen Ethik und Recht noch zahlreiche ungelöste Fragen. Aber auch strukturelle Überlegungen fehlen noch, zum Beispiel, wie die langfristige Sicherung von Forschungsoutput aus zeitlich befristeten Drittmittelprojekten finanziert werden kann. Unter anderem nennt Knoche im Bereich Repositorien folgende unerledigte Aufgaben auf gesamtstaatlicher Ebene in Deutschland, die auch für Österreich gelten: Die Langzeitarchivierung der digitalen Medien, die Aufgabenteilung bei den Forschungsdaten und eine Strategie zur Retrodigitalisierung. (Knoche 2018, 120f.) Um all diese Fragen in einem größeren Zusammenhang betrachten zu können und Einzellösungen zu vermeiden, ist es heute mehr denn je wichtig, dass die RepositorienbetreiberInnen eng mit internen und externen Serviceeinrichtungen zusammenarbeiten und auch zunehmend die WissenschaftlerInnen in Diskussionen miteinzubeziehen. Mag.a Dr.in Susanne Blumesberger ORCID: http://orcid.org/0000-0001-9018-623X Universität Wien, Bibliotheks- und Archivwesen E-Mail: susanne.blumesberger@univie.ac.at Literatur Bauer, Bruno; Ferus, Andreas; Gorraiz, Juan; Gründhammer, Veronika; Gumpenberger, Christian; Maly, Nikolaus; Mühlegger, Johannes Michael; Preza, José Luis; Sánchez Solís, Barbara; Schmidt, Nora; Steineder, Christian (2015): Forschende und ihre Daten. Ergebnisse einer österreichweiten Befragung – Report 2015. Version 1.2. DOI: http://doi. org/10.5281/zenodo.32043. Online auch unter: http://phaidra.univie. ac.at/o:407513 Bayer, Florian; Gorraiz, Juan; Gumpenberger, Christian; Mitterauer, Lukas; Reding, Steve (2017): Sichtbarkeitssteigerung in den Geistes-, Sozial- und Kulturwissenschaften (GSK). Ergebnisse einer Befragung an der Universität Wien. Version 1.0. DOI: http://doi.org/10.5281/zenodo.400965. Online auch unter: http://phaidra.univie.ac.at/o:526603 Mitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich 159 Blumesberger, Susanne (2009): Wissen intelligent und sicher archivieren, verbreiten und nutzbar machen. Phaidra – Das innovative digitale Langzeitarchivierungssystem der Universität Wien. In: Mitteilungen der Vereinigung Österreichischer Bibliothekarinnen und Bibliothekare 62(2), S. 7–17. Blumesberger, Susanne (2009): Das kulturelle Erbe – sicher und langfristig in Phaidra. Digitale Langzeitarchivierung an der Universität Wien. In: B.I.T. online. Zeitschrift für Bibliothek, Information und Technologie 12(3), S. 294–296. Online unter: http://www.b-i-t-online.de/archiv/2009-03/nach2.htm Blumesberger, Susanne (2010): Sicher archivieren – grenzenlos recherchieren – intelligent nutzen. Phaidra – digitale Langzeitarchivierung an der Universität Wien. In: Bergner, Ute; Erhard Göbel (Hg.): The ne(x) t Generation. Das Angebot der Bibliotheken. 30. 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Online unter: http://phaidra.univie.ac.at/o:245909 Fecher, Benedikt (2018): Eine Reputationsökonomie. Der Wert der Daten in der akademischen Forschung. Wiesbaden: Springer VS. DOI: http:// doi.org/10.1007/978-3-658-20895-0 Knoche, Michael (2018): Die Idee der Bibliothek und ihre Zukunft. Göttingen: Wallstein Verlag. Longo, Dan L.; Drazen, Jeffrey M. (2016). Data Sharing. New England Journal of Medicine 374(3), p. 276–277. DOI: http://doi.org/10.1056/ NEJMe1516564 1 Bibliotheken: Weg damit! NZZ am Sonntag, 7.2.2016. https://nzzas. nzz.ch/hintergrund/bibliotheken-und-buecher-weg-damit-meint-rafael-ball-ld.147683 2 https://www.force11.org/group/fairgroup/fairprinciples 3 https://phaidra.univie.ac.at/ 4 https://ubifo.at/images/repmannet.pdf 5 Permanent Hosting, Archiving and Indexing of Digital Resources and Assets. 6 https://phaidra.cab.unipd.it 7 Das Vorarlberger Landesrepositorium basiert auf Phaidra. https://pid. volare.vorarlberg.at 8 https://e-book.fwf.ac.at 9 Eine Übersicht über die derzeitigen PHAIDRA-Partner befindet sich unter: https://www.phaidra.org/community/phaidra-partners/ 10 http://e-infrastructures.univie.ac.at/home/ 11 EOSC Declaration. https://ec.europa.eu/research/openscience/pdf/ eosc_declaration.pdf#view=fit&pagemode=none 12 EOSC Declaration. https://ec.europa.eu/research/openscience/pdf/ eosc_declaration.pdf#view=fit&pagemode=none 13 EOSC Declaration, S. 2. Mitteilungen der VÖB 71 (2018) Nr. 1: Repositorien in Österreich 161
https://openalex.org/W2942734148	https://europepmc.org/articles/pmc6539519?pdf=render	English	null	Alternative Calibration of Cup Anemometers: A Way to Reduce the Uncertainty of Wind Power Density Estimation	Sensors	2,019	cc-by	13,345	Received: 3 April 2019; Accepted: 27 April 2019; Published: 30 April 2019 Abstract: This study presents a procedure to reduce the uncertainty of wind power density estimations, which is useful to improve the energy production predictions of wind farms. Power density is usually determined from the wind speed measured by a cup anemometer and the air density value (conventional procedure). An alternative procedure based on wind speed and dynamic pressure estimations provided by a cup anemometer is proposed. The dynamic pressure is obtained by means of a calibration curve that relates the anemometer rotation frequency and the dynamic pressure measured by a Pitot tube. The quadratic regression, used to deﬁne the calibration curve, and its uncertainty are both detailed. A comparison between the alternative procedure and the conventional one points out the advantage of the proposed alternative since results show a high reduction of the indirect measurement uncertainty of wind power density. Keywords: wind speed sensor; cup anemometer; quadratic regression uncertainty; wind dynamic pressure; wind power density; wind tunnel; anemometer calibration; pitot tube sensors sensors sensors Article Francisca Guerrero-Villar, Rubén Dorado-Vicente , Gustavo Medina-Sánchez and Eloísa Torres-Jiménez * Department of Mechanical and Mining Engineering; Universidad de Jaén, 23071 Jaén, Spain; mgvillar@ujaen.es (F.G.-V.); rdorado@ujaen.es (R.D.-V.); gmedina@ujaen.es (G.M.-S.) * Correspondence: etorres@ujaen.com; Tel.: +34-953212867 Received: 3 April 2019; Accepted: 27 April 2019; Published: 30 April 2019 1. Introduction Cup anemometer is a robust and reliable device. From the ﬁrst cup anemometer, invented by Robinson [1], this device has been investigated and improved [2,3]. Current research provides analytical models and numerical studies related to its performance [4]. The use of cup anemometers is widely extended in ﬁelds such as meteorology and wind energy production. In the wind energy ﬁeld, their use is increasing as the installed wind power capacity does [5]. In fact, wind speed metrology is key to assess the energy potential of wind turbines [6,7]. Accurate estimations of power curves are required to determine performance and the Annual Energy Production (AEP) of wind turbines [8]. The calibration of these devices is growing in importance, since regulations, regarding the determination of potential wind power production, establish a calibration of anemometers before and after each ﬁeld data collection. In the wind energy ﬁeld, power delivered by wind turbines is calculated from wind speed measurement. Even more, according to the international standard IEC 61400-12-1 [9], wind speed is needed to calculate the Annual Energy Production (AEP) and the power coeﬃcient curve (CP): CP = P 1 2· ρ·S· V3 = P Pw· S , (1) (1) , 2029; doi:10.3390/s19092029 www.mdpi.com/journal/sensors Sensors 2019, 19, 2029; doi:10.3390/s19092029 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors Sensors 2019, 19, 2029 2 of 18 where P is the electrical power produced by the wind turbine, ρ is the air density, S is the blade swept area, and V is the wind speed. Pw is known as “wind power density” [10]: this magnitude allows the assessment of CP, which depends on the air conditions at the location of the turbine. Sensors 2019, 19, x 2 of 18 where P is the electrical power produced by the wind turbine, ρ is the air density, S is the blade swept area, and V is the wind speed. Pw is known as “wind power density” [10]: this magnitude allows the assessment of CP, which depends on the air conditions at the location of the turbine. Sensors 2019, 19, x 2 of 18 In the calibration process of a cup anemometer, its rotational frequency fr, which provides the rotational speed, is usually related to the wind speed. Previous studies demonstrated that this is a linear relationship [11] (see Figure 1a), which is explained in detail by Pindado et al. [12]. 1. Introduction (3) nd wind speed measured by a cup anemometer: Hereafter, the cup anemometer used to measure ∆p will be called as “Dynamic Pressure (DYP)-Cup Anemometer”, meanwhile if the anemometer is used to measure V it will be called as “Air Speed (AS)-Cup Anemometer” (see Figure 1). Pw=p V. (3) Hereafter, the cup anemometer used to measure Δp will be called as “Dynamic Pressure (DYP)- Cup Anemometer”, meanwhile if the anemometer is used to measure V it will be called as “Air Speed (AS) C A ” ( Fi 1) The aerodynamic movement of the cup anemometer is mainly due to drag force [17], and this force is proportional to air dynamic pressure [18], which leads us to think that the relationship between air dynamic pressure and cup anemometer rotation frequency is quadratic (see Figure 1b). (AS)-Cup Anemometer” (see Figure 1). The aerodynamic movement of the cup anemometer is mainly due to drag force [17], and this force is proportional to air dynamic pressure [18], which leads us to think that the relationship between air dynamic pressure and cup anemometer rotation frequency is quadratic (see Figure 1b) (a) (b) Figure 1. Calibration of a cup anemometer by means of a Pitot tube: (a) Conventional calibration: Air Speed Cup Anemometer (“AS-Cup Anemometer”), (b) Alternative calibration: Dynamic Pressure Cup Anemometer (“DYP-Cup Anemometer”). Figure 1. Calibration of a cup anemometer by means of a Pitot tube: (a) Conventional calibration: Air Speed Cup Anemometer (“AS-Cup Anemometer”), (b) Alternative calibration: Dynamic Pressure Cup Anemometer (“DYP-Cup Anemometer”). (a) (b) (b) (a) Figure 1. Calibration of a cup anemometer by means of a Pitot tube: (a) Conventional calibration: Air Speed Cup Anemometer (“AS-Cup Anemometer”), (b) Alternative calibration: Dynamic Pressure Cup Anemometer (“DYP-Cup Anemometer”). Figure 1. Calibration of a cup anemometer by means of a Pitot tube: (a) Conventional calibration: Air Speed Cup Anemometer (“AS-Cup Anemometer”), (b) Alternative calibration: Dynamic Pressure Cup Anemometer (“DYP-Cup Anemometer”). 1. Introduction Due to its reliability, the Measuring Network of Wind Energy Institutes (MEASNET) [13], the European Association of National Metrology Institutes EUROMET in their project titled “Comparison for Air speed Measurements” [14], and other authors such as Ghaemi-Nasab et al. [15] use the Pitot tube as the standard device in the cup anemometer calibration process. In the calibration process of a cup anemometer, its rotational frequency fr, which provides the rotational speed, is usually related to the wind speed. Previous studies demonstrated that this is a linear relationship [11] (see Figure 1a), which is explained in detail by Pindado et al. [12]. Due to its reliability, the Measuring Network of Wind Energy Institutes (MEASNET) [13], the European Association of National Metrology Institutes EUROMET in their project titled “Comparison for Air speed Measurements” [14], and other authors such as Ghaemi-Nasab et al. [15] use the Pitot tube as Pitot tube provides the ﬂuid ﬂow velocity based on Bernoulli’s equation [16]. This device measures the diﬀerence between total and static pressure, which is call dynamic pressure and represents the ﬂuid kinetic energy per unit volume. This diﬀerence is proportional to the square of ﬂuid ﬂow velocity multiplied by air density: the standard device in the cup anemometer calibration process. Pitot tube provides the fluid flow velocity based on Bernoulli’s equation [16]. This device measures the difference between total and static pressure, which is call dynamic pressure and represents the fluid kinetic energy per unit volume. This difference is proportional to the square of ∆p = ptotal −pstatic = 1 2· ρ· V2. (2) air density: 1 ଶ (2) (2) Since Pitot tube measures dynamic pressure instead of ﬂuid velocity, it is proposed to calibrate the cup anemometer rotation frequency respect to the dynamic pressure. This calibration allows to estimate Pw using the dynamic pressure and wind speed measured by a cup anemometer: ∆𝑝= 𝑝௧௢௧௔௟−𝑝௦௧௔௧௜௖= 2 · 𝜌· 𝑉ଶ. (2) Since Pitot tube measures dynamic pressure instead of fluid velocity, it is proposed to calibrate the cup anemometer rotation frequency respect to the dynamic pressure. This calibration allows to Pw =∆p ·V. (3) p y p nd wind speed measured by a cup anemometer: (3) Pw =∆p ·V. 1. Introduction The targets of the present study are as follows: (a) show that Pw can be obtained using a cup anemometer with a double calibration, so that fr provides Δp and V, (b) determine the goodness and the uncertainty of the quadratic regression between the wind dynamic pressure and the cup anemometer rotation frequency, (c) quantify and compare the uncertainty of Δp measurements, with the uncertainty of V measurements obtained according to the conventional method developed in annex F of IEC 61400-12-1 international standard [9], and (d) determine if the uncertainty of wind power density calculation decreases when the proposed method, based on dynamic pressure, is applied. The targets of the present study are as follows: (a) show that Pw can be obtained using a cup anemometer with a double calibration, so that fr provides ∆p and V, (b) determine the goodness and the uncertainty of the quadratic regression between the wind dynamic pressure and the cup anemometer rotation frequency, (c) quantify and compare the uncertainty of ∆p measurements, with the uncertainty of V measurements obtained according to the conventional method developed in annex F of IEC 61400-12-1 international standard [9], and (d) determine if the uncertainty of wind power density calculation decreases when the proposed method, based on dynamic pressure, is applied. decreases when the proposed method, based on dynamic pressure, is applied. Because cup-anemometers are usually calibrated in wind tunnels, uncertainties calculated from the application of the conventional and proposed calibration are obtained using a wind tunnel. Note that in-field calibration has no clear advantages over conventional calibration [19]. Although it is Because cup-anemometers are usually calibrated in wind tunnels, uncertainties calculated from the application of the conventional and proposed calibration are obtained using a wind tunnel. Note that in-ﬁeld calibration has no clear advantages over conventional calibration [19]. Although it is 3 of 18 Sensors 2019, 19, 2029 possible to assess the measurement variability at a speciﬁc site, such as Aquila et al. [20] propose, it is diﬃcult to compare those uncertainties to the conventional calibration method performed within a wind tunnel. In other studies, such as those with the target of determining the average ﬂow velocity overestimation [21], cup anemometer uncertainty is analyzed under time-varying ﬂow, nevertheless, the present study is focused on steady state ﬂows because this is the appropriate procedure for the calibration of cup anemometers. 2.1. Experimental Set-up and Measurement Strategy 2.1. Experimental Set-up and Measurement Strategy The experimental results presented in next sections are obtained by means of a cup anemometer taken from a commercial meteorological station Auriol model IAN 114435. This anemometer has three semi-spherical shape cups of 26 mm diameter each one, and 52 mm distance from the rotor axis to the center of the cups. This small cup anemometer with a rotor diameter of 130 mm (see Figure 2), has an appropriate size to be installed inside the tunnel available in the department of Mechanical and Mining Engineering of the University of Jaén. This cup anemometer has a vane, which has not been removed, because it does not disturb the experiment and it will be useful in future ﬁeld applications. The experimental results presented in next sections are obtained by means of a cup anemometer taken from a commercial meteorological station Auriol model IAN 114435. This anemometer has three semi-spherical shape cups of 26 mm diameter each one, and 52 mm distance from the rotor axis to the center of the cups. This small cup anemometer with a rotor diameter of 130 mm (see Figure 2), has an appropriate size to be installed inside the tunnel available in the department of Mechanical and Mining Engineering of the University of Jaén. This cup anemometer has a vane, which has not been removed, because it does not disturb the experiment and it will be useful in future field applications Figure 2. Cup anemometer used in the present study. Figure 2. Cup anemometer used in the present study. Figure 2. Cup anemometer used in the present study. Figure 2. Cup anemometer used in the present study. The cup anemometer was tested in a closed jet-type wind tunnel (see Figure 3), whose testing transversal area is 40 cm by 40 cm. The uniform main flow in the tunnel is produced by a 5 kW rotating fan with a variable speed drive engine, which provides a flow velocity between 1 m/s and 30 m/s. The wind tunnel turbulence levels are less than 2%, and the ratio of flow uniformity is high (within 0.2% for x, y, and z axis flows) thanks to several wire meshes, one honeycomb type filter and a convergent nozzle. Note that, according to the standard IEC 61400-12-1 [9], the calibration of a cup- anemometer should be accomplished at low turbulence conditions (axial turbulence below 2%). 1. Introduction Sensors 2019, 19, x 3 of 18 the present study is focused on steady state flows because this is the appropriate procedure for the calibration of cup anemometers. 2.1. Experimental Set-up and Measurement Strategy 2.1. Experimental Set-up and Measurement Strategy For the wind speeds tested [4 m/s to 16 m/s], the Reynolds number goes from 1.015 × 105 to 4.063·× 105. Although turbulence and density variations influence in-field measurements [3,22], since this work presents a calibration procedure, we only considered wind tunnel measures at low turbulence levels. The cup anemometer was tested in a closed jet-type wind tunnel (see Figure 3), whose testing transversal area is 40 cm by 40 cm. The uniform main ﬂow in the tunnel is produced by a 5 kW rotating fan with a variable speed drive engine, which provides a ﬂow velocity between 1 m/s and 30 m/s. The wind tunnel turbulence levels are less than 2%, and the ratio of ﬂow uniformity is high (within 0.2% for x, y, and z axis ﬂows) thanks to several wire meshes, one honeycomb type ﬁlter and a convergent nozzle. Note that, according to the standard IEC 61400-12-1 [9], the calibration of a cup-anemometer should be accomplished at low turbulence conditions (axial turbulence below 2%). For the wind speeds tested [4 m/s to 16 m/s], the Reynolds number goes from 1.015 × 105 to 4.063 × 105. Although turbulence and density variations inﬂuence in-ﬁeld measurements [3,22], since this work presents a calibration procedure, we only considered wind tunnel measures at low turbulence levels. 4 of 18 sents a Sensors 2019, 19, 2029 turbulence and de lib i d Figure 3. Wind tunnel used in the present study. Figure 3. Wind tunnel used in the present study. Figure 3. Wind tunnel used in the present study. Figure 3. Wind tunnel used in the present study. The standard reference device in this work is a Pitot tube: Testo stainless steel, 500 mm length, reference 06352045 and Pitot tube factor = 1. It is connected to a Testo 512 2hPa diﬀerential micro-manometer, with 0.1 Pa of resolution, a precision of ±0.01 hPa in a measuring range from 2 to 17.5 m/s, and it is capable to provide two lectures per second. The pitot tube is placed upstream at the same height as the cups, as shown in Figure 4. A digital Photo Tachometer model RM-1501, with a sampling frequency of 1 Hz and a precision of ±0.2 rpm, is used to obtain the anemometer rotation frequency. 2.1. Experimental Set-up and Measurement Strategy 2.1. Experimental Set-up and Measurement Strategy In order to determine the air conditions, three thermometers are used: a Tecpel 305 placed inside the tunnel to obtain the dry bulb temperature, another Tecpel 305 placed outside the tunnel to obtain the ambient temperature and ﬁnally, one alcohol thermometer placed inside the tunnel to obtain the wet bulb temperature. The setup of these elements is schematically shown in Figure 4. The barometer placed at the roof of the Higher Polytechnic School building of the University of Jaén is used to obtain the ambient barometric pressure. These results are published on the MATRAS webpage [23], and they have been corrected taken in account the height between the building roof and the laboratory where the wind tunnel is located, according to the standard atmosphere formulation speciﬁed in ISO 2533:1975. The testing methodology consists on placing the cup anemometer inside the wind tunnel, testing several wind speeds in a range from 4 m/s to 16 m/s and registering the cup anemometer rotation frequency together with the dynamic pressure. A steady state operating condition is set every 0.43 m/s. This sampling interval satisﬁes the requirements recommended by MEASNET [13] and it is usually taken by other authors in the ﬁeld of anemometers calibration [24]. For each operating condition tested, the anemometer rotation frequency is registered for at least 50 s with a sampling frequency of 1 Hz (mf = 50 measurements for each operating condition). The dynamic pressure is recorded for 10 s with a sampling frequency of 0.5 Hz (mp = 20 measurements for each operating condition). The actual value of rotation frequency and dynamic pressure for a speciﬁc operating condition is taken as the mean value. A number of n = 29 operating conditions were tested. About half of the operating conditions were tested by increasing wind speed (15 operating conditions in odd steps) and the rest of them by decreasing the wind speed (14 operating conditions in even steps). 5 of 18 5 of 18 Sensors 2019, 19, 2029 Sensors 2019 19 x Figure 4. Schematic diagram of the experimental setup. Upper: front view. Lower: top view. Figure 4. Schematic diagram of the experimental setup. Upper: front view. Lower: top view. Figure 4. Schematic diagram of the experimental setup. Upper: front view. Lower: top view. Figure 4. Schematic diagram of the experimental setup. Upper: front view. Lower: top view. 2.1. Experimental Set-up and Measurement Strategy 2.1. Experimental Set-up and Measurement Strategy The wind dynamic pressure at the anemometer position for a k operating condition Δpk is determined as the mean of mp = 20 measurements of the dynamic pressure provided by the Pitot tube Δpi at the reference position, corrected by the factors previously discussed: ௠ The wind tunnel calibration factor kc is determined by comparing the measurements of the Pitot tube placed at the reference position and the measurements at the anemometer position (Figure 4). This procedure allows obtaining the relationship between both measurements and its uncertainty. At the anemometer position, air ﬂow velocity is lightly higher than at the reference position. ∆𝑝௞= 𝑘௖ 𝐶௛ · 𝑘௙ ଶ· 1 𝑚௣ · ෍∆𝑝௜ ௠೛ ௜ୀଵ . (5) Finally, because the anemometer acquisition system obtains mf = 50 measurements for each k The Pitot tube head coeﬃcient Ch is related to the alignment accuracy of the Pitot tube with the wind ﬂow direction inside the tunnel. The ISO 3966, about measurement of ﬂuid ﬂow velocity using Pitot static tubes [25], suggests a Ch value. This value and its corresponding uncertainty is given in Section 3. operating condition, the anemometer rotation frequency fk is determined as follows: 𝑓௞= 1 ෍𝑓௜ ௠೑ (6) On the other hand, the blockage correction factor kf quantiﬁes the inﬂuence of the anemometer shape on the mean ﬂow ﬁeld velocity Vb. This correction factor is calculated through Maskell theorem [26]: V 1 𝑓௞= 𝑚௙ ෍𝑓௥,௜ ௜ୀଵ . (6) kf = Vb V = 1 + 1 2 a·c f ·b, (4) ( ) (4) 2.2. Quadratic Regression Uncertainty In order to determine if the proposed calibration improves Pw predictions, the uncertainty components of Pw have to be determined. This section is devoted to explain how to quantify the uncertainty of the quadratic regression between air dynamic pressure and anemometer rotation where the blockage ratio b (anemometer front area divided by tunnel cross section) is equal to 0.025, and the product of the shape factor a and the force coeﬃcient cf is a·cf = 0.5 as is recommended for unusual shapes tested within a wind tunnel [27]. The value of kf has been checked in the wind tunnel by comparing the measurements of the Pitot tube placed at the anemometer section with and without the cup anemometer inside the tunnel. frequency (Equation 7). Authors such as Ilic et al. 2.2. Quadratic Regression Uncertainty In order to determine if the proposed calibration improves Pw predictions, the uncertainty components of Pw have to be determined. This section is devoted to explain how to quantify the uncertainty of the quadratic regression between air dynamic pressure and anemometer rotation frequency (Equation (7)). Authors such as Ilic et al. [28], who measure stagnation pressure inside wind tunnel, point out the high nonlinearity of the measurement process of dynamic pressure. The calculation of the quadratic regression uncertainty is more complex than the linear one. In fact, the “Guide to the expression of uncertainty in measurement”(GUM) [29] only provides simpliﬁed equations for the linear case. Let the following equation be the quadratic regression for a DYP-Cup anemometer, which provides the wind dynamic pressure estimation ˆ∆p: ˆ∆p = y1 + y2·fr + y3·f 2 r . (7) (7) The quadratic regression coeﬃcients y = [y1, y2, y3] (y1 intercept: anemometer’s oﬀset, y2 slope and y3 curvature) are determined by the least-squared method minimizing the total of the squared residuals for n observations. The resulted system of equations provides the regression coeﬃcients: y = S−1∆x, (8) (8) where: S =   n P k fr,k P k f 2 r,k P k fr,k P k f 2 r,k P k f 3 r,k P k f 2 r,k P k f 3 r,k P k f 4 r,k   andx = [x1, x2, x3] =   X k ∆pk, X k fr,k·∆pk , X k f 2 r,k·∆pk   S =   n P k fr,k P k f 2 r,k P k fr,k P k f 2 r,k P k f 3 r,k P k f 2 r,k P k f 3 r,k P k f 4 r,k   andx = [x1, x2, x3] =   X k ∆pk, X k fr,k·∆pk , X k f 2 r,k·∆pk   Applying the law of propagation of uncertainty to Equation (7), we obtain the regression combined standard uncertainty. This uncertainty is function of the y coeﬃcients’ variances and covariances. Applying the law of propagation of uncertainty to Equation (7), we obtain the regression combined standard uncertainty. This uncertainty is function of the y coeﬃcients’ variances and covariances. In order to obtain the aforementioned variances and covariances, we applied the law of propagation of uncertainty to Equation (8). 2.1. Experimental Set-up and Measurement Strategy 2.1. Experimental Set-up and Measurement Strategy [28], who measure stagnation pressure inside wind tunnel, point out the high nonlinearity of the measurement process of dynamic pressure. The calculation of the quadratic regression uncertainty is more complex than the linear one. In The wind dynamic pressure at the anemometer position for a k operating condition ∆pk is determined as the mean of mp = 20 measurements of the dynamic pressure provided by the Pitot tube ∆pi at the reference position, corrected by the factors previously discussed: n of uncertainty in measurement (GUM) [29] only provides case. Let the following equation be the quadratic regression for a ides the wind dynamic pressure estimation ∆𝑝 ෢: ෢ ଶ ∆pk = kc Ch · k2 f · 1 mp · mp X i=1 ∆pi. (5) or a (5) ∆𝑝= 𝑦ଵ+ 𝑦ଶ· 𝑓௥+ 𝑦ଷ· 𝑓௥ ଶ. (7) The quadratic regression coefficients y = [y1, y2, y3] (y1 intercept: anemometer’s offset, y2 slope and y u atu e) a e dete i ed by the lea t ua ed ethod i i i i the total of the ua ed Finally, because the anemometer acquisition system obtains mf = 50 measurements for each k operating condition, the anemometer rotation frequency fk is determined as follows: ∆𝑝= 𝑦ଵ+ 𝑦ଶ· 𝑓௥+ 𝑦ଷ· 𝑓௥ ଶ. (7) The quadratic regression coefficients y = [y1, y2, y3] (y1 intercept: anemometer’s offset, y2 slope and Finally, because the anemometer acquisition system obtains mf = 50 measurements for each k operating condition, the anemometer rotation frequency fk is determined as follows: east squared method minimizing the total of the squared d system of equations provides the regression coefficients: 𝐲= 𝐒ିଵ∙𝐱, (8) fk = 1 mf mf X i=1 fr,i. (6) east squared metho d system of equatio 𝐲= 𝐒ିଵ∙𝐱, fk = 1 mf mf X i=1 fr,i. ts: (8) (6) Sensors 2019, 19, 2029 6 of 18 2.2. Quadratic Regression Uncertainty The authors considered this approach easier to extend to complex regressions than the conventional solution used in linear regressions, as well as simpler than the method proposed by Hilbert [30], who modiﬁed the calibration equation and then applied the law of propagation of uncertainty. According to the GUM [29], for correlated variables, the variances and covariances for the y coeﬃcients can be obtained as follows: u(yl, ym) = s(yl, ym) = 3 X i=1 3 X j=1 ∂yl ∂xi ·∂ym ∂xj ·u xi, xj , (9) (9) where l and m go from 1 to 3. where l and m go from 1 to 3. where l and m go from 1 to 3. For the x covariances, we used the previous Equation (9); however, the variables are the dynamic pressure observations. Thus, assuming a zero covariance between diﬀerent observations, the estimation of the deviation of each observation can be determined by the regression error as follows: u(xl, xm) = s(xl, xm) = n X k=1 ˆ∆pk −∆pk 2·f l+m−2 k . (10) (10) Sensors 2019, 19, 2029 7 of 18 Finally, the application of the “law of propagation of uncertainty” to Equation (7) yields the combined standard uncertainty for the quadratic regression. Let C be the covariance matrix and F = [∂ˆ∆p/∂y1, ∂ˆ∆p/∂y2, ∂ˆ∆p/∂y3] then: u2 ˆ∆p = F C FT = u2(y1) + f 2 r ·u2(y2) + f 4 r ·u2(y3) + 2·fr· u(y1, y2) + fr·u(y1, y3) + f 2 r ·u(y2, y3) . (11) u2 ˆ∆p = F C FT = u2(y1) + f 2 r ·u2(y2) + f 4 r ·u2(y3) + 2·fr· u(y1, y2) + fr·u(y1, y3) + f 2 r ·u(y2, y3) . (11) 2 3 Wind Speed and Dynamic Pressure Uncertainty (11) 2.3. Wind Speed and Dynamic Pressure Uncertainty 2.3. Wind Speed and Dynamic Pressure Uncertainty According to Annex F of IEC 61400-12-1 international standard [9], in order to obtain the measurement uncertainty of a cup anemometer to measure wind speed (conventional measurement procedure), the following equations are used (“Wind speed equations”): Vs = kf · r kc Ch · s 2 . ∆pPt ρ , (12) (12) ˆV = A· fr + B, (13) (13) where Vs is the wind speed determined by the dynamic pressure ∆pPt measured with the Pitot tube, and ˆV is the predicted wind speed by means of a linear regression. where Vs is the wind speed determined by the dynamic pressure ∆pPt measured with the Pitot tube, and ˆV is the predicted wind speed by means of a linear regression. p p y g In the present paper, the alternative measurement procedure is based on the following equations the present paper, the alternative measurement procedure is based on the following equations: ∆ps = kc Ch · k2 f ·∆pPt, (14) (14) The Equations (7) and (14) are the “Wind dynamic pressure equations” and can be used to calibrate cup anemometers. Equation (14) is a generalization of Equation (5). ∆pPt and ∆ps are the dynamic pressures measured with the Pitot tube at the reference position and at the anemometer position, respectively. In order to obtain the combined uncertainty for wind speed and dynamic pressure measurements, the standard uncertainty and the sensibility coeﬃcient for each parameter included in the calibration equations have to be computed according to Table 1. Table 1. Standard uncertainty and sensibility coeﬃcients for each parameter included in the calibration equations. Table 1. Standard uncertainty and sensibility coeﬃcients for each parameter included in the calibration equations. Table 1. Standard uncertainty and sensibility coeﬃcients for each parameter included in the calibration equations. Parameters Standard Uncertainty Sensibility Coeﬃcients: Equation (12) Sensibility Coeﬃcients: Equation (14) Blockage correction factor (kf) u kf ∂Vs/∂kf ∂∆ps/∂kf Wind tunnel calibration factor (kc) u(kc) ∂Vs/∂kc ∂∆ps/∂kc Pitot tube head coeﬃcient (Ch) u(Ch) ∂Vs/∂Ch ∂∆ps/∂Ch Air density (ρ) u(ρ) ∂Vs/∂ρ Wind Dynamic pressure (Pitot tube) (∆pPt) u(∆pPt) ∂Vs/∂∆pPt ∂∆ps/∂∆pPt Linear regression (A, B) ulr(V) = p u2(B) + fr2·u2(A) + 2fr· u(A, B) - - Quadratic regression (y1, y2, y3) u ˆ∆p Equation (11) - - Note: kf, kc, Ch values and their uncertainties are provided in Section 3. Note: kf, kc, Ch values and their uncertainties are provided in Section 3. 2.3. Wind Speed and Dynamic Pressure Uncertainty Note: kf, kc, Ch values and their uncertainties are provided i The moist air density is obtained by means of the equation proposed in Annex F of IEC 61400-12-1 international standard [9]: 1 P M M M ρ = 1 T PB·Ma R −∅·Pvs Ma R −Mv R , (15) Pvs = 0.0000205 e0.0631846·T, (16) (15) (16) where PB is the barometric pressure (Pa), T is the absolute dry bulb temperature (K), R is the ideal gas constant (J/mol·K), Pvs is the saturated vapour pressure (Pa), Ma is the air molar mass, and Mv is the where PB is the barometric pressure (Pa), T is the absolute dry bulb temperature (K), R is the ideal gas constant (J/mol·K), Pvs is the saturated vapour pressure (Pa), Ma is the air molar mass, and Mv is the S 19 f 8 of 18 Sensors 2019, 19, 2029 water vapour molar mass (kg/mol). The relative humidity ∅is determined by applying the following equation: ∅= Pvs(Tv) −A·P·(T −Tv) Pvs(T) = Pvs(Tv) −A0·(1 + 0.00115·(Tv −273.15))·P·(T −Tv) Pvs(T) , (17) (17) where Tv is the absolute wet bulb temperature (K), A is the so-called psychometric constant pertinent to the standard wet-bulb temperature, A0 is the so-called psychometric constant pertinent to the standard wet-bulb temperature of 0 ◦C, which was experimentally adjusted by Ferrel [31]. Therefore, the moist air density can be determined via barometric pressure, dry and wet bulb temperatures, as shown in the experimental set up description. A similar procedure to that described by Picard et al. [32] was applied to the last three equations for determining the moist air density uncertainty u(ρ). The determination of the moist air dynamic viscosity is required to assess the Reynolds number in the wind tunnel. The viscosity can be calculated using the formulation recommended by Zuckerwar and Meredith [33] for air applications considering atmospheric pressure, a temperature between 10 ◦C and 50 ◦C, and a relative humidity in the range from 0.3% to 92%. Regarding the conventional linear regression (Equation (13)), the slope A and oﬀset B variances and covariances can be obtained via the simpliﬁed formulation proposed by the GUM [29]. The quadratic formulation presented in this work (described in Section 2.2) can be used, as well, to assess the linear regression by replacing y2 by A, y1 by B, and ∆pk by Vk, and making y3 null. 2.3. Wind Speed and Dynamic Pressure Uncertainty In the present work, both methods, the conventional linear regression and the quadratic formulation, were applied and the results show similar values. Finally, the squared combined uncertainty of the calibration based on “Wind speed Equations (12) and (13)” (conventional measurement procedure) was obtained taking into account the terms of the second and third columns of Table 1. u2 c(V) = u2 kf · ∂Vs ∂kf 2 + u2(kc)· ∂Vs ∂kc 2 + u2(Ch)· ∂Vs ∂Ch 2 + u2(ρ)· ∂Vs ∂ρ 2 + u2(∆pPt) · ∂Vs ∂∆pPt 2 + 2·u2 ˆV . (18) (18) The squared combined uncertainty of the wind dynamic pressure measurements ∆p based on Equations (7) and (14) (alternative measurement procedure) was obtained taking into account the terms of the second and fourth columns of Table 1. u2 c(∆p) = u2 kf · ∂∆ps ∂kf !2 + u2(kc)· ∂∆ps ∂kc !2 + u2(Ch)· ∂∆ps ∂Ch !2 + u2(∆pPt)· ∂∆ps ∂∆pPt !2 + 2·u2 ˆ∆p . (19) (19) In the last two equations, the regression uncertainty applies twice; one by the calibration process and another one for in-ﬁeld measurements. In the last two equations, the regression uncertainty applies twice; one by the calibration process and another one for in-ﬁeld measurements. 2.4. Wind Power Density Uncertainty The wind power density can be obtained by means of the air density and wind speed measurements (conventional method) or by means of wind dynamic pressure and wind speed measurements (alternative method, Equation (3)). Note that although both equations estimate Pw, they have diﬀerent uncertainties (Table 2). The conventional procedure to estimate the wind power density with an “AS-Cup anemometer” requires to specify ρ: the air density measured by the user where the turbine is located. In the present study, a standard uncertainty of ±0.03 kg/m3 for the user air density is considered. The IEC 61400-12-1 international standard [9] recommends the air density normalization for power measurements, in an eﬀort to compensate for the atmospheric variations of air density at the wind turbine location. During AEP assessment, a measurement campaign was performed, which included the measurement 9 of 18 Sensors 2019, 19, 2029 of air density in the range from 4 m/s to 16 m/s (at least 30 measurements must be recorded in each step of 0.5 m/s). Each air density measure is the mean value of those observations obtained in a continuous interval of 10 minutes and keeps a linear relationship with the turbine power output during that interval. The normalization determines the output power during each interval of 10 minutes considering a reference air density. The IEC standard proposes as the reference air density the mean value of all air density measures collected during the measurement campaign, or alternatively, a nominal air density pre-deﬁned for the location. The previous version of the IEC standard establishes that the normalization is not necessary if the actual average value of air density is within the reference density ±0.05 kg/m3, so that a value of ±0.03 kg/m3 (that we used in Pw examples) means a density measure with low uncertainty, and therefore, it is a favorable situation for the conventional measurement method. The squared combined uncertainty of the wind power density measured with an AS-Cup anemometer is: 1 9 u2 AS(Pw) = u2(ρ)·1 4V6 + u2 c(V)·9 4·ρ2·V4, (20) (20) On the other hand, the squared combined uncertainty of the wind power density measured with a DYP-Cup anemometer is: On the other hand, the squared combined uncertainty of the wind power density measured with a DYP-Cup anemometer is: u2 DYP(Pw) = u2 c(V)·∆p2 + u2 c(∆p)· V2. (21) u2 DYP(Pw) = u2 c(V)·∆p2 + u2 c(∆p)· V2. (21) Table 2. 2.4. Wind Power Density Uncertainty Standard uncertainty and sensibility coeﬃcient of the wind power density equations. Parameters Standard Uncertainty Sensibility Coeﬃcients: Equation (20) Sensibility Coeﬃcients: Equation (3) User air density ρ u(ρ) ∂Pw/∂ρ = 1/2 V3 Wind speed V uc(V) ∂Pw/∂V = 3/2 ρ V2 ∂Pw/∂V= ∆p Anemometer dynamic pressure ∆p uc (∆p) ∂Pw/∂∆p = V Table 2. Standard uncertainty and sensibility coeﬃcient of the wind power density equations. 3.1. Cup Anemometer Regression: Quadratic vs Linear While wind speed and cup anemometer rotation frequency are linearly related, dynamic pressure and rotation frequency has a quadratic relation. An ANOVA study of the experimental data suggests that a quadratic polynomial calibration curve is the best solution to ﬁt the wind dynamic pressure as a function of the anemometer rotation frequency. Sensors 2019, 19, x 10 of 18 greater than 0.99995 for both measurement methods (Figure 5), which is the limit established by the annex F of the IEC 61400 12 1 international standard [9] for a high quality regression The idea is to compare the quadratic regression to the linear regression. To reach this target, we compute the correlation coeﬃcients and the uncertainty of both regressions. The same 29 experimental measurements were assessed for both regressions. The correlation coeﬃcient r is greater than 0.99995 for both measurement methods (Figure 5), which is the limit established by the annex F of the IEC 61400-12-1 international standard [9] for a high-quality regression. annex F of the IEC 61400-12-1 international standard [9] for a high-quality regression. For both methods, the values of relative uncertainty are lower than 0.4% and decrease to 0.05% at the maximum rotational frequency tested. The DYP-cup anemometer shows higher relative uncertainty at low rotational frequency (0.4% DYP vs 0.2% AS at 30 rad/s). On the other hand, regression uncertainty curves are not monotone functions: they decrease at low fr and grow at high fr. The differences observed in the regression uncertainty shape are consequence of the variance and b co a ia ce co t ibutio s ( igu e 6) (a) (b) Figure 5. Regression uncertainty: (a) Linear regression between “Wind speed” and “Anemometer rotation frequency” (AS-Cup anemometer measurement method), (b) Quadratic regression between “Wind dynamic pressure” and “Anemometer rotation frequency” (DYP-Cup anememometer measurement method). 3. Results and Discussion This paper proposes a novel calibration for a cup-anemometer based on a quadratic regression. Firstly, this Section discusses how good the regression is compared with that of a conventional calibration (Section 3.1). Secondly, it shows the uncertainty for the ∆p and V estimations obtained with the proposed and the conventional calibration, respectively (Section 3.2). Finally, we demonstrate that a double calibration leads to an uncertainty reduction in Pw measurements, as density does not appear in the proposed indirect measurement procedure (Section 3.3). All computations showed in this Section have been performed using Excel® and the scientiﬁc computational software Mathematica® in a 2 kernels Intel Core i7 2.9 GHz CPU. The calibration process was done via 29 experimental measurements of the variables: wind dynamic pressure and cup anemometer rotation frequency. To correct the dynamic pressure lectures obtained with the Pitot tube, the following parameters were deﬁned according the IEC 61400-12-1 international standard [9] recommendations: kf =1.00625 u kf = 2.4 10−4; kc =1.004 u(kc) = 7 10−4; Ch = 0.997 u(Ch) = 10−3 In order to compare the new measurement method with the conventional one, it is necessary to determine the wind speed corresponding to every wind dynamic pressure value. To reach this target, the last three parameters are used in the Equation (12), where the air density measured within the wind tunnel was obtained during the experiment from ambient temperatures measures and the ambient pressure, resulting in: ρ = 1.168 kg/m3; u(ρ) = 1.45 × 10−3 kg/m3. The air dynamic viscosity measured within the wind tunnel was obtained as well: µ =1.84 × 10-5 kg/m·s. It is noteworthy that Sensors 2019, 19, 2029 10 of 18 the results take into account the previously deﬁned air density value and a wind speed of V ∈[4 m/s, 16 m/s], recommended in the annex F of the IEC 61400-12-1 international standard [9]. the results take into account the previously deﬁned air density value and a wind speed of V ∈[4 m/s, 16 m/s], recommended in the annex F of the IEC 61400-12-1 international standard [9]. We assumed a constant air density in our proposed calibration. The reason for this assumption is that the standard about anemometers [9] only requires the density value during calibration, and we were focused on the calibration procedure. 3. Results and Discussion For in-ﬁeld Pw measures, it is normal practice to consider a constant density value, as this simpliﬁes the measurement procedure and the proposed approach. Because of that, we also assumed a constant density for the Pw examples showed in this Section. This assumption helps to understand the advantages of the proposed double calibration; however, note that recent studies, such as the work of Ulazia et al. [34], have warned about the inﬂuence of density variations in ﬁeld Pw assessment. Since the proposed calibration has been tested for one density condition, further works could extend it to the case of a variable density, helping to understand its inﬂuence in ﬁeld Pw measurements. 3.1. Cup Anemometer Regression: Quadratic vs Linear 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% 0.30% 0.35% 0.40% 2 4 6 8 10 12 14 16 18 20 30 40 50 60 70 80 90 100 110 Relative regression uncertainty Wind speed [m/s] Anemometer rotation frequency [rad/s] Linear regression Relative uncertainty 20 30 40 50 60 70 80 90 100 110 0 0.002 0.004 0.006 0.008 0.01 0.012 Linear regression uncertainty [m/s] 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% 0.30% 0.35% 0.40% 0 20 40 60 80 100 120 140 160 180 20 30 40 50 60 70 80 90 100 110 Relative regression uncertainty Wind Dynamic pressure [Pa] Anemometer rotation frequency [rad/s] Quadratic regression Relative uncertainty + 1.1482 20 30 40 50 60 70 80 90 100 110 0 0.02 0.04 0.06 0.08 0.1 0.12 Quadratic regression uncertainty [Pa] Figure 5. Regression uncertainty: (a) Linear regression between “Wind speed” and “Anemometer rotation frequency” (AS-Cup anemometer measurement method), (b) Quadratic regression between “Wind dynamic pressure” and “Anemometer rotation frequency” (DYP-Cup anememometer measurement method). 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% 0.30% 0.35% 0.40% 0 20 40 60 80 100 120 140 160 180 20 30 40 50 60 70 80 90 100 110 Relative regression uncertainty Wind Dynamic pressure [Pa] Anemometer rotation frequency [rad/s] Quadratic regression Relative uncertainty + 1.1482 0.00% 0.05% 0.10% 0.15% 0.20% 0.25% 0.30% 0.35% 0.40% 2 4 6 8 10 12 14 16 18 20 30 40 50 60 70 80 90 100 110 Relative regression uncertainty Wind speed [m/s] Anemometer rotation frequency [rad/s] Linear regression Relative uncertainty Anemometer rotation frequency [rad/s] (a) 20 30 40 50 60 70 80 90 100 110 0 0.002 0.004 0.006 0.008 0.01 0.012 Linear regression uncertainty [m/s] (b) 20 30 40 50 60 70 80 90 100 110 0 0.02 0.04 0.06 0.08 0.1 0.12 Quadratic regression uncertainty [Pa] (b) (a) Figure 5. Regression uncertainty: (a) Linear regression between “Wind speed” and “Anemometer rotation frequency” (AS-Cup anemometer measurement method), (b) Quadratic regression between “Wind dynamic pressure” and “Anemometer rotation frequency” (DYP-Cup anememometer measurement method). Figure 5. Regression uncertainty: (a) Linear regression between “Wind speed” and “Anemometer rotation frequency” (AS-Cup anemometer measurement method), (b) Quadratic regression between “Wind dynamic pressure” and “Anemometer rotation frequency” (DYP-Cup anememometer measurement method). 11 of 18 Sensors 2019, 19, 2029 For both methods, the values of relative uncertainty are lower than 0.4% and decrease to 0.05% at the maximum rotational frequency tested. 3.1. Cup Anemometer Regression: Quadratic vs Linear Wind Dynamic Pressure and Wind Speed Uncertainty 3.2. Wind Dynamic Pressure and Wind Speed Uncertain 3.2. Wind Dynamic Pressure and Wind Speed Uncertainty 3.1. Cup Anemometer Regression: Quadratic vs Linear The DYP-cup anemometer shows higher relative uncertainty at low rotational frequency (0.4% DYP vs 0.2% AS at 30 rad/s). On the other hand, regression uncertainty curves are not monotone functions: they decrease at low fr and grow at high fr. The diﬀerences observed in the regression uncertainty shape are consequence of the variance and covariance contributions (Figure 6). Sensors 2019, 19, x 11 of 18 (a) (b) Figure 6. Regression uncertainty budget. (a) Linear regression (AS-Cup anemometer measurement method), (b) quadratic regression (DYP-Cup anemometer measurement method). -0.0008 -0.0006 -0.0004 -0.0002 0 0.0002 0.0004 0.0006 20 30 40 50 60 70 80 90 100 110 BUDGET of squared uncertainty for WIND SPEED LINEAR regression [m2/s2] Anemometer rotation frequency [rad/s] Contribution A variance Contribution A,B covariance Contribution B variance -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 20 30 40 50 60 70 80 90 100 110 BUDGET of squared uncertainty for WIND DYNAMIC PRESSURE QUADRATIC regression [Pa2] Anemometer rotation frequency [rad/s] Contribution y1 variance Contribution y1,y2 covariance Contribution y2 variance Contribution y1,y3 covariance Contribution y3 variance Contribution y2,y3 covariance Figure 6. Regression uncertainty budget. (a) Linear regression (AS-Cup anemometer measurement method), (b) quadratic regression (DYP-Cup anemometer measurement method). (a) -0.0008 -0.0006 -0.0004 -0.0002 0 0.0002 0.0004 0.0006 20 30 40 50 60 70 80 90 100 110 BUDGET of squared uncertainty for WIND SPEED LINEAR regression [m2/s2] Anemometer rotation frequency [rad/s] Contribution A variance Contribution A,B covariance Contribution B variance (b) -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 20 30 40 50 60 70 80 90 100 110 BUDGET of squared uncertainty for WIND DYNAMIC PRESSURE QUADRATIC regression [Pa2] Anemometer rotation frequency [rad/s] Contribution y1 variance Contribution y1,y2 covariance Contribution y2 variance Contribution y1,y3 covariance Contribution y3 variance Contribution y2,y3 covariance (b) -2.5 -2.0 -1.5 -1.0 -0.5 0.0 20 30 40 50 60 70 80 90 100 110 BUDGET of squared uncertainty for WIND DYNAMIC PRESSURE QUADRATIC regressi Anemometer rotation frequency [rad/s] Contribution y1 variance Contribution y1,y2 covariance Contribution y2 variance Contribution y1,y3 covariance Contribution y3 variance Contribution y2,y3 covariance (b) (a) Figure 6. Regression uncertainty budget. (a) Linear regression (AS-Cup anemometer measurement method), (b) quadratic regression (DYP-Cup anemometer measurement method). Figure 6. Regression uncertainty budget. (a) Linear regression (AS-Cup anemometer measurement method), (b) quadratic regression (DYP-Cup anemometer measurement method). 3.2. Wind Dynamic Pressure and Wind Speed Uncertain 3.2. 3.2. Wind Dynamic Pressure and Wind Speed Uncertain 3.2. Wind Dynamic Pressure and Wind Speed Uncertainty (a) 0.11 0.09 0.08 0.07 0.06 0.06 0.05 0.05 0.05 0.05 0.05 0.06 0.06 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 2 4 6 8 10 12 14 16 4 5 6 7 8 9 10 11 12 13 14 15 16 Wind speed uncertainty [m/s] Wind speed [m/s] Wind speed [m/s] Calibration uncertainty with the WIND SPEED equations Wind speed Sensors 2019, 19, x Fi 8 h h i f (b) 0.55 0.55 0.56 0.58 0.60 0.62 0.64 0.67 0.69 0.72 0.75 0.78 0.82 0 0.5 1 1.5 2 2.5 3 3.5 4 0 20 40 60 80 100 120 140 160 4 5 6 7 8 9 10 11 12 13 14 15 16 Wind dynamic pressure uncertainty [Pa] Wind dynamic pressure [Pa] Wind speed [m/s] Calibration uncertainty with the WIND DYNAMIC PRESSURE equations Wind dynamic Pressure 12 of 18 Wi d d ti ” d Fi 9 h th (b) 0.55 0.55 0.56 0.58 0.60 0.62 0.64 0.67 0.69 0.72 0.75 0.78 0.82 0 0.5 1 0 20 40 4 5 6 7 8 9 10 11 12 13 14 15 16 Wind dyna Wi Wind speed [m/s] Calibration uncertainty with the WIND DYNAMIC PRESSURE equations Wind dynamic Pressure 12 of 18 Wi d d ti ” d Fi 9 h th (b) (a) Figure 7. Measurement uncertainty: (a) “Wind speed equations” (AS-Cup anemometer measurement method), (b) “Wind dynamic pressure equations” (DYP-Cup anemometer measurement method). Figure 7. Measurement uncertainty: (a) “Wind speed equations” (AS-Cup anemometer measurement method), (b) “Wind dynamic pressure equations” (DYP-Cup anemometer measurement method). Figure 8 shows the parameters uncertainty for “Wind speed equations”, and Figure 9 shows the ameters uncertainty for “Wind dynamic pressure equations”. It is observed that, in both cases, the nometer uncertainty (uncertainty of the standard: u(pPt)) is the main contribution. The calibration uncertainty for both methods (conventional and alternative) was obtained rom the uncertainties combination of several parameters that appear in the applied measurement method equations. (a) (b) Figure 8. Calibration by means of “Wind speed equations” (AS-Cup anemometer measurement procedure): (a) wind speed uncertainty and its budget, (b) components of the squared uncertainty. 3.2. Wind Dynamic Pressure and Wind Speed Uncertain 3.2. Wind Dynamic Pressure and Wind Speed Uncertainty Figures 8b and 9b portrait the speciﬁc budget in logarithmic scale and allow knowing how each uncertainty component changes with the wind velocity. (a) (b) Figure 7. Measurement uncertainty: (a) “Wind speed equations” (AS-Cup anemometer measurement method), (b) “Wind dynamic pressure equations” (DYP-Cup anemometer measurement method). 0.11 0.09 0.08 0.07 0.06 0.06 0.05 0.05 0.05 0.05 0.05 0.06 0.06 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 2 4 6 8 10 12 14 16 4 5 6 7 8 9 10 11 12 13 14 15 16 Wind speed uncertainty [m/s] Wind speed [m/s] Wind speed [m/s] Calibration uncertainty with the WIND SPEED equations Wind speed 0.55 0.55 0.56 0.58 0.60 0.62 0.64 0.67 0.69 0.72 0.75 0.78 0.82 0 0.5 1 1.5 2 2.5 3 3.5 4 0 20 40 60 80 100 120 140 160 4 5 6 7 8 9 10 11 12 13 14 15 16 Wind dynamic pressure uncertainty [Pa] Wind dynamic pressure [Pa] Wind speed [m/s] Calibration uncertainty with the WIND DYNAMIC PRESSURE equations Wind dynamic Pressure Figure 7. Measurement uncertainty: (a) “Wind speed equations” (AS-Cup anemometer measurement method), (b) “Wind dynamic pressure equations” (DYP-Cup anemometer measurement method). Sensors 2019, 19, x 12 of 18 Figure 8 shows the parameters uncertainty for “Wind speed equations”, and Figure 9 shows the parameters uncertainty for “Wind dynamic pressure equations”. It is observed that, in both cases, the manometer uncertainty (uncertainty of the standard: u(pPt)) is the main contribution. 3.2. Wind Dynamic Pressure and Wind Speed Uncertain 3.2. Wind Dynamic Pressure and Wind Speed Uncertainty The measurement uncertainty provided by a cup anemometer with a calibration based on the aforementioned quadratic relationship is observed in Figure 7b. The order of magnitude of the values of its relative uncertainty is similar to that obtained by means of the annex F found in the IEC 61400- 12-1 international standard [9], whose graphic is shown in Figure 7a. The measurement uncertainty provided by a cup anemometer with a calibration based on the aforementioned quadratic relationship is observed in Figure 7b. The order of magnitude of the values of its relative uncertainty is similar to that obtained by means of the annex F found in the IEC 61400-12-1 international standard [9], whose graphic is shown in Figure 7a. g p g 0.35 0.4 14 16 3.5 4 140 160 a] The calibration uncertainty for both methods (conventional and alternative) was obtained from the uncertainties combination of several parameters that appear in the applied measurement method equations. 0.25 0.3 10 12 ertainty [m/s] ed [m/s] 2.5 3 100 120 re uncertainty [Pa c pressure [Pa] Figure 8 shows the parameters uncertainty for “Wind speed equations”, and Figure 9 shows the parameters uncertainty for “Wind dynamic pressure equations”. It is observed that, in both cases, the manometer uncertainty (uncertainty of the standard: u(∆pPt)) is the main contribution. 12 of 18 e of the 12 of 18 e of the Sensors 2019, 19, 2029 aforementioned q alue of it elati (a) (b) Figure 7. Measurement uncertainty: (a) “Wind speed equations” (AS-Cup anemometer measurement method), (b) “Wind dynamic pressure equations” (DYP-Cup anemometer measurement method). The calibration uncertainty for both methods (conventional and alternative) was obtained from the uncertainties combination of several parameters that appear in the applied measurement method equations. 3.2. Wind Dynamic Pressure and Wind Speed Uncertain 3.2. Wind Dynamic Pressure and Wind Speed Uncertainty 0.11 0.09 0.08 0.07 0.06 0.06 0.05 0.05 0.05 0.05 0.05 0.06 0.06 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 2 4 6 8 10 12 14 16 4 5 6 7 8 9 10 11 12 13 14 15 16 Wind speed uncertainty [m/s] Wind speed [m/s] Wind speed [m/s] Calibration uncertainty with the WIND SPEED equations Wind speed 0.55 0.55 0.56 0.58 0.60 0.62 0.64 0.67 0.69 0.72 0.75 0.78 0.82 0 0.5 1 1.5 2 2.5 3 3.5 4 0 20 40 60 80 100 120 140 160 4 5 6 7 8 9 10 11 12 13 14 15 16 Wind dynamic pressure uncertainty [Pa] Wind dynamic pressure [Pa] Wind speed [m/s] Calibration uncertainty with the WIND DYNAMIC PRESSURE equations Wind dynamic Pressure Figure 7. Measurement uncertainty: (a) “Wind speed equations” (AS-Cup anemometer measurement method), (b) “Wind dynamic pressure equations” (DYP-Cup anemometer measurement method). Sensors 2019, 19, x 12 of 18 Figure 8 shows the parameters uncertainty for “Wind speed equations”, and Figure 9 shows the parameters uncertainty for “Wind dynamic pressure equations”. It is observed that, in both cases, the manometer uncertainty (uncertainty of the standard: u(pPt)) is the main contribution. (a) (b) Figure 8. Calibration by means of “Wind speed equations” (AS-Cup anemometer measurement procedure): (a) wind speed uncertainty and its budget, (b) components of the squared uncertainty. Figures 8a and 9a show the calibration uncertainty budget in percentage. Figures 8b and 9b portrait the specific budget in logarithmic scale and allow knowing how each uncertainty component changes with the wind velocity. 84% 86% 88% 90% 92% 94% 96% 98% 100% 0 0.02 0.04 0.06 0.08 0.1 0.12 4.0 7.0 10.0 13.0 16.0 Uncertainty budget for cup anemometer calibration by means of "Wind speed equations" Wind speed uncertainty [m/s] Wind Speed [m/s] Manometer: Pressure differential Linear Regression Air Density Pitot tube Head Coefficient Wind Tunnel Calibration Blockage Wind Tunnel Wind speed uncertainty 0.0000001 0.000001 0.00001 0.0001 0.001 0.01 0.1 4.0 7.0 10.0 13.0 16.0 Budget of logarithmic squared uncertainty for "Wind speed equations" [m2/s2] Wind speed [m/s] Manometer: Pressure differential Linear regression Air density Pitot tube Head Coefficient Figure 8. Calibration by means of “Wind speed equations” (AS-Cup anemometer measurement procedure): (a) wind speed uncertainty and its budget, (b) components of the squared uncertainty. Figures 8a and 9a show the calibration uncertainty budget in percentage. 3.2. Wind Dynamic Pressure and Wind Speed Uncertain 3.2. Wind Dynamic Pressure and Wind Speed Uncertainty 84% 86% 88% 90% 92% 94% 96% 98% 100% 0 0.02 0.04 0.06 0.08 0.1 0.12 4.0 7.0 10.0 13.0 16.0 Uncertainty budget for cup anemometer calibration by means of "Wind speed equations" Wind speed uncertainty [m/s] Wind Speed [m/s] Manometer: Pressure differential Linear Regression Air Density Pitot tube Head Coefficient Wind Tunnel Calibration Blockage Wind Tunnel Wind speed uncertainty 0.0000001 0.000001 0.00001 0.0001 0.001 0.01 0.1 4.0 7.0 10.0 13.0 16.0 Budget of logarithmic squared uncertainty for "Wind speed equations" [m2/s2] Wind speed [m/s] Manometer: Pressure differential Linear regression Air density Pitot tube Head Coefficient Figure 8. Calibration by means of “Wind speed equations” (AS-Cup anemometer measurement procedure): (a) wind speed uncertainty and its budget, (b) components of the squared uncertainty. onventional and alternative) was obtaine rs that appear in the applied measuremen (b) 0.0000001 0.000001 0.00001 0.0001 0.001 0.01 0.1 4.0 7.0 10.0 13.0 16.0 Budget of logarithmic squared uncertainty for "Wind speed equations" [m2/s2] Wind speed [m/s] Manometer: Pressure differential Linear regression Air density Pitot tube Head Coefficient The calibration uncertainty for both methods rom the uncertainties combination of several param method equations. (a) 84% 86% 88% 90% 92% 94% 96% 98% 100% 0 0.02 0.04 0.06 0.08 0.1 0.12 4.0 7.0 10.0 13.0 16.0 Uncertainty budget for cup anemometer calibration by means of "Wind speed equations" Wind speed uncertainty [m/s] Wind Speed [m/s] Manometer: Pressure differential Linear Regression Air Density Pitot tube Head Coefficient Wind Tunnel Calibration Blockage Wind Tunnel Wind speed uncertainty (b) (a) Figure 8. Calibration by means of “Wind speed equations” (AS-Cup anemometer measurement procedure): (a) wind speed uncertainty and its budget, (b) components of the squared uncertainty. Figure 8. Calibration by means of “Wind speed equations” (AS-Cup anemometer measurement procedure): (a) wind speed uncertainty and its budget, (b) components of the squared uncertainty. Figures 8a and 9a show the calibration uncertainty budget in percentage. Figures 8b and 9b portrait the specific budget in logarithmic scale and allow knowing how each uncertainty component changes with the wind velocity. Figures 8a and 9a show the calibration uncertainty budget in percentage. Figures 8b and 9b portrait the speciﬁc budget in logarithmic scale and allow knowing how each uncertainty component changes with the wind velocity. Sensors 2019, 19, 2029 Figures 8a an portrait the specific 13 of 18 and 9b ponent (a) (b) Figure 9. 3.2. Wind Dynamic Pressure and Wind Speed Uncertain 3.2. Wind Dynamic Pressure and Wind Speed Uncertainty Calibration by means of “Wind dynamic pressure equations” (DYP-Cup anemometer measurement method): (a) wind speed uncertainty and its budget, (b) components of the squared uncertainty. 84% 86% 88% 90% 92% 94% 96% 98% 100% 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 4.0 7.0 10.0 13.0 16.0 Uncertainty budget for cup anemometer calibration by means of "Wind dynamic pressure equations" Wind dynamic Pressure uncertainty [Pa] Wind Speed [m/s] Manometer: Pressure differential Quadratic Regression Pitot tube Head Coefficient Wind Tunnel Calibration Blockage Wind Tunnel Wind dynamic pressure uncertainty 0.000001 0.00001 0.0001 0.001 0.01 0.1 1 4.0 7.0 10.0 13.0 16.0 Budget of logarithmic squared uncertainty for "Wind dynamic pressure equations" [Pa2] Wind speed [m/s] Manometer: Pressure differential Quadratic regression Pitot tube Head Coefficient Wind Tunnel Calibration Figure 9. Calibration by means of “Wind dynamic pressure equations” (DYP-Cup anemometer measurement method): (a) wind speed uncertainty and its budget, (b) components of the squared uncertainty. 0.000001 0.00001 0.0001 0.001 0.01 0.1 1 4.0 7.0 10.0 13.0 16.0 Budget of logarithmic squared uncertainty for "Wind dynamic pressure equations" [Pa2] Wind speed [m/s] 84% 86% 88% 90% 92% 94% 96% 98% 100% 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 4.0 7.0 10.0 13.0 16.0 Uncertainty budget for cup anemometer calibration by means of "Wind dynamic pressure equations" Wind dynamic Pressure uncertainty [Pa] Wind Speed [m/s] (a) Manometer: Pressure differential Quadratic Regression Pitot tube Head Coefficient Wind Tunnel Calibration Blockage Wind Tunnel Wind dynamic pressure uncertainty (b) (a) Figure 9. Calibration by means of “Wind dynamic pressure equations” (DYP-Cup anemometer measurement method): (a) wind speed uncertainty and its budget, (b) components of the squared uncertainty. Figure 9. Calibration by means of “Wind dynamic pressure equations” (DYP-Cup anemometer measurement method): (a) wind speed uncertainty and its budget, (b) components of the squared uncertainty. The evolution of the relative uncertainty of each parameter that contributes to the calibrations is similar (Figures 8b and 9b), except for the case of density, which only appears in the conventional measurement procedure. Density contribution is less than 3% inside the wind speed measurement interval. It must be noted that all these uncertainties are computed considering that the air density The evolution of the relative uncertainty of each parameter that contributes to the calibrations is similar (Figures 8b and 9b), except for the case of density, which only appears in the conventional measurement procedure. 3.2. Wind Dynamic Pressure and Wind Speed Uncertain 3.2. Wind Dynamic Pressure and Wind Speed Uncertainty Density contribution is less than 3% inside the wind speed measurement interval. It must be noted that all these uncertainties are computed considering that the air density value during the user measurement process is the same as the one in the calibration process. In other words, a data normalization is required. Inside the wind speed measurement range, it is important to note that the regression uncertainty is less than 3% for the AS-Cup anemometer measurement method and it is less than 6% for the DYP-Cup anemometer measurement method. Because the correlation coeﬃcient is greater than 0.99995, the contribution of the regression to the calibration uncertainty is less than that of the standard device. 3.3. Power Density Uncertainty: Alternative and Conventional Procedures In the present section, we are going to determine the combined uncertainty of the indirect measure of the air power density for both methods under consideration (AS-Cup anemometer and DYP-Cup anemometer). Once the calibration uncertainty is known, it is possible to compare the eﬀect of using the DYP-Cup anemometer (Equation (3)) to determine the wind power density Pw, respect to the conventional computation based on air density and wind speed (Pw = 0.5·ρ·V3). According to Figure 10, the ratio of Pw uncertainty obtained with an AS-Cup anemometer to the Pw uncertainty obtained with a DYP-Cup anemometer evolves linearly with the air speed by a factor of 1/4. Let the density uncertainty be the main factor in Equation (20), and let the dynamic pressure uncertainty be the main factor in Equation (21) then, u2 AS ≃u2(ρ)· 1 4·V6, u2 DYP ≃u2 c(∆p)·V2, where u(ρ) = 0.03 kg/m3 and u(∆p) = 0.06·V2 Pa. Therefore, uAS/uDYP ≃V/4, which means that uAS is two times higher for 8 m/s, three times higher for 12 m/s and four times higher for 16 m/s than uDYP. We conclude that, in general, uAS ~ V3 and uDYP ~ V2; thus, the DYP-Cup anemometer provides less uncertainty than the AS-Cup anemometer. 14 of 18 an 𝑢஽௒௉. provides Sensors 2019, 19, 2029 two times higher f We conclude that, y p Figure 10. Wind power density uncertainty by means of the alternative and the conventional method. 3 4 6 8 10 14 18 23 29 36 45 55 67 2 3 4 4 5 6 7 8 9 11 12 14 16 0 20 40 60 80 100 120 140 160 180 200 0 500 1000 1500 2000 2500 4 5 6 7 8 9 10 11 12 13 14 15 16 Wind power density uncertainty with two different measurement methods [W/m2] Wind power density [W/m2] Wind Speed [m/s] Uncertainty for AS-Cup anemometer measurement system Uncertainty for DYP-Cup anemometer measurement system Wind power density Figure 10. Wind power density uncertainty by means of the alternative and the conventional method. Figure 10. Wind power density uncertainty by means of the alternative and the conventional method. Figure 10. Wind power density uncertainty by means of the alternative and the conventional method. When wind power in a field site is determined using the conventional method, it is necessary to determine the air density at that specific location, “user air density”. 3.3. Power Density Uncertainty: Alternative and Conventional Procedures In the present work, this value is considered equal to the air density during the wind tunnel tests; however, the user air density When wind power in a ﬁeld site is determined using the conventional method, it is necessary to determine the air density at that speciﬁc location, “user air density”. In the present work, this value is considered equal to the air density during the wind tunnel tests; however, the user air density uncertainty is greater, as is explained in Section 2.4. Therefore, ρ = 1.168 kg/m3 with u(ρ) = 0.03 kg/m3. ρ g The magnitude of the sensitivity coeﬃcients (mainly the sensitivity coeﬃcient of density) is the reason for the large diﬀerence, in terms of the squared uncertainty, between the Pw measurement procedure based on a conventional calibration and the alternative procedure based on a double calibration. A system based on a conventional calibration requires the computation of the user air density uncertainty (Table 2 and Equation (20)), whose sensitivity coeﬃcient grows rapidly with air speed because it is a function of wind speed raised to the third power (sixth power in a squared uncertainty budget). Figure 11 displays Pw uncertainty desegregated to evaluate the parameters contribution using the conventional and the proposed estimation procedure. It is noteworthy that density rapidly becomes the highest contribution to uncertainty with the AS-cup anemometer, while dynamic pressure contribution slowly decreases for the DYP-cup anemometer. This result points out that, even without other inﬂuence variables, density plays a key role in Pw measurements and explains the uncertainty reduction with the proposed approach. Moreover, this result could support future works to focus on other phenomena that appear in ﬁeld measurements, such as turbulence. The alternative procedure provides a chance to improve energy production predictions of wind farms, which supports the recommendations of Tindal et al. [35], as they foster researchers to validate advanced measurement techniques, since they observed, for a data base of 156 wind farms, that the average actual production was 93.3 % of pre-construction projections. 15 of 18 validate that the Sensors 2019, 19, 2029 farms, which supp advanced measure g p p p j Figure 11. Power density uncertainty budget by means of “power density equations based on wind speed” (AS-Cup, conventional method) and “power density equations based on wind dynamic pressure” (DYP-Cup, alternative method). 4 Conclusions 4. Conclusions 4. Conclusions This work shows an alternative calibration for cup-anemometers that reduces the uncertainty of the wind power density measurements. We demonstrated that a cup anemometer can measure dynamic pressure as well as wind speed via a double calibration. Therefore, unlike the conventional method, which calculates the wind power density as a function of air density and wind speed (Pw = 0.5··V3), the double calibration provides the wind power density as a function of dynamic pressure and wind speed (Pw = p·V) obtained from the anemometer rotation frequency. The main advantage of this measurement method is that the wind power density equation does not require measuring the moist air density (considering the repeatability condition of measurement) This work shows an alternative calibration for cup-anemometers that reduces the uncertainty of the wind power density measurements. We demonstrated that a cup anemometer can measure dynamic pressure as well as wind speed via a double calibration. Therefore, unlike the conventional method, which calculates the wind power density as a function of air density and wind speed (Pw = 0.5·ρ·V3), the double calibration provides the wind power density as a function of dynamic pressure and wind speed (Pw = ∆p·V) obtained from the anemometer rotation frequency. The main advantage of this measurement method is that the wind power density equation does not require measuring the moist air density (considering the repeatability condition of measurement). moist air density (considering the repeatability condition of measurement). The relation of dynamic pressure and rotation frequency p(fr) follows a quadratic function: “dynamic pressure calibration”; to determine its uncertainty, it is required to assess the uncertainty of a quadratic regression. While the uncertainty computation of a linear regression, such as that used in the “wind speed calibration”, is described in the “Guide to the expression of uncertainty in The relation of dynamic pressure and rotation frequency ∆p(fr) follows a quadratic function: “dynamic pressure calibration”; to determine its uncertainty, it is required to assess the uncertainty of a quadratic regression. While the uncertainty computation of a linear regression, such as that used in the “wind speed calibration”, is described in the “Guide to the expression of uncertainty in measurement”, this is not the case of the quadratic one. In order to facilitate the reproducibility of the proposed method, the computation of the uncertainty for a second order polynomial has been included in detail. 3.3. Power Density Uncertainty: Alternative and Conventional Procedures 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 4.0 7.0 10.0 13.0 16.0 BUDGETS of wind power density uncertainty by means of two different measurement methods [W/m2] Uncertainty of wind power density [W/m2] Wind Speed [m/s] User air density Wind speed (AS-Cup system) Wind dynamic pressure Wind speed (DYP-Cup system) AS-Cup anemometer measurement system DYP-Cup anemometer measurement system Figure 11. Power density uncertainty budget by means of “power density equations based on wind speed” (AS-Cup, conventional method) and “power density equations based on wind dynamic pressure” (DYP-Cup, alternative method). Figure 11. Power density uncertainty budget by means of “power density equations based on wind speed” (AS-Cup, conventional method) and “power density equations based on wind dynamic pressure” (DYP-Cup, alternative method). Figure 11. Power density uncertainty budget by means of “power density equations based on wind speed” (AS-Cup, conventional method) and “power density equations based on wind dynamic pressure” (DYP-Cup, alternative method). 4 Conclusions 4. Conclusions We have demonstrated that the relative uncertainty values of the linear and quadratic regressions are similar (Figure 5). The uncertainty components common to the wind speed measurements based on conventional calibration and the dynamic pressure measurements based on the proposed calibration are similar. The regression uncertainties represent less than 6% of the uncertainty budget, although they depend on the quality of the regression. In this study, the correlation coeﬃcients are greater than 0.99995. The manometer uncertainty (Pitot tube, which is the calibration standard) used to measure the diﬀerence between total and static pressure, represents more than 90% in the calibration uncertainty budget, regardless of the measurement system used for the calibration process. Once the double cup anemometer calibration is accomplished, the product of ∆p and V provides the wind power density Pw. The new measurement procedure does not need any cup anemometer modiﬁcation. Double calibration can be done in the same wind tunnel. Knowing the calibration functions for ∆p and V, in-ﬁeld measurements only need the anemometer rotation frequency determination. This result can be useful to improve the energy production predictions of wind farms. Sensors 2019, 19, 2029 16 of 18 16 of 18 Noteworthy is the high reduction of power density uncertainty addressed by the alternative measurement method. For those applications where a cup anemometer is used to measure wind power, the proposed indirect measurement method provides less uncertainty than the conventional one. The results showed that Pw uncertainty for an AS-cup anemometer is V/4 times the Pw uncertainty obtained with a DYP-Cup anemometer, so that above 4 m/s the proposed method provides lower uncertainty than the conventional one. Note that, while the alternative measurement method does not require density uncertainty estimation, the conventional procedure does. The results showed in Section 3 highlight that density has an important contribution in Pw uncertainty. Pw estimation based on the proposed double calibration could reduce this uncertainty and transfer this drawback from in-ﬁeld measurements (with diﬀerent density conditions than those during calibration) to the lab calibration. Density could implicitly inﬂuence the proposed calibration and, in this sense, future studies should aim to extend the proposed method to diﬀerent density conditions, since the present study assumes a no-signiﬁcant density variation. 4 Conclusions 4. Conclusions Additional work could be based on the comparison of the actual power provided by a wind turbine to the estimated power provided by the new calibration procedure, as well as by the conventional calibration method. It will be interesting to take into consideration several factors that are not involved in the calibration procedure, since they can signiﬁcantly inﬂuence the results, such as wind farm density and in-ﬁeld turbulence. Author Contributions: conceptualization, F.G.-V., E.T.-J., and R.D.-V.; methodology, F.G.-V. and R.D.-V.; validation, F.G.-V. and R.D.-V.; formal analysis, F.G.-V.; investigation, F.G.-V. and G.M.-S.; resources, F.G.-V. and E.T.-J.; data curation, F.G.-V., E.T.-J., and G.M.-S.; writing—original draft preparation, F.G.-V., E.T.-J., and R.D.-V.; writing—review and editing, E.T.-J. and R.D.-V.; visualization, F.G.-V. and G.M.-S.; supervision, E.T.-J. and R.D.-V.; project administration, E.T.-J. Funding: This research received no external funding. Acknowledgments: The authors would like to acknowledge the usage of the wind tunnel provided by the ﬂuid mechanics area belonging to the department of Mechanical and Mining Engineering of the University of Jaén. Conﬂicts of Interest: The authors declare no conﬂict of interest. Nomenclature Fluid kinetic energy per volume unit [Pa] ˆ∆p Wind dynamic pressure predicted via quadratic regression [Pa] ∆pPt Wind dynamic pressure function measured with the Pitot tube at the reference position [Pa] ∆ps Wind dynamic pressure determined by the Pitot tube [Pa] ρ Moist air density [kg/m3] ∅ Relative humidity ψ and ϑ Ferrel coeﬃcients R2 Coeﬃcient of determination S Blade swept area [m2] T Absolute dry bulb temperature [K] Tv Absolute wet bulb temperature [K] u Standard uncertainty uc Combined standard uncertainty V Wind speed [m/s] ˆV Wind speed predicted via linear regression [m/s] Vb Mean ﬂow ﬁeld velocity inside wind tunnel, with blockage eﬀect [m/s] Vs Wind speed determined by the Pitot tube [m/s] x1 P k ∆pk the ﬁrst variable change to assess the quadratic regression uncertainty [Pa] x2 P k ( fk·∆pk) the second variable change to assess the quadratic regression uncertainty [Pa/s] x3 P k f 2 k .∆pk the third variable change to assess the quadratic regression uncertainty [Pa/s2] y1 Intercept or oﬀset for quadratic regression [Pa] y2 Slope for quadratic regression [Pa·s] y3 Curvature for quadratic regression [Pa·s2] ∆p Wind dynamic pressure. Fluid kinetic energy per volume unit [Pa] ˆ∆p Wind dynamic pressure predicted via quadratic regression [Pa] ∆pPt Wind dynamic pressure function measured with the Pitot tube at the reference position [Pa] ∆ps Wind dynamic pressure determined by the Pitot tube [Pa] ρ Moist air density [kg/m3] ∅ Relative humidity ψ and ϑ Ferrel coeﬃcients Nomenclature A Slope for linear regression [m] a Factor depending on the shape of the structure within the wind tunnel B Intercept or oﬀset for linear regression [m/s] b Blockage ratio c f Force coeﬃcient of the structure within the wind tunnel Ch Pitot tube head coeﬃcient CP Power coeﬃcient fr Anemometer rotation frequency [rad/s] kc Wind tunnel calibration factor kf Blockage correction factor m f Number of measurements of fri for each operating condition mp Number of measurements of ∆pi for each operating condition Ma Air molar mass [kg/mol] Mv Water vapour molar mass [kg/mol] n Total number of operating conditions P Electrical power [W] PB Barometric pressure [Pa] pstatic Static pressure ﬂuid ﬂow [Pa] ptotal Total pressure ﬂuid ﬂow [Pa] Pvs Saturated vapour pressure [Pa] Pw Wind power density [W/m2] R Ideal gas constant [J/(mol·K)] r Correlation coeﬃcient A Slope for linear regression [m] a Factor depending on the shape of the structure within the wind tun B Intercept or oﬀset for linear regression [m/s] b Blockage ratio c f Force coeﬃcient of the structure within the wind tunnel Ch Pitot tube head coeﬃcient CP Power coeﬃcient fr Anemometer rotation frequency [rad/s] kc Wind tunnel calibration factor kf Blockage correction factor m f Number of measurements of fri for each operating condition mp Number of measurements of ∆pi for each operating condition Ma Air molar mass [kg/mol] Mv Water vapour molar mass [kg/mol] n Total number of operating conditions P Electrical power [W] PB Barometric pressure [Pa] pstatic Static pressure ﬂuid ﬂow [Pa] ptotal Total pressure ﬂuid ﬂow [Pa] Pvs Saturated vapour pressure [Pa] Pw Wind power density [W/m2] R Ideal gas constant [J/(mol·K)] r Correlation coeﬃcient 17 of 18 Sensors 2019, 19, 2029 R2 Coeﬃcient of determination S Blade swept area [m2] T Absolute dry bulb temperature [K] Tv Absolute wet bulb temperature [K] u Standard uncertainty uc Combined standard uncertainty V Wind speed [m/s] ˆV Wind speed predicted via linear regression [m/s] Vb Mean ﬂow ﬁeld velocity inside wind tunnel, with blockage eﬀect [m/s] Vs Wind speed determined by the Pitot tube [m/s] x1 P k ∆pk the ﬁrst variable change to assess the quadratic regression uncertainty [Pa] x2 P k ( fk·∆pk) the second variable change to assess the quadratic regression uncertainty [Pa/s] x3 P k f 2 k .∆pk the third variable change to assess the quadratic regression uncertainty [Pa/s2] y1 Intercept or oﬀset for quadratic regression [Pa] y2 Slope for quadratic regression [Pa·s] y3 Curvature for quadratic regression [Pa·s2] ∆p Wind dynamic pressure. 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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/W4323653721	https://jasbsci.biomedcentral.com/counter/pdf/10.1186/s40104-023-00832-5	English	null	Antimicrobial peptides act on the rumen microbiome and metabolome affecting the performance of castrated bulls	Journal of Animal Science and Biotechnology/Journal of animal science and biotechnology	2,023	cc-by	11,766	Abstract Background Many countries have already banned the use of antibiotics in animal husbandry, making it extremely difficult to maintain animal health in livestock breeding. In the livestock industry, there is an urgent need to develop alternatives to antibiotics which will not lead to drug resistance on prolonged use. In this study, eighteen castrated bulls were randomly divided into two groups. The control group (CK) was fed the basal diet, while the antimicrobial peptide group (AP) was fed the basal diet supplemented with 8 g of antimicrobial peptides in the basal diet for the experimental period of 270 d. They were then slaughtered to measure production performance, and the ruminal contents were isolated for metagenomic and metabolome sequencing analysis. Result The results showed that antimicrobial peptides could improve the daily weight, carcass weight, and net meat weight of the experimental animals. Additionally, the rumen papillae diameter and the micropapillary density in the AP were significantly greater than those in the CK. Furthermore, the determination of digestive enzymes and fermen- tation parameters showed that the contents of protease, xylanase, and β-glucoside in the AP were greater than those in the CK. However, lipase content in the CK was greater than that in the AP. Moreover, the content of acetate, propi- onate, butyrate, and valerate was found to be greater in AP than those in CK. The metagenomic analysis annotated 1993 differential microorganisms at the species level. The KEGG enrichment of these microorganisms revealed that the enrichment of drug resistance-related pathways was dramatically decreased in the AP, whereas the enrichment of immune-related pathways was significantly increased. There was also a significant reduction in the types of viruses in the AP. 187 probiotics with significant differences were found, 135 of which were higher in AP than in CK. It was also found that the antimicrobial mechanism of the antimicrobial peptides was quite specific. Seven low-abundance microorganisms (Acinetobacter_sp._Ac_1271, Aequorivita soesokkakensis, Bacillus lacisalsi, Haloferax larsenii, Lysinibacil- lus_sp._3DF0063, Parabacteroides_sp._2_1_7, Streptomyces_sp._So13.3) were found to regulate growth performance of the bull negatively. Metabolome analysis identified 45 differentially differential metabolites that significantly different between the CK and the AP groups. Seven upregulated metabolites (4-pyridoxic acid, Ala-Phe, 3-ureidopropion- ate, hippuric acid, terephthalic acid, L-alanine, uridine 5-monophosphate) improve the growth performance of the experimental animals. © 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. Open Access Open Access Abstract To detect the interactions between the rumen microbiome and metabolism, we associated the rumen microbiome with the metabolome and found that negative regulation between the above 7 microorganisms and 7 metabolites. Correspondence: Zhaomin Lei leizm@gsau.edu.cn Full list of author information is available at the end of the article Correspondence: Zhaomin Lei leizm@gsau.edu.cn Full list of author information is available at the end of the article Correspondence: Zhaomin Lei leizm@gsau.edu.cn Full list of author information is available at the end of the article Antimicrobial peptides act on the rumen microbiome and metabolome affecting the performance of castrated bulls Jinping Shi1, Yu Lei2, Jianping Wu3, Zemin Li1, Xiao Zhang1, Li Jia1, Ying Wang1, Yue Ma1, Ke Zhang2, Qiang Cheng4, Zhao Zhang5, Yannan Ma3 and Zhaomin Lei1 Jinping Shi1, Yu Lei2, Jianping Wu3, Zemin Li1, Xiao Zhang1, Li Jia1, Ying Wang1, Yue Ma1, Ke Zhang2, Qiang Cheng4, Zhao Zhang5, Yannan Ma3 and Zhaomin Lei1* Shi et al. Journal of Animal Science and Biotechnology (2023 https://doi.org/10.1186/s40104-023-00832-5 Shi et al. Journal of Animal Science and Biotechnology (2023 https://doi.org/10.1186/s40104-023-00832-5 Shi et al. Journal of Animal Science and Biotechnology https://doi.org/10.1186/s40104-023-00832-5 Journal of Animal Science and Biotechnology Journal of Animal Science and Biotechnology Shi et al. (2023) 14:31 Introduction In 1950, the Food and Drug Administration (FDA) approved using antibiotics as feed additives. In 1994, the Ministry of Agriculture of China issued the “List of Allowable Species of Feed Drug Additives”, listing anti- biotics as feed additives. Since then, antibiotics have been extensively used as growth promoters, substantially reducing production costs, morbidity, and mortality and promote animal production performance [1]. However, with the extensive use of antibiotics in the production of livestock and poultry, problems of antibiotic residues in animals and bacterial resistance have become increas- ingly severe. These issues posed immense threats to the development of animal husbandry and food safety for human consumption [2]. It is for these reasons that the European Union (2006), Japan (2008), the United States (2012), China (2019), and other countries have succes- sively issued bans on the addition of antibiotics in feeds to prevent the damages caused by antibiotics abuse and to maintain food safety from animal sources and public health safety. Since most antibiotics are used in livestock farming, these bans have had a significant economic impact [3]. The pursuit of ecologically friendly and safe antibiotic alternatives has become a key subject of study in the farming industry [4]. In recent years, many dif- ferent alternatives to antibiotics such as essential oils [5], organic acids [6], antimicrobial peptides [7, 8], pro- biotics [9], and bacteriocins [10] have also been stud- ied. Many experiments have been done on pigs [11, 12], poultry [13], and other animals to test and develop these alternatives. In ruminants, the rumen acts as a bioreactor, ena- bling them to obtain nutrients from plants that humans cannot digest [20]. The rumen microbiota can directly or indirectly influence the growth and health of its host. Therefore, it is essential to determine the meta- bolic function of the rumen microbiome. The metabolic functions of the rumen microbiome reported to date have been primarily based on metagenomics [21] and transcriptomics [20, 22]. Few reports have integrated metagenomics and metabolomics to study the meta- bolic functions of the microbiome. This study explores the answers to the following two fundamental questions through metagenomics and metabolomics studies: Can antimicrobial peptides dis- rupt the rumen microbiota through their antibacterial activities? Furthermore, is there a regulatory relation- ship between rumen microbes and metabolites, and do they contribute to growth performance and meat production? © 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. Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 Page 2 of 17 Conclusions This study shows that antimicrobial peptides can improve the growth performance of animals while resisting viruses and harmful bacteria and are expected to become healthy alternatives to antibiotics. We demon- strated a new antimicrobial peptides pharmacological model. We demonstrated low-abundance microorganisms may play a role by regulating the content of metabolites. Keywords Antimicrobial peptides, Castrated bull, Growth performance, Metabolites, Microorganisms, Rumen entrapping (Antimicrobial aggregates) [16]. Although there has been significant research on the mechanism of action of antimicrobial peptides, investigations on the use of antimicrobial peptides as animal additives have been limited to in vitro tests or examination of simple microbial diversity, such as using β-hairpin anti- microbial peptide in improving the average daily gain (ADG) of piglets and average daily feed intake (ADFI) and can effectively reduce diarrhea [17]. In mouse stud- ies, antimicrobial peptides were found to protect and reduce the lethality of E. coli in mice by damaging the E. coli membrane [18]. A study reported that feeding chicks with the antimicrobial peptide microcin J25 sig- nificantly increased the body weight and was accompa- nied by a reduction in the Salmonella infection rate in feces and an increase in the length of intestinal villi and the depth of crypts [19]. Materials and methods meat percentage were measured and calculated according to previously reported methods [24, 25]. A rumen tissue specimen of approximately 2 cm × 2 cm was immediately removed from the left dorsal sac, fixed in glutaraldehyde solution, and stored at 4 °C for examination under scan- ning electron microscopy (SEM). Then, 5 mL of the liquid and solid mixture was collected from the left dorsal sac of each test animal, transferred to sterile tubes, and imme- diately frozen with liquid nitrogen. Six randomly selected samples from each group were sent back to the labora- tory the same day and stored at −80 °C for metagenomic and metabolomic sequencing. A further 15 mL of rumen content (18 bulls) was collected and stored in a steri- lized container at −20 °C for measurement of digestive enzymes and fermentation parameters. Animals, feeding management and experimental design All experiments in this study were approved by the Ani- mal Care Committee of Gansu Agricultural University (Lanzhou, People’s Republic of China) with approval number GSAU-Eth-AST-2022–035, and the experi- ments were performed according to the regulations and guide-lines established by this committee. The experi- ments were conducted in Huarui Ranch, Minle County, Zhangye, Gansu Province. Forty healthy Holstein bulls with no significant difference in body weight were cas- trated at 2 months of old. Bulls were fed a total mixed ration (TMR) consisting of corn silage and grain mix- tures to meet or exceed their nutritional requirements outlined by the National Research Council (NRC 2000) [23]. At 10 months old, 18 animals (351.62 ± 4.69 kg BW) were selected and randomly distributed into two treatments, with nine replicates per treatment (3 bulls in each enclosure, and each bull were separated by a fence). The control group (CK) was fed the basal diet while the antimicrobial peptide group (AP) was fed the basal diet supplemented with 8 g/(d·head) antimicrobial peptides (50% each of cecropin and apidaecin). The Api- daecin (chemical structure: NH2-KWKLFKKIEKVGQRV RDAVISAGPAVATVAQATALAK) was from the pat- ent product of Gansu Aolinbeier Biotechnology Group Co., Ltd., Patent No. CN201310067480.99 (Zhangye, Gansu, China), and the cecropin (chemical structure: NH2-PRVRRVYIPQPRPPHPRL) was from the patent product of Zhangye Aopu Biotechnology Co., Ltd., Pat- ent No. CN20141065433.x (Zhangye, Gansu, China). The appropriate amount of antimicrobial peptide was accurately weighed, mixed with 1 kg corn daily, and top- dressed to the feed bunk. Determining the activity of digestive enzymesh Determining the activity of digestive enzymes The rumen contents and homogenate mixture were placed in an ultrasonic beater to obtain a 10% homog- enization buffer. The supernatant was collected, and sub- sequent processed was done in accordance with the kit’s (Biosino Biotechnology Co., Ltd., Beijing, China) instruc- tions. The enzyme activities (Lipase, Cellulase, Protease, Xylanase and β-glucosidase) were determined by colorim- etry assays [5]. Determining fermentation parameters Following animal slaughter, concentrations of volatile fatty acids (VFAs) were measured using the method described in the literature [5]. Briefly, the rumen contents were first centrifuged at 5400 r/min for 10 min. One milliliter of the supernatant was mixed with 0.2 mL of a 25% metaphos- phate solution containing 2-ethylbutyrate as an internal standard and mixed uniformly in a new centrifuge tube. After cooling in an ice bath for 30 min, the reaction tube was centrifuged at 10,000 r/min for 10 min. The super- natant was passed through a 0.22-micron organic filter and stored in a 2-mL bottle for subsequent analysis. The Introduction Based on these two main questions, the effects of antimicrobial peptides on rumen micro- organisms and metabolites of castrated bulls will be compared. Further, the effects of the above two omics levels on the growth performances of the bull will be evaluated. Taken together, the goal of this study was to understand how the microbiome and metabolic changes brought about by antibacterial peptide feed supplementation influence growth performance and production in bulls. Antibacterial peptides are a class of small proteins with diverse structures and broad anti-inflammatory and growth-promoting functions. An advantage of using these proteins is that resistance cannot be eas- ily developed against them and is commonly used in animal production. It was originally believed that anti- microbial peptides kill bacteria directly or indirectly, mainly through physical adsorption, rapid penetration, and destruction of the cell membrane [14]. Studies in recent years have demonstrated that antimicrobial peptides inhibit microorganisms through vital life pro- cesses like metabolism, biosynthesis, and translation [15]. Recent studies have also reported that antimicro- bial peptides can trap bacteria by forming nanonets Page 3 of 17 Page 3 of 17 Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 Shi et al. Journal of Animal Science and Biotechnology Materials and methods The pre-trial period was 30 d and the positive trial period was 270 d. According to the feeding standard of bulls, the diet was adjusted every 30 d and weighed the bulls (fasting). The basic diet for fatten- ing cattle consisted of a TMR consisting of corn, silage, and grain (Table S1). All bulls were fed twice daily at 07:00 and 15:00. The remaining feed in the feed tank is collected at 6:00 every morning and weighed to measure the daily feed intake of each bull. During the experiment, all animals had ad libitum access to feed and free water, ensuring that they all received the same nutrient levels and management conditions. Sample collection Scanning electron microscope of rumen papillah g The diameter of the rumen papilla and the density of the micropapillary (microscopic bumps on the rumen papilla) were investigated by SEM. The sample prepara- tion steps were as follows: 1 cm × 1 cm rumen tissue was washed twice with water for 5 min each. Next, the tissues were dehydrated with 30%, 50%, 70%, 80%, 90%, 95%, and 100% gradient alcohol, respectively for10 min each. The samples were lightly adhered to using a conductive adhesive, ion-sputtered, and finally imaged with a scan- ning electron microscope (Inspect, Hillsboro, TX, USA). Rumen papilla diameter and micropapillary density sta- tistics (n ≥ 50) were performed using Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA) software. Metagenome sequencing and bioinformatics analysis Metagenome sequencing and bioinformatics analysis DNA was extracted from the rumen contents using Soil DNA Kit (MOBIO, Carlsbad, CA, USA). The concentra- tion and purity of the extracted DNA were determined with TBS-380 fluorometer (Turner Biosystems, Sunnyvale, CA, USA) and NanoDrop 2000 (NanoDrop Technologies, Wilmington, DE, USA), respectively. The DNA was frag- mented to fragments of approximately 400 bp using Cova- ris M220 (Gene Company Limited, Hongkong, China) for library construction. Sequencing was performed using the Illumina NovaSeq6000 (Illumina Inc, San Diego, CA, USA) sequencing platform. Sequencing adapters were removed with FAST software (version 0.20.0), and low- quality reads (length < 50 bp in length or quality value < 20 or with N bases) were removed [26]. Reads were aligned to the Bos Taurus reference genome assembly using BWA 0.7.9a (http://bio-bwa.sourceforge.net). Metagenomic sequencing data were assembled with MULTIPLE MEG- AHIT (Version 1.1.2) [27]. Overlapping sequences with lengths ≥ 300 bp were selected as the final assembly results and used for further gene annotation. Metagenes were used to predict the best candidate open reading frames (ORFs) [28]. The predicted ORFs with length ≥ 100 bp were retrieved. A cluster analysis of a non-redundant gene catalog with sequence homology and 90% coverage was constructed using CD-HIT (version 4.6.1) [29]. Sub- sequently, representative sequences of the non-redundant gene catalog were aligned with the NCBI NR database using BLASTP (version 2.2.28 +) (the e-value cutoff for the best match was 1e −5) to obtain annotation results and species abundance degree [30]. Finally, a BLAST search (version 2.2.28 +) with an optimization criterion cutoff of 1e −5, annotated against the KEGG database [31], was performed using USEARCH (http://www.drive5.com/ usearch/) CAZY annotation [32]. Data statistics and analysis SPSS version 22.0 (IBM Corp., Armonk, New York, USA) was used for independent variance t-test and correlation analysis. Data are presented as mean ± standard deviation. Using ImageJ (National Institutes of Health, Bethesda, MD, USA) to calculate the papillae’s diameter and the micropapillary density. Metabolome statistics were ana- lyzed based on retention time and ion current strength using the MultiQuant software to calculate the relative content of each compound. Orthogonal projections to latent structures-discriminate analysis (OPLS-DA) were used to determine metabolic differences between the two groups. Enrichment analysis of metabolic pathways (MPEA) was performed by MetOrigin [40]. The Omic- Share Tools [41] were employed to perform the two-way orthogonal partial least squares (O2PLS) analysis. Effects of antimicrobial peptides on growth performance of castrated bulls Average daily gain (ADG), dry matter intake (DMI), net- meat weight, feed/gain ratio (F/G), and carcass weight were measured for statistical analysis (Table 1). The Metagenome sequencing and bioinformatics analysis More- over OriginPro 9.1 (OriginLab, Northampton, US, USA) were used to draw statistical maps. Sample collection h On the 270th d, all experimental animals fasted for 12 h and were weighed. Next then the 18 animals were trans- ported, without mixing treatment groups, to a commer- cial abattoir and slaughtered within 1 h of their arrival by bolt stunning followed by exsanguination from the jugu- lar vein. Following slaughter, the carcass weight and net Page 4 of 17 Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 of rumen contents was analyzed by ultra-performance liquid chromatography (UPLC) and Tandem mass spec- trometry (MS/MS) (QTRAP® 6500+, SCIEX, Framing- ham, MA, USA) [33–35]. After obtaining the LC/MS data of different samples, the extracted ion chromatographic peaks of all metabolites were integrated using Multi- Quant software (Applied Biosystems, Foster, MA, USA) and the MetWare database (MWDB) database, respec- tively. The chromatographic peaks of the metabolites in different samples were corrected by integration [36–38]. The relative concentrations of rumen metabolites were screened by FC (FC ≥ 2 and FC ≤ 0.5) and VIP (VIP ≥ 1) to identify the different metabolites. The identified metabolites were annotated using the Kyoto Encyclope- dia of Genes and Genomes (KEGG) compound database, and the annotated metabolites were then mapped to the KEGG Pathway database [39]. volatile fatty acid content was determined by gas chro- matography (Agilent, Palo Alto, CA, USA). The column temperature was maintained at 60 °C for 1 min, raised to 115 °C at 5 °C/min without reservation, and increased to 180 °C at 15 °C/min. Notably, the detector and injector temperatures were 260 and 250 °C, respectively. Metabolome sequencing and bioinformatics analysis Table 1 Comparative analysis of production performance ADG Average daily gain, DMI Dry matter intake, F/G Feed/gain ratio Item CK AP P-value ADG, kg 0.96 ± 0.07 1.17 ± 0.05 0.021 DMI, kg/d 10.45 ± 0.25 11.00 ± 0.25 0.134 Carcass weight, kg 323.26 ± 11.34 382.80 ± 11.30 0.001 F/G 9.12 ± 1.01 10.55 ± 1.09 0.349 Net meat weight, kg 258.55 ± 7.93 307.65 ± 8.07 0.001 Table 1 Comparative analysis of production performance Fifty milligrams of rumen contents were thawed on ice, and 500 µL of 70% methanol internal standard extract was added at 4 °C. After shaking for 3 min, the mixture was left to stand at −20 °C for 30 min, followed by cen- trifugation at 12,000 r/min for 10 min at 4 °C. Next, 250 µL of the supernatant is centrifuged at 12,000 r/min for 5 min at 4 °C. Next, 150 µL of this supernatant is taken in the liner of the corresponding injection bottle for data acquisition and further analysis. The metabolome Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 Page 5 of 17 results showed that the ADG gain in the AP was signifi- cantly greater than that in the CK (P = 0.021). Carcass weight and net-meat weight was extremely significantly greater than those in the CK (P = 0.001). However, the DMI and F/G in the AP were greater than that in the CK, but not significantly different (P > 0.05). microscopy using rumen tissue (Fig. 1A and Fig. S1). From the electron microscopy images, it can be observed that the development of rumen papillae in the AP is bet- ter than that in the CK. In order to understand these changes more objectively, the ruminal papillae’s diameter and the micropapillary density were analyzed (Fig. 1B). The diameter of rumen papillae in AP was larger than that in CK (P = 0.31). The density of the micropapillary in the AP was also more than that in CK (P = 0.004). Genome profiling of rumen microorganisms Genome profiling of rumen microorganisms Metagenomic analysis of rumen contents in CK and AP showed that 74,792,855 and 70,449,158 raw reads were obtained on average. After excluding low-quality and n-containing reads, CK and AP acquired 73,283,668 and 69,227,622 clean reads, accordingly. Furthermore, opti- mized reads obtained for subsequent analysis after removing the host genome sequence were 61,891,630 and 58,924,630, accounting for 82.79% and 83.82% of RAW reads for CK and AP, respectively. The underlined results indicated that the sequencing data are credible and can be used for subsequent bioinformatics investigation. Archaeal (Fig. 2A), bacterial (Fig. 2B), and viral (Fig. 2C) species had isolates across the two groups, according to Principal Coordinates Analysis (PCoA) at the domain level, but no isolates with eukaryotic species or unclassi- fied microorganisms were found. Therefore, the compar- ative analysis of rumen microbes between the two groups only focused on bacteria, archaea, and viruses. Effect of antimicrobial peptides on the rumen epitheliumh Effect of antimicrobial peptides on the rumen epithelium The growth and development of the rumen epithelium in castrated bulls were studied by scanning electron The effect of antimicrobial peptides on the activity of digestive enzymesh The contents of digestive enzymes were examined to compare their digestibility between the two groups (Table 2). The results showed that the lipase content in the CK was greater than that in the AP (P = 0.025), but the content of protease, xylanase, and β-glucosidase in the AP was vary significantly greater than that in the CK (P = 0.001). However, there was no different in cellulase (P = 0.974). The effect of antimicrobial peptides on rumen fermentation parameters Herein, we assessed the presence of four major VFAs in the rumen (Table 3 and Table S2). The results showed that the acetate and valerate were greater than in the CK (P = 0.039, P = 0.018). Moreover, the level of propionate and butyrate in the AP was significantly greater than that in the CK (P = 0.004, P = 0.001). Differences in rumen microbial taxonomyh The dominant bacterial phyla in the rumen were Bac- teroidetes (CK: 44.47%, AP: 38.98%), Firmicutes (CK: 36.07%, AP: 40.62%), and unclassified Bacteria (CK: 8.515%, AP: 7.011%). According to the comparative anal- ysis of differential abundance at the genus level revealed that among the top 15 genera with significant variations in abundance, the abundance of 7 species in the CK was greater than that in the AP (P < 0.05), and 8 genera were significantly lower than those in the AP group (P < 0.05, Fig. 2D). At the species level, the dominant bacteria in the CK and AP were Clostridiales bacterium (CK: 8.126%, AP: 8.98%), Bacteroidales bacterium (CK: 5.212%, AP: 4.328%), Rikenellaceae bacterium (CK: 4.224%, AP: 2.596 %); bacterium P3 (CK: 1.794%, AP: 2.949%); Prevotella ruminicola (CK: 2.066%, AP: 2.508%) (Fig. 2E) Although these species were dominant, the abundances did not dif- ferent between the two groups.f Table 2 Effects of antibacterial peptide on rumen digestive enzyme activities in bull Table 2 Effects of antibacterial peptide on rumen digestive enzyme activities in bull Item CK AP P-value Lipase, U/mg 8.50 ± 0.37 6.94 ± 0.10 0.020 Cellulase, U/m 89.36 ± 7.46 88.97 ± 6.77 0.974 Protease, U/mg 1.77 ± 0.09 2.84 ± 0.08 0.000 Xylanase, U/mg 3.92 ± 0.07 6.64 ± 0.30 0.000 β-glucosidase, U/mg 21.66 ± 0.62 32.43 ± 1.61 0.001 Table 3 Effects of antibacterial peptide on rumen fermentation parameters in bull Item CK AP P-value Acetate, mg/kg 20.01 ± 0.41 21.34 ± 0.38 0.039 Propionate, mg/kg 10.2408 ± 0.1909 11.52 ± 0.28 0.004 Butyrate, mg/kg 9.41 ± 0.26 11.09 ± 0.22 0.001 Valerate, mg/kg 0.67 ± 0.01 0.82 ± 0.04 0.018 Table 3 Effects of antibacterial peptide on rumen fermentation parameters in bull Differential analysis revealed that there were a total of 1993 distinct microorganisms. Further, the archaeal dif- ferential analysis revealed that the abundance of 32 spe- cies with differential abundance in the rumen of AP Page 6 of 17 Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 Fig. 1 Scanning electron microscope and analysis of rumen papilla. A Scanning electron microscope image of rumen papilla and rumen micropapillary. B Rumen papilla diameter and ruminal micropapillary density. P < 0.05, P < 0.01 Fig. 1 Scanning electron microscope and analysis of rumen papilla. A Scanning electron microscope image of rumen papilla and rumen micropapillary. B Rumen papilla diameter and ruminal micropapillary density. Differences in rumen microbial taxonomyh In particular, the drug resistance of the antimicrobial pathway in the CK was greater than in the AP group. In contrast, the immunological pathway was significantly higher in the AP group than in the CK, confirming that the antimicrobial peptides reduce drug resistance while enhancing immunity. 44 third-level pathways were significantly enriched in the rumen microbiota of AP bulls (Table S7, Fig. 3C): 3 “cellular process”, 4 “genetic information processing”, 3 “environmental information Processing”, 16 “Metabolic Pathways”, 11 “Human Dis- eases” and 7 “Organismal Systems”. On the other hand, 26 pathways were significantly enriched in the rumen animals was greater than that of CK (P < 0.05), including 5 species of Methanobrevibacter and 2 species of Methano- sphaera sp. The abundance of species such as 3 Methano- brevibacter and 2 Methanosarcinales in the rumen of CK animals was greater than that of AP (P < 0.05) (Table S3). A comparative analysis of the bacterial differential abundance showed that there are 1691 kinds of bacteria with significant difference (P < 0.05). Out of these 1691 species, 826 were greater in the CK than in the AP, and 865 in the AP were greater than in the CK (Table S4). A comparison of the differential abundance of viruses revealed that 65 viruses showed differences (P < 0.05). However, only 3 viruses were more abundant in the AP than in the CK, while the remaining 62 viruses were more abundant in the CK. The abundance was signifi- cantly higher than that of the AP, which indicated that antimicrobial peptides could effectively kill most viruses and that the effect was significant (Table S5). Differences in rumen microbial taxonomyh P < 0.05, **P < 0.01 Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 Page 7 of 17 Fig. 2 Domain-level PCoA and genus and species level microbial analysis A Archaea PCoA analysis chart. B Bacteria PCoA analysis chart. C Virus PCoA analysis chart. D Differences between CK and AP at the genus level. E The dominant strains of CK and AP at the species level Fig. 2 Domain-level PCoA and genus and species level microbial analysis A Archaea PCoA analysis chart. B Bacteria PCoA analysis chart. C Virus PCoA analysis chart. D Differences between CK and AP at the genus level. E The dominant strains of CK and AP at the species level For KEGG analysis, a total of 6 pathways were anno- tated at the first level (Fig. 3A), namely “metabolism”, “genetic information processing”, “environmental infor- mation processing”, “cellular processes”, “organizational systems”, and “human diseases”. At the second level, 46 species were observed (Fig. 3A, Table S6). Out of these 46 global and overview maps, carbohydrate metabo- lism, amino acid metabolism, metabolism of cofactors and vitamins, energy metabolism, and replication and repair were the most abundant. There were 13 path- ways with significant differences (P < 0.05, Fig. 3B). In particular, the drug resistance of the antimicrobial pathway in the CK was greater than in the AP group. In contrast, the immunological pathway was significantly higher in the AP group than in the CK, confirming that the antimicrobial peptides reduce drug resistance while enhancing immunity. 44 third-level pathways were significantly enriched in the rumen microbiota of AP bulls (Table S7, Fig. 3C): 3 “cellular process”, 4 “genetic information processing”, 3 “environmental information Processing”, 16 “Metabolic Pathways”, 11 “Human Dis- eases” and 7 “Organismal Systems”. On the other hand, 26 pathways were significantly enriched in the rumen For KEGG analysis, a total of 6 pathways were anno- tated at the first level (Fig. 3A), namely “metabolism”, “genetic information processing”, “environmental infor- mation processing”, “cellular processes”, “organizational systems”, and “human diseases”. At the second level, 46 species were observed (Fig. 3A, Table S6). Out of these 46 global and overview maps, carbohydrate metabo- lism, amino acid metabolism, metabolism of cofactors and vitamins, energy metabolism, and replication and repair were the most abundant. There were 13 path- ways with significant differences (P < 0.05, Fig. 3B). Rumen metabolome analysis A total of 662 compounds were identified in the rumen metabolites. The OPLS-DA score map showed that both groups could separate rumen metabolites (Fig. 4A). After screening the relative concentrations of rumen metabo- lites by FC (FC ≥ 2 and FC ≤ 0.5) and VIP (VIP ≥ 1), the expression of 45 metabolites were significantly different between the CK and AP. These included 27 upregulated and 18 downregulated metabolites (Fig. 4C). Analysis of the 45 differential metabolites showed that 14 were derived from amino acids and their derivatives, 6 were from nucleotides and their metabolites and 25 belong to others. KEGG pathway analysis revealed that of these 45 differential metabolites, 15 were significantly enriched in 22 pathways (Fig. 4B). The metabolites with significant differences were screened according to the screening criteria and analyzed by the Pearson correlation analysis method. According to the obtained results, the highest correlation coefficients of three metabolites (7-ketolitho- cholic acid, apocholic acid, and 12-ketolithocholic acid) Comparison of probiotics and VFDBh There were substantial variations between the AP and CK for 187 probiotics. Furthermore, 135 probiotics in the AP were greater than that in the CK, especially the Propioni- bacterium and Acetobacter in the AP were more than 10 times higher than in the CK, on average. The 4 probiotics, Carnobacterium, Ellulomonas, Lactobacillus, and Kleb- siella, were not detected in the CK but were present in the AP (Table S12). The virulence factor analysis of the Viru- lence Factor Database (VFDB) revealed that 364 strains produced 1151 virulence factors. The abundance of bacte- ria producing these virulence factors was not significantly different between the two groups, indicating that adding antimicrobial peptides did not contribute to the virulence factors while increasing the beneficial bacteria. Combined metagenome and metabolome analysish Combined metagenome and metabolome analysis The data of the two omics were analyzed to examine whether there is a linkage effect between the two omics. The O2PLS analysis revealed the top 25 microorganisms and metabolites with the largest linkage effect (Fig. 5A). The data was analyzed by MPEA via MetOrigin to further screen the differential metabolites related to microorganisms. The analysis eliminated the metabolites from the host and only 17 metabolites from the microorganisms or shared by both the microorganisms and the host were retained (Fig. 5B). Top 25 microorganisms obtained by O2PLS analysis were classified, and 17 microorganisms did not have any clas- sification. Viruses and unclassified microorganisms were removed, and only 17 microorganisms belonging to archaea and bacteria were retained. Venn diagrams of differential metabolite enrichment pathways and differential microbial enrichment pathways were explored, and it was found that four pathways are common enrichment pathways in metab- olites and microorganisms (ABC transporters, Metabolic pathways, Taurine, and Hypotaurine Metabolism, Sulfur relay system). Screening 17 metabolites again with FC ≥ 2.5, 7 differential metabolites (4-pyridoxic acid, Ala-Phe, 3-urei- dopropionate, hippuric acid, terephthalic acid, L-alanine, uridine 5-monophosphate), all upregulated, were obtained. Similarly, screening 17 microorganisms with P ≤ 0.005, 7 microorganisms (Acinetobacter_sp._Ac_1271, Aequorivita soesokkakensis, Bacillus lacisalsi, Haloferax larsenii, Lysini- bacillus_sp._3DF0063, Parabacteroides_sp._2_1_7, Strep- tomyces_sp._So13.3) were obtained. The abundance of these seven microorganisms was down-regulated in the AP group. In order to further explore the relationship between these 7 metabolites and 7 microorganisms, a Pearson corre- lation analysis was done, and the results showed a negative regulatory correlation between these 7 microorganisms and 7 metabolites (Fig. 5C). Functional maps and functional differences of the rumen microbiome The function of the rumen microbiome was deter- mined by KEGG map and genes encoding CAZyme. Page 8 of 17 Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 CK and AP enrichment analysis in KEGG. A First and second level enrichment. B Second level differentially significant pathw ntially significant pathway ig. 3 CK and AP enrichment analysis in KEGG. A First and second level enrichment. B Second level differentially significant pathway. C Thi ifferentially significant pathway Fig. 3 CK and AP enrichment analysis in KEGG. A First and second level enrichment. B Second level differentially significant pathway. C Third level differentially significant pathway Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 Page 9 of 17 of the CK. The list included 4 “cellular process path- ways”, 16 “metabolic pathways”, 2 “human diseases”, and 4 “organic systems “ (Table S8, Fig. 3C). were down-regulated, and one metabolite (phenyl ace- tate) was upregulated in the AP. It is particularly note- worthy that 7-ketolithocholic acid, apocholic acid, and 12-ketolithocholic acid showed a robust and positive cor- relation with each other (r = 1), while phenyl acetate and the above mentioned three metabolites (7-ketolithocholic acid, apocholic acid, and 12-ketolithocholic acid) showed a moderate, negative correlation (r = −0.694) (Fig. 4D). g y ( g ) The CAZyme map identified a total of 534 genes encod- ing CAZyme (Table S9), including 17 accessory activities (AA), 69 carbohydrate-binding modules (CBMs), 16 car- bohydrate esterase (CE), 263 glycoside hydrolases (GH), 92 glycosyltransferases (GT) and 77 polysaccharide lyases (PL). Out of all the genes encoding CAZyme, involved in the breakdown of carbohydrates (including cellulose, hemi- cellulose, starch, protein, and lignin), 26 were enriched in the rumen of AP cattle (19 GH, 5 PL, 2 AA, and 1 CBM, Table S10), while 23 genes were enriched in the rumen of CK cattle (17 GH, 3 PL, 2 AA, 2 CBM, and 1 CE, Table S11). Among the GTs involved in carbohydrate synthesis, 4 were enriched in the rumen in both AP and CK. Correlation analysis of microorganisms, metabolites, and phenotypesf To understand the effects of the screened seven metab- olites and seven microorganisms on the growth per- formance of castrated bulls, a correlation analysis was performed. The results revealed that the seven upregu- lated metabolites showed a positive correlation (r ≥ 0.3, Fig. 6A) with feed conversion ratio, average daily gain, Page 10 of 17 Journal of Animal Science and Biotechnology (2023) 14:31 Shi et al. Journal of Animal Science and Biotechnology (202 Change in rumen content metabolite levels in castrated bulls with AP diet. A OPLS-DA analysis. B Pathways of differential met ment. C Differential metabolites. D Correlation between 7-ketolithocholic acid, apocholic acid, 12-ketolithocholic acid, and phe Fig. 4 Change in rumen content metabolite levels in castrated bulls with AP diet. A OPLS-DA analysis. B Pathways of differential metabolite enrichment. C Differential metabolites. D Correlation between 7-ketolithocholic acid, apocholic acid, 12-ketolithocholic acid, and phenylacetate Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 Page 11 of 17 A B C Microorganism Metabolite 4-Pyridoxic acid Ala-Phe 3-Ureidopropionic acid Hippuric acid Terephthalic acid L-Alanine Uridine 5 -monophosphate First joint loadings s g nid a ol t nioj d n o c e S Fig. 5 Combined metagenome and metabolome analysis. A Metagenome and metabolome O2PLS analysis. B Metabolite MPEA Analysis. C Correlation analysis between target metabolites and microorganisms A First joint loadings s g nid a ol t nioj d n o c e S A s g nid a ol t nioj d n o c e S Microorganism Metabolite First joint loadings B C 4-Pyridoxic acid Ala-Phe 3-Ureidopropionic acid Hippuric acid Terephthalic acid L-Alanine Uridine 5 -monophosphate Fig. 5 Combined metagenome and metabolome analysis. A Metagenome and metabolome O2PLS analysis. B Metabolite MPEA Analysis. C Correlation analysis between target metabolites and microorganisms B C C B Fig. 5 Combined metagenome and metabolome analysis. A Metagenome and metabolome O2PLS analysis. B Metabolite MPEA Analysis. C Correlation analysis between target metabolites and microorganisms Page 12 of 17 Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 Page 12 of 17 Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 0.01 0.05 0.1 Acinetobacter_sp._Ac_1271 Streptomyces_sp._So13.3 Parabacteroides_sp._2_1_7 Lysinibacillus_sp._SDF0063 Haloferax larsenii Bacillus lacisalsi Aequorivita soesokkakensis B A 4-Pyridoxic acid Ala-Phe 3-Ureidopropionic acid Hippuric acid Terephthalic acid L-Alanine Uridine 5 -monophosphate Fig. 6 Correlation analysis between microorganisms, metabolites, and production performance. A Correlation analysis between metabolites and production performance. Correlation analysis of microorganisms, metabolites, and phenotypesf It is also important to note that through the correlation analysis of microor- ganisms and production performance, a negative corre- lation was observed between the seven down-regulated microorganisms and dry matter intake, feed conversion rate, average daily gain, net meat weight, and slaugh- ter rate (r ≤ −0.3). The Parabacteroides_sp._2_1_7 was negatively correlated (r ≤ −0.7) with carcass weight, feed conversion ratio, and the average daily gain. The average daily gain was moderately negatively correlated (r ≤ −0.6) with Haloferax larsenii, Acinetobacter_sp._Ac_1271, and Aequorivita soesokkakensis. DMI and Acinetobacter_sp._ Ac_1271, Haloferax larsenii were moderately negatively correlated (r ≤ −0.6). Furthermore, Streptomyces_sp._ So13.3, meat weight and carcass weight were moderately negatively correlated (r ≤ −0.6, Fig. 6B). interacted and played a role together, which may be the main reason why the growth performance of AP was bet- ter than that of CK. Similar to many previous studies that have assessed rumen microbiomes using metagenomics, bacteria were the most abundant rumen microbial kingdom in the rumen of bull and the differences in the rumen microbial features between CK and AP castrated bulls were mainly found in bacteria [38, 48]. Bacteria are key players in most of the feed biopolymer degradation and fermenta- tion [49], which suggests that the bacteria play more sig- nificant roles in contributing to host growth performance than other microbial kingdoms. CAZyme degrades diet structural polysaccharides to provide nutrient substances for absorption by rumen epithelium. The enrichment of genes encoding CAZyme, which are involved in the breakdown of carbohydrates (GH and PL) in the rumen microbiota of AP bull, further demonstrated that the addition of antimicrobial peptides provided bulls with a greater ability to degrade complex substrates. There was no difference in the abundance of genes encoding CAZyme involved in carbohydrate synthesis (GT) in the rumen of the CK and AP. The primary role of VFAs in ruminants is to provide energy. The main function of rumen microorganisms is to decompose nutrients into VFAs and ammonia and then perform re-biosynthesis or energy metabolism. The abundance of genes encod- ing CAZyme involved in carbohydrate synthesis (GT) was similar in the rumens belonging to the CK and AP, but the concentration of major VFAs was higher in the AP. This result indicated that the rumen microbiota in the AP was probably more efficient in producing VFAs, thereby providing more energy for the growth and meat production of the host bull. Correlation analysis of microorganisms, metabolites, and phenotypesf Feed-efficient animals pro- duce more VFAs and less methane [50]. The AP had higher VFAs content in the rumen and more methano- trophic species than the CK (CK: 12, AP: 18, Table S3), indicating that the CK may have higher feed utilization than the AP group. In fact, although the F/G of CK was higher than AP, there was no difference between the two groups (P = 0.349), probably because AP methanogens can use microbial fermentation to convert H2 and CO2 in the rumen into methane which is then excreted [51]. Thus, the fermentation process reduces the accumulation of H2 in the rumen, preventing the inhibition of enzymes involved in metabolism, promote the activity of digestive enzymes [51–53], improving AP feed utilization. The antibacterial peptide could selectively kill micro- Correlation analysis of microorganisms, metabolites, and phenotypesf B Correlation analysis between microorganisms and production performance A 4-Pyridoxic acid Ala-Phe 3-Ureidopropionic acid Hippuric acid Terephthalic acid L-Alanine Uridine 5 -monophosphate A 0.01 0.05 0.1 Acinetobacter_sp._Ac_1271 Streptomyces_sp._So13.3 Parabacteroides_sp._2_1_7 Lysinibacillus_sp._SDF0063 Haloferax larsenii Bacillus lacisalsi Aequorivita soesokkakensis B Fig. 6 Correlation analysis between microorganisms, metabolites, and production performance. A Correlation analysis between metabolites and B Fig. 6 Correlation analysis between microorganisms, metabolites, and production performance. A Correlation analysis between metabolites and production performance. B Correlation analysis between microorganisms and production performance Fig. 6 Correlation analysis between microorganisms, metabolites, and production performance. A Correla production performance. B Correlation analysis between microorganisms and production performance Fig. 6 Correlation analysis between microorganisms, metabolites, and production performance. A Correlation analysis between metabolites and production performance. B Correlation analysis between microorganisms and production performance Fig. 6 Correlation analysis between microorganisms, metabolites, and production performance. A Correlation analysis between metabolites and production performance. B Correlation analysis between microorganisms and production performance Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 Page 13 of 17 Page 13 of 17 Shi et al. Journal of Animal Science and Biotechnology net meat weight, and slaughter rate. Especially hippu- ric acid and uridine 5-monophosphate showed a posi- tive correlation with average daily gain (r > 0.5), Ala-Phe with feed conversion ratio (r > 0.5). It is also important to note that through the correlation analysis of microor- ganisms and production performance, a negative corre- lation was observed between the seven down-regulated microorganisms and dry matter intake, feed conversion rate, average daily gain, net meat weight, and slaugh- ter rate (r ≤ −0.3). The Parabacteroides_sp._2_1_7 was negatively correlated (r ≤ −0.7) with carcass weight, feed conversion ratio, and the average daily gain. The average daily gain was moderately negatively correlated (r ≤ −0.6) with Haloferax larsenii, Acinetobacter_sp._Ac_1271, and Aequorivita soesokkakensis. DMI and Acinetobacter_sp._ Ac_1271, Haloferax larsenii were moderately negatively correlated (r ≤ −0.6). Furthermore, Streptomyces_sp._ So13.3, meat weight and carcass weight were moderately negatively correlated (r ≤ −0.6, Fig. 6B). net meat weight, and slaughter rate. Especially hippu- ric acid and uridine 5-monophosphate showed a posi- tive correlation with average daily gain (r > 0.5), Ala-Phe with feed conversion ratio (r > 0.5). Discussion abundance of Propionibacterium and Cetobacter was more than 10 times greater in the AP compared to the CK, on average. Further, four probiotics, Carnobacte- rium, Ellulomonas, Lactobacillus and Klebsiella were unique to the AP group (Table S12). These probiotics improve growth and development. For example, Ceto- bacter can produce vitamin B2 [54], which is associ- ated with the metabolism of carbohydrates in the body [55]. On the other hand, Lactobacillus is a probiotic that is often used in livestock breeding because it promotes growth. Many studies have also confirmed that adding Lactobacillus can promote growth performance and ADG [56]. Based on the high efficiency of antibacterial peptides in killing viruses without affecting probiot- ics and even though the antimicrobial peptides reduced the abundance and species of microorganisms in the rumen, but the dominant bacteria did not change. it can be assumed that the antibacterial function of anti- bacterial peptides is specific. However, this assumption is contrary to the classic antimicrobial peptides phar- macodynamic model [57], which holds that antimicro- bial peptides exert a microbial killing effect by broadly targeting microorganisms, thereby establishing a broad inhibitory mechanism against microorganisms. How- ever, a recent re-evaluation of this model revealed that different antimicrobial peptides could act synergistically, exhibiting specificity toward inhibiting microorganisms [14]. In this study, equal amounts of cecropin and api- daecin were mixed to prepare the antimicrobial peptide, combined with self-produced antimicrobial peptides. This may be the main cause for the sterilization speci- ficity of adding antimicrobial peptides. Our experimen- tal results also confirm that the new model is correct. This study also reveals that the antimicrobial peptides improved immunity without developing drug resistance (Fig. 3B). Which is a simple and efficient killing mecha- nism of antimicrobial peptides, effectively preventing the evolution of bacterial resistance [14, 58], while the efficient killing of the virus directly improves immunity. Seven down-regulated microbes and seven upregu- lated metabolites that may play an essential role in the production performance of bulls were identified. It was further observed that a negative regulatory relation- ship existed between the metabolites and the microbes. It has been reported that feeding parabacteroides dista- sonis to mice can promote the production of secondary bile acids, thereby inhibiting weight gain [63]. Keystone cholic acid and Parabacteroides were significantly down- regulated in the AP, while the ADG and net meat weight in the AP were significantly higher than those in the CK. Discussion By integrating the rumen microbiome and metabolites, the effect of antimicrobial peptides on growth perfor- mance was studied in castrated bulls. Additionally, the effect of rumen microbes and metabolites on production performance and their composition were estimated. p p Acetate, propionate and butyrate, which are fermen- tation products of feed in the rumen, were beneficial to the growth and development of the rumen [42, 43], VFAs infusions stimulate cellular proliferation in the rumi- nal epithelial tissue of ruminants [44]. Interestingly, the present study established that antibacterial peptide feed supplementation increases the papillae diameter and micropapillary density of bulls, this may be correlated with high VFAs concentration in the rumen. This result is important as the rumen papilla facilitates the absorption of nutrients. Further, the content of digestive enzymes (protease, xylanase, and β-glucosidase) in the rumen con- tents of the AP was significantly higher than that of the CK. Xylanase can degrade xylan (a type of hemicellulose) and convert it into a monosaccharide for absorption and utilization by the body [45]. Similarly, β -glucosidase can break down cellulose into glucose to provide energy for the growth and development of the body [46]. Cellulose and hemicellulose are the primary nutrients in the rumen of ruminants broken down by xylanase and β-glucosidase into monosaccharides that can be directly absorbed and utilized by the rumen papilla. The increase in the rumen mucosal surface area (Fig. 1) promotes the absorption of these energy substances [47], contributing to the growth and development of bulls. The addition of antimicrobial peptides increased the surface area of rumen mucosa, the content of digestive enzymes and VFAs, these factors The antibacterial peptide could selectively kill micro- organism extremely effectively. As a result, the AP had only three species, while the CK had as many as 62 species of virus. Furthermore, out of a total of 187 identified probiotics with different, 135 probiotics were greater in the AP than in the CK group. Especially, the Shi et al. Journal of Animal Science and Biotechnology (2023) 14:31 Page 14 of 17 acetate in AP is 114% higher than CK. Many esterase can be produced by microorganisms. Esterase is the key enzyme for the synthesis of phenylethyl acetate, and ace- tic acid is the substrate for the synthesis of phenylethyl acetate [61]. Interestingly, phenyl acetate has antibacte- rial activity [62], which is essentially consistent with the results of this study. Discussion Journal of Animal Science and Biotechnology (2023) 14:31 Shi et al. Journal of Animal Science and Biotechnology desulfurization bacteria has a significant contribution to the overall sulfate reduction [71]. Additional file 4: Table S3. CK and AP significant difference in archaea. Additional file 5: Table S4. CK and AP significant difference in bacteria. Additional file 6: Table S5. CK and AP significant difference in virus. Additional file 7: Table S6. KEGG second level differentially significant pathway. Additional file 8: Table S7. KEGG third level differentially significant pathway. Additional file 9: Table S8. Pathways in which microorganisms in the CK group were significantly enriched in third level pathways. Additional file 10: Table S9. CAZy map annotated genes. Additional file 11: Table S10. Genes significantly enriched by CAZy in AP rumen. Additional file 12: Table S11. Genes significantly enriched by CAZy in CK rumen. Additional file 13: Table S12. Comparison of CK and AP probiotics. Availability of data and materials The metagenomic data are available in the NCBI database under accession PRJNA854757. Conflict of interest Th h d l h The authors declare that the research was conducted in the absence of any commer- cial or financial relationships that could be construed as a potential conflict of interest. Ethics approval and consent to participate The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the Gansu Agricultural University under permit number No. GSAU-Eth-AST-2022–035. Conclusion This study identifies taxonomic features, functions, metabolites of rumen microbes, and their interac- tions with metabolites that contribute to host growth performance. The antibacterial mechanism of anti- microbial peptides is specific and did not affect pro- biotics in the rumen. Additionally, antimicrobial peptides could efficiently destroy viruses. While enhancing immunity, problems related to drug resist- ance are also not associated with them. Low-abun- dance microorganisms play a growth-promoting role by regulating metabolites. Finally, in this study, 7 microorganisms were screened that negatively regu- lated growth performances and 7 metabolites that had a growth-promoting effect. And we found that low-abundance microorganisms may play a role in improving the production performance of bulls by regulating metabolites. Additional file 11: Table S10. Genes significantly enriched by CAZy in AP rumen. Additional file 12: Table S11. Genes significantly enriched by CAZy in CK rumen. Additional file 13: Table S12. Comparison of CK and AP probiotics. Authors’ contributions JS, ZL and JW conceived and designed the experiments. JS, YL, LJ, YW, XZ, YM, ZZ, QC, ML, YM and KZ conducted the experiments and performed the statisti- cal analysis of the experimental data. Finally, the paper was written JS, and was modified by ZL and KZ. All authors read and approved the final manuscript. Abbreviations AA Accessory activities ADFI Average daily feed intake ADG Average daily gain AP Antimicrobial peptide group CAZYme Carbohydrate-active enzyme CBM Carbohydrate-binding modules CE Carbohydrate esterase CK Control group DMI Dry matter intake F/G Feed/gain FC Fold change FDA Food and Drug Administration FDR False discovery rate GH Glycoside hydrolases Gt Glycosyl transferases KEGG Kyoto Encyclopedia of Genes and Genomes NCBI National Center for Biotechnology Information O2PLS Two-way orthogonal partial least squares OPLS-DA Orthogonal projections to latent structures discrimination analysis PCoA Principal coordinates analysis PL Polysaccharide lyases TMR Total mixed ration UPLC Ultra-performance liquid chromatography VFA Volatile fatty acid VFDB Virulence Factor Database VIP Variable importance in the projection Competing interests The authors declare that there are no conflicts of interest. Supplementary Information Funding g This research was financially supported by Research and application of corn straw forage and beef cattle high-efficiency and quality production technology (Provincial Education Science and Technology Innovation Project) (GSSYLXM-02), the Gansu beef cattle quality fattening project (GSA- XMLZ-2021–01), the Application of Pingliang Red Bull Planting and Breeding Combined with High-efficiency Circular Production System Construction Technology Application (2020C-08), and the local funding (GSSLCSX-2020–1). The funding agencies did not participate in study design, data collection, analysis and interpretation or writing of the manuscript. Discussion These results were consistent with those reported for mice. Streptomyces_sp._So13.3 (Streptomyces genus) pro- duces a bicyclic 19-peptide compound BI-32169, which potently inhibits the glucagon receptor and hence, can act as a glucagon antagonist [64]. Glucagon is a hor- mone that promotes catabolism, reduces body weight, and increases energy expenditure [65]. Furthermore, a negative correlation was found in this study between Aci- netobacter_sp._Ac_1271 and terephthalic acid because Acinetobacter sp. can degrade terephthalic acid [66]. It is necessary to perform further studies to determine the correlation between variety of other microorganisms and metabolites. In order to view these bacterial populations in numer- ous samples, it has been standard protocol to show organisms at abundances greater than 1%, or group the low-abundant organisms into an “other” category [67– 70]. While this is a widely accepted method, it may miss the key role of low abundance populations. Conjoint analysis and correlation analysis revealed that Strepto- myces_sp._So13.3 exhibited a weak correlation with ADG (Fig. 6B), Streptomyces_sp._So13.3 also moderately corre- lated with hippuric acid (Fig. 5C), and highly correlated with hippuric acid and ADG (Fig. 6A). This result is con- sistent with reports on several other strains and metab- olites. It is worth noting that the relative abundance of these strains is less than 0.0001%. Previous studies only focused on some high-abundance microorganisms that may not present the entire picture properly. This study demonstrates that metabolites may amplify low-abun- dance species, resulting in improved production perfor- mance. The importance of low abundance organisms has been reported in other systems. For example, in peat- land communities, 0.006% of the 16S rRNA reading of fi Bile acids are synthesized from cholesterol in the liver and are further hydrolyzed by bacterial bile salt hydrolase to form toxic free bile acids. 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https://openalex.org/W4323075367	https://journal.formosapublisher.org/index.php/eajmr/article/download/3024/2817	English	null	Elements of Vandalism in Law Number 1 of 2023 Concerning the Criminal Code	East Asian Journal of Multidisciplinary Research	2,023	cc-by	5,375	Elements of Vandalism in Law Number 1 of 2023 Concerning the Criminal Code Murshal Senjaya1* Universitas Pasundan Corresponding Author: Murshal Senjaya Murshal.sanjaya@unpas.ac.id A R T I C L E I N F O Keywords: Elements of Crime, Vandalism, Criminal Code A R T I C L E I N F O Keywords: Elements of Crime, Vandalism, Criminal Code A B S T R A C T The element of vandalism in Law Number 1 of 2023 concerning the Criminal Code, vandalism is included in the form of delinquency. Crimes related to delinquency are regulated in Article 331. In this article, it is explained that perpetrators of delinquency can be punished with category II fines or as much as IDR 10 million. "Any person who in a public place commits delinquency against people or goods that can cause harm, loss or distress, shall be punished with a maximum fine of category II," reads Article 331. In the explanation section, the example of delinquency in question is scribbling wall on a public street. The factors that cause vandalism are users, factors from the library, other factors including the environment, stress and communication blockages. The effort to deal with vandalism is the need for an intellectual mentality of users so they don't vandalize a collection of library materials. Attitudes and behavior of librarians/library officers who always control users both in the room and the exit. For certain types of collections, closed access services can be performed. Make written rules and clear sanctions for those who commit violations. Controlling the use of manual and automated membership cards so that they do not use other people's membership cards. The entrance is always closed, only users who have a card can enter the library. Received : 15, December Revised : 16, January Accepted: 18, February ( DOI: https://doi.org/10.55927/eajmr.v2i2.3024 ISSN-E: 2828-1519 https://journal.yp3a.org/index.php/eajmr East Asian Journal of Multidisciplinary Research (EAJMR) Vol. 2, No. 2, 2023: 673-682 East Asian Journal of Multidisciplinary Research (EAJMR) Vol. 2, No. 2, 2023: 673-682 East Asian Journal of Multidisciplinary Research (EAJMR) Vol. 2, No. 2, 2023: 673-682 INTRODUCTION g Based on the description above, the formulation of the problem is: 1. What is the element of vandalism in Law Number 1 of 2023 concerning the Criminal Code? 1. What is the element of vandalism in Law Number 1 of 2023 concerning the Criminal Code? 2. What are the factors and efforts to deal with graffiti vandalism behaviour (doodle)? ( DOI: https://doi.org/10.55927/eajmr.v2i2.3024 ISSN-E: 2828-1519 https://journal.yp3a.org/index.php/eajmr 673 Senjaya INTRODUCTION Vandalism is the intentional destruction of property that is carried out viciously and causes losses. Vandalism is the destruction of public property. The Big Indonesian Dictionary defines vandalism as acts of damaging and destroying works of art and other valuable items (damage to nature, etc.) or destruction and destruction in a rough and vicious way. According to Cohen quoted by Ajeng Triani, categorizing the types of Vandalism based on the motivations that encourage acts of Vandalism are as follows: First Aquistive Vandalism, namely Vandalism committed with the motivation to obtain money or property. For example: placing advertisements, banners, posters, billboards or other forms of marketing that damage the environment in which they are located. Second, tactical vandalism, namely vandalism carried out with the motivation of achieving certain goals such as introducing an ideology. An example is what Pong Harjianto did, who wrote the sentence "honest, fair, firm" in the roof of the DPR building (People's Representative Council) to notify members of the DPR that the performance of a DPR representative must be based on honesty, fairness and firmness. The third is Malicious vandalism, namely vandalism that is carried out because the perpetrator of vandalism gets pleasure by giving disturbance to other people, or feels amused when destroying other people's property. Fourth, play vandalism, namely vandalism that is carried out with the motivation to show or demonstrate the abilities he has, not for the purpose of to annoy other people. AL Wilde also identified vandalism into 3 (three) main types as follows: First, Indiscriminate vandalism includes destructive actions that have no purpose and do not generate immediate profits. This is a common act of vandalism, "unclear" destruction by teenagers for fun. Both means of Predatoris vandalism include destructive acts for profit, such as "trashing" or destroying vending machines to steal their contents. The third is revenge vandalism, namely actions taken as an expression of hatred towards certain racial or ethnic groups. Concerning vandalism itself is regulated in the Criminal Code. As a whole there are efforts, but in fact cases of vandalism and scribbles are still commonly found in urban areas. Therefore, it is necessary to look at the efforts of law enforcement in dealing with criminal acts of graffiti vandalism (scribbling) and the legal policies that apply in regulating these actions. Then reveal how the effectiveness of these efforts, based on the facts that cases of vandalism are increasing. THEORETICAL REVIEW 1. Definition and Elements of a Criminal Act 1. Definition and Elements of a Criminal Act Formal elements: 1) The act of something 2) The act was done or not done 3) The act is stated by law as a prohibited act 4) This regulation is threatened by statutory regulations criminal. a. Formal elements: a. Formal elements: 1) The act of something 1) The act of something 2) The act was done or not done 3) The act is stated by law as a prohibited act 4) This regulation is threatened by statutory regulations criminal. 1. Definition and Elements of a Criminal Act 1. Definition and Elements of a Criminal Act The term crime is a translation of the Dutch term "Strafbaar Feit" or "delict". In Indonesian, as a translation of strafbaar feit or delict, there are several terms such as: f f The term crime is a translation of the Dutch term "Strafbaar Feit" or "delict". In Indonesian, as a translation of strafbaar feit or delict, there are several terms such as: a. Criminal act 674 East Asian Journal of Multidisciplinary Research (EAJMR) Vol. 2, No. 2, 2023: 673-682 Among the six terms mentioned above, the most appropriate and appropriate term to use is the term "crime", on the grounds that this term, apart from containing a precise and clear meaning as a legal term, is also very easy to pronounce. Crime is a term that is often used to translate the term strafbaarfeit in Dutch language. Criminal acts are one of the important joints of criminal law besides mistakes and crimes. A criminal act is an act that is defined in criminal legislation as a prohibited act. If this act is committed by mistake, the person who committed the act can be subject to criminal sanctions. As for the definition of a crime according to scholars, one of them is Wirjono Prodjodikoro, that is a crime is an act whose perpetrators can be subject to criminal penalties. Muljatno is of the opinion that the criminal act is an act prohibited by a rule of law which prohibition is accompanied by a threat (sanction) in the form of a certain punishment, for whoever violates this prohibition. R. Tresna stated that a criminal event is an act or series of human actions, which is contrary to laws or other regulations, for which acts of punishment are carried out, and then Simons in Mustafa Abdullah, Ruben Achmad argues, that: "Criminal events is "Een Strafbaargestelde, onrechtmatige, met schuld in verband staande handeling van een toerekeningsva baar person". The free translation is: wrongdoing and resistance law, which is punishable by crime and carried out by someone who is capable of being responsible. p g p According to the definition of the National Criminal Code are: According to the definition of the National Criminal Code are: a Formal elements: g a. b. Material elements: The act must be contrary to the law, that is, it must be truly perceived by society as an act that should not be done. The elements of a crime can be viewed from two aspects, namely the subjective and objective aspects. The act must be contrary to the law, that is, it must be truly perceived by society as an act that should not be done. The elements of a crime can be viewed from two aspects, namely the subjective and objective aspects. 1) From a subjective point of view, a criminal event is an act that someone did wrong. It is the elements of the perpetrator's fault that result in a criminal event. This element of error arises from the intention or will of the perpetrator. So, as a result of the act it is known that it is prohibited by law and is threatened with punishment. So, there is indeed an element of intentionality. 2) From an objective point of view, people's actions, the visible consequences of those actions, there may be certain circumstances that accompany the action. 675 Senjaya Judging from these criminal elements, an act committed by a person must meet the requirements so that it can be declared a criminal event. The conditions that must be met as a criminal event are as follows: a) There must be an action. That is, there really is an activity carried out by a person or several people. The activity is seen as a certain action that can be understood by others as an event. b) The act must be in accordance with what is described in the legal provisions. It means action as thing legal events comply with the contents of the legal provisions in effect at the time. The culprit really did do as it happened. Mandatory doer take responsibility. Actions that cannot be blamed can be committed by someone or some people in carrying out their duties, defend themselves from the threats of other people who interfere with their safety and emergencies. y g c) It must be proven that there was a mistake that can be accounted for. It means that the actions committed by a person or several persons can be proven as an act that is condemned by legal provisions d) Must be against the law. b. Material elements: That is, an action that is against the law is meant if the action is clearly contrary to the rules law. There must be a legal threat. This means that if there are provisions that regulate prohibitions or obligations in certain actions, those provisions contain sanctions and threats of punishment. The threat of punishment was clearly stated the maximum punishment that must be carried out by the perpetrators. If in a provision there is no threat of punishment for a particular act, in an event/crime, the perpetrator does not need to carry out a specific sentence. 2. Violation Violations are actioning whose character is against the law, which can only be known after there is a decisive decree thus. 25 Violation is a statute delict, namely an act whose nature is against the law can only be known after there is a law that regulates it. Violation in English is called misdemeanour. The penalty is usually a fine. The difference between crimes and violations is no longer used as a criterion to determine which court has the authority to try them, as before, because now all are tried by district courts. Nonetheless, there are differences in the adjudication. 2. Violation Violations are actioning whose character is against the law, which can only be known after there is a decisive decree thus. 25 Violation is a statute delict, namely an act whose nature is against the law can only be known after there is a law that regulates it. Violation in English is called misdemeanour. The penalty is usually a fine. The difference between crimes and violations is no longer used as a criterion to determine which court has the authority to try them, as before, because now all are tried by district courts. Nonetheless, there are differences in the adjudication. 3. Definition of Vandalism The Big Indonesian Dictionary defines vandalism as acts of damaging and destroying works of art and other valuable items (damage to nature, etc.) or destroying and destroying things violently and viciously. Vandalism is an act of destroying objects or items that are public property or needed by the public. Vandalism comes from the word vandal or vandalus, which refers to the name of an ancient Germanic tribe that inhabited the area south of the Baltic between the Vistula and the Oder. 4. Forms of Vandalism f According to Cohen quoted by Ajeng Triani, categorizing the types of vandalism based on the motivations that encourage acts of vandalism are as follows: f According to Cohen quoted by Ajeng Triani, categorizing the types of vandalism based on the motivations that encourage acts of vandalism are as follows: a. Aquistive Vandalism is vandalism that is carried out with the motivation to obtain money or property. Example: ad placement, banners, posters, billboards or other forms of marketing that damage the environment in which they exist. y b. Tactical Vandalism is vandalism done with motivation to achieve certain goals such as introducing an ideology. An example is what Pong Harjiatno did who wrote the sentence "honest, fair, firm" on the roof of the DPR People's Representative Council) building to notify members of the DPR that the performance of a DPR representatives must be based on honesty, fairness and firmness. c. Malicious Vandalism is vandalism that is carried out because the perpetrators of vandalism get pleasure from disturbing other people, or feel entertained when destroying other people's property. Play Vandalism is vandalism that is done with the motivation to show or demonstrate abilities possessed, not old to disturb others. AL Wilde also identified vandalism into 3 mains (types), namely: 1) Indiscriminate vandalism includes destructive acts that have no purpose and produce no monetary gain. Scientific acts of vandalism that are commonly carried out, “obscure” destruction that teenagers do for fun. 2) Predatory vandalism includes destructive acts for profit, such as "rubbing up" or destroying vending machines to steal their contents. g p y g g 3) Revenge vandalism is an act committed as an expression of hatred against a certain racial or ethnic group. According to the Big Indonesian Dictionary (KBBI) graffiti is an ancient painting on a wall or rock (derived from Italian). Scratches on cave walls in the past were used as graffiti or graffiti on city walls in various forms and the like as graffiti. Graffiti is a branch of art that can be enjoyed visually. Graffiti is graffiti containing writing, symbols or sentences in which there is a combination of elements of line, colour, shape and volume. Not all scribbles or pictures on the wall can be categorized as graffiti that contains artistic value. A Graffiti contains artistic value if it can give a decorative and ornamental impression. b. Material elements: In the fourth and fifth centuries AD the vandals expanded their territory to reach Spain and South Africa. In 455 AD the vandals entered the city of Rome and destroyed works of Roman art and literature that existed at that time. 676 East Asian Journal of Multidisciplinary Research (EAJMR) Vol. 2, No. 2, 2023: 673-682 East Asian Journal of Multidisciplinary Research (EAJMR) Vol. 2, No. 2, 2023: 673-682 From the behaviour of the vandal tribe, vandal is then given the meaning of someone who deliberately destroys or destroy something beautiful. another definition of vandalism is an action or act that disturbs or damages physical and man-made objects, both private property and public facilities. Commonly encountered vandalism is scribbling on school walls, tables, chairs, bridges, bus stops, damaging public facilities such as public telephones, buses, public toilets, and plants. METHODOLOGY The research method is analytical descriptive in nature, namely describing the problems and facts that occur based on positive legal norms, namely the laws related to this research. Approach method with normative juridical namely using positive legal norms related to the element of vandalism in Law Number 1 of 2023 concerning the Criminal Code. Data analysis was carried out qualitatively, meaning without using numbers and statistical formulas. 4. Forms of Vandalism But there are times when a graffiti is expressions of disappointment, anger, frustration, and hatred. The disclosure of negative 677 Senjaya feelings gives the impression of being vulgar so that it is considered as vandalism. The graffiti referred to in this study is illegal graffiti that does not have legality in its licensing and carried out by teenagers who do not have the aim of seeking profit from something that is used by the perpetrator as a place strike out but solely to emphasize how the existence of a gang or a group to control an area certain. Gangs that usually become perpetrators of graffiti vandalism are unorganized gangs or just teenage gangs that have the intention of defending their territory from other gangs, unlike mafia syndicates or organized gangs. The behaviour of graffiti vandalism is very disturbing to residents because the action that started as a fad developed into anarchic behaviour. For example, destroying public facilities, big or small, light or heavy, in the end causing disturbance and loss to the orderliness of the surrounding environment. The previously clean city was littered with scribbles and random pictures made by pranksters. Discussion of the Element of Vandalism in Law Number 1 of 2023 Concerning the KUHP Discussion of the Element of Vandalism in Law Number 1 of 2023 Concerning th KUHP Discussion of the Element of Vandalism in Law Number 1 of 2023 Concerning the KUHP The new Criminal Code regulates punishment for people who are considered to have committed vandalism by scribbling on walls. In the new Criminal Code, vandalism is included in the form of delinquency. Crimes related to delinquency are regulated in Article 331. In this article, it is explained that perpetrators of delinquency can be punished with category II fines or as much as IDR 10 million. "Any person who in a public place commits delinquency against people or goods that can cause harm, loss or distress, shall be punished with a maximum fine of category II," reads Article 331. In the explanation section, the example of delinquency in question is scribbling wall on a public street. y g Vandalism behaviours in the library can occur due to several factors, both originating from the users themselves and those from the library. The factors that led to the act of vandalism were: 1. Factors From Users a. Lack of user awareness Lack of user awareness of the importance of information in library materials that actually belongs together where the collections they vandalize can cause other people to be unable to access them anymore, they are not aware of the possibility that the information 678 East Asian Journal of Multidisciplinary Research (EAJMR) Vol. 2, No. 2, 2023: 673-682 needed has also been vandalized by other users. If library users realize that the vandalism, they are doing can harm themselves, then the collection of library materials in the library will be sustainable. needed has also been vandalized by other users. If library users realize that the vandalism, they are doing can harm themselves, then the collection of library materials in the library will be sustainable. b. Disappointment with library services. This disappointment factor can also cause users to vandalize library collections. Disappointment can occur because the information they are looking for is not met, so they feel disappointed and commit vandalism. Disappointment of users can also occur due to librarians who are less friendly to users, ignorant, and do not want to help with their difficulties. This factor usually occurs because librarians are too rigid with existing rules and there is no tolerance. Discussion of the Element of Vandalism in Law Number 1 of 2023 Concerning the KUHP For example, users who are late in returning books for a long time are subject to quite a lot of fines where the amount of the fine exceeds the price of the book, they borrowed but the officer has no discretion. Enforcement of rules like this can actually cause users to get angry and can vandalize other collections. c. There is an opportunity "Opportunity" is also a factor for users to commit acts of vandalism. Actually, the user has no intention to commit vandalism, but due to lack of or there is no supervision so they just fad do vandals. If the opportunity first they feel safe not being known by the officers, then next they will look for another opportunity, even after a long time they will look for opportunities. 2. Factors from Libraries a. Weak Oversight. Factors that cause acts of vandalism and book theft include the result of weak supervision of officers both towards users and against the collection. Loose supervision of users and collections that will be taken out of the library or collections that are returned is the cause of many missing books. Likewise, the weak supervision of users on collection shelves or on tables reading and places can cause the user to freely tear up some of the pages of the book. p p g b. Unprofessional Officers Service to users who are too bureaucratic can cause service to be slow so users feel difficult and impatient which can eventually result in users taking shortcuts by taking books out without going through legal procedures. In addition, due to colliding with the applicable rules/rules of the library, such as for example not being able to photocopy, certain collections may not be borrowed, and the provision for the maximum number of books borrowed also causes users to commit acts of vandalism. Officers who are less professional in providing services such as unsympathetic, low quality of service, officers cannot help users with difficulties so they are dissatisfied. The dissatisfaction of the users is that they can destroy library materials. b. Unprofessional Officers Service to users who are too bureaucratic can cause service to be slow so users feel difficult and impatient which can eventually result in users taking shortcuts by taking books out without going through legal procedures. Discussion of the Element of Vandalism in Law Number 1 of 2023 Concerning the KUHP In addition, due to colliding with the applicable rules/rules of the library, such as for example not being able to photocopy, certain collections may not be borrowed, and the provision for the maximum number of books borrowed also causes users to commit acts of vandalism. Officers who are less professional in providing services such as unsympathetic, low quality of service, officers cannot help users with difficulties so they are dissatisfied. The dissatisfaction of the users is that they can destroy library materials. 679 Senjaya 3. Other Factors 3. Other Factors a. Environmental factor Environmental factors include the economic conditions of users, social factors society, the library layout environment is not quite right. b. Stress People who are stressed, frustrated, confused and angry and disappointed can be taken out by destroying collections. Acts of vandalism caused by these factors are usually not because of the information needed but actually to damage it by not thinking about the consequences. a. Environmental factor Environmental factors include the economic conditions of users, social factors society, the library layout environment is not quite right. b. Stress People who are stressed, frustrated, confused and angry and disappointed can be taken out by destroying collections. Acts of vandalism caused by these factors are usually not because of the a. Environmental factor Environmental factors include the economic conditions of users, social factors society, the library layout environment is not quite right. y, y y q g b. Stress People who are stressed, frustrated, confused and angry and disappointed can be taken out by destroying collections. Acts of vandalism caused by these factors are usually not because of the information needed but actually to damage it by not thinking about the consequences. c. Blockage of Communication Disharmony in the relationship between librarians and users can cause the a priori attitude of the user towards the library which in the end the user does not have a sense of ownership of the collection. The librarian in a library is a mediator who connects the library as a communicator to the user as a communicant. For this reason, librarians must be able to help the interests of users so that communication between users and the library is not blocked. Efforts to Prevent Vandalism of library material collections can occur in all types and forms of libraries, whether the library is small or large. Discussion of the Element of Vandalism in Law Number 1 of 2023 Concerning the KUHP To overcome various forms of vandalism which are very detrimental to the library and other library users, prevention and security measures need to be taken. Things that need to be done for prevention (preventive) can be in the form of actions as follows: c. Blockage of Communication Disharmony in the relationship between librarians and users can cause the a priori attitude of the user towards the library which in the end the user does not have a sense of ownership of the collection. The librarian in a library is a mediator who connects the library as a communicator to the user as a communicant. For this reason, librarians must be able to help the interests of users so that communication between users and the library is not blocked. Efforts to Prevent Vandalism of library material collections can occur in all types and forms of libraries, whether the library is small or large. To overcome various forms of vandalism which are very detrimental to the library and other library users, prevention and security measures need to be taken. Things that need to be done for prevention (preventive) can be in the form of actions as follows: 1) Physical Security: First, it prohibits users from wearing jackets and bringing bags, stop maps or the like into the collection room. Second, provide special officers to check when users leave the library. 2) Security Against the System: First, provide control tools in the system. Second installing security systems such as matrix sensors and alarm sensors at the exit of the library. Prevention and security against vandalism in the library can be in the form of actions as follows: 1. There needs to be an intellectual mentality of users so as not to vandalize the collection of library materials. 2. Attitudes and behaviour of librarians/ library officers who always hold control towards users both indoors and out. 3. For certain types of collections, closed access services can be performed. y 4. Make written rules and clear sanctions for those who commit violations. 5. Control the use of membership cards, both manual and automated so that they do not use other people's membership cards. 6. The entrance is always closed, only users who have a card can enter the library. 7. Provide a representative reading room that is separate from collection shelves. 8. Installation of convex mirrors (cannex mirrors) in certain places. 9. y Recommendations 1. The effort to deal with vandalism is to provide a representative reading room which is separate from collection shelves. 1. The effort to deal with vandalism is to provide a representative reading room which is separate from collection shelves. 2. Installation of convex mirrors (cannex mirrors) in certain places. Installation of CCTV cameras in the collection room to control the perpetrators of vandalism. 2. Installation of convex mirrors (cannex mirrors) in certain places. Installation of CCTV cameras in the collection room to control the perpetrators of vandalism. CONCLUSIONS AND RECOMMENDATIONS 1. The element of vandalism in Law Number 1 of 2023 concerning the Criminal Code, vandalism is included in the form of delinquency. Crimes related to delinquency are regulated in Article 331. In this article, it is explained that perpetrators of delinquency can be punished with category II fines or as much as IDR 10 million. "Any person who in a public place commits delinquency against people or goods that can cause harm, loss or distress, shall be punished with a maximum fine of category II," reads Article 331. In the explanation section, the example of delinquency in question is scribbling wall on a public street. 2. 2. The factors that cause vandalism are users, factors from the library, other factors including the environment, stress and communication blockages. The effort to deal with vandalism is the need for an intellectual mentality of users so they don't vandalize a collection of library materials. Attitudes and behaviour of librarians /library officers who always control users both in the room and the exit. For certain types of collections, closed access services can be performed. Make written rules and clear sanctions for those who commit violations. Controlling the use of manual and automated membership cards so that they do not use other people's membership cards. The entrance is always closed, only users who have a card can enter the library. Discussion of the Element of Vandalism in Law Number 1 of 2023 Concerning the KUHP Installation of CCTV cameras in the collection room. 680 East Asian Journal of Multidisciplinary Research (EAJMR) Vol. 2, No. 2, 2023: 673-682 East Asian Journal of Multidisciplinary Research (EAJMR) Vol. 2, No. 2, 2023: 673-682 East Asian Journal of Multidisciplinary Research (EAJMR) Vol. 2, No. 2, 2023: 673-682 REFERENCES Abdullah Marlang and Irwansyah and Emperoruddin Kamaruddin, Introduction to Indonesian Law, Aspublishing, Makassar, 2011. Nature, Introduction to Criminology, Reflections, Makassar, 2010. Andi Sofyan and Nur Azisa, Criminal Law, Pustaka Pena Press, Makassar, 201 Andi Sofyan and Nur Azisa, Criminal Law, Pustaka Pena Press, Makassar, 2016 Bambang Poernomo, Principles of Indonesian Criminal Law. Ghalia, Yogyakarta, 2015. Barda Nawawi Arief, Theories and Criminal Policy. Alumni, Bandung, 2018. 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Efendi, Introduction to Indonesian Law (PHI), PT Rajagrafindo Persada, Jakarta, 2014 I Ketut Mertha, et al, Textbook of Criminal Law, Udayana Faculty of Law, Denpasar, 2016 Ida Yeni R and Safrina Arifiani F and Diyan Fatimatuz Zahra, “Wall Art as an Educational Media to Prevent Vandalism in SMA Negeri 5 Yogyakarta, Pelita Journal, Faculty of Languages and Arts, Yogyakarta State University, Vol. IV, Number 1 April 2009 Ismu Gunadi and Jonaedi Efendi, Understanding Criminal Law Quickly & Easily, Prenadamedia Group, Jakarta, 2014 Isran Elnadi, "Vandalism of Collections at the Technical Service Unit (UPT) of the Bengkulu University Library", Journal of Library and Information Science, Librarian of the Bengkulu University Library, Vol 2, Number 1 2018 Khairunnisa Lutfi, "Vandalism in the Perspective of Islamic Criminal Law", Thesis, Bachelor of Law, Faculty of Sharia and Law, State Islamic University of North Sumatra, Medan, 2020. 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https://openalex.org/W2109435591	https://academiccommons.columbia.edu/doi/10.7916/D8FJ2TBR/download	English	null	Impact of adding a limitations section to abstracts of systematic reviews on readers’ interpretation: a randomized controlled trial	BMC Medical research methodology	2,014	cc-by	6,921	Abstract Background: To allow an accurate evaluation of abstracts of systematic reviews, the PRISMA Statement recommends that the limitations of the evidence (e.g., risk of bias, publication bias, inconsistency, imprecision) should be described in the abstract. We aimed to evaluate the impact of adding such limitations sections on reader’s interpretation. Method: We performed a two-arm parallel group randomized controlled trial (RCT) using a sample of 30 abstracts of systematic reviews evaluating the effects of healthcare intervention with conclusions favoring the beneficial effect of the experimental treatments. Two formats of these abstracts were derived: one reported without and one with a standardized limitations section written according to the PRISMA statement for abstracts. The primary outcome was readers’ confidence in the results of the systematic review as stated in the abstract assessed by a Likert scale from 0, not at all confident, to 10, very confident. In total, 300 participants (corresponding authors of RCT reports indexed in PubMed) were randomized by a web-based randomization procedure to interpret one abstract with a limitations section (n = 150) or without a limitations section (n = 150). Participants were blinded to the study hypothesis. Results: Adding a limitations section did not modify readers’ interpretation of findings in terms of confidence in the results (mean difference [95% confidence interval] 0.19 [−0.37–0.74], p = 0.50), confidence in the validity of the conclusions (0.07 [−0.49–0.62], p = 0.80), or benefit of the experimental intervention (0.12 [−0.42–0.44], p = 0.65). This study is limited because the participants were expert-readers and are not representative of all systematic review readers. Conclusion: Adding a limitations section to abstracts of systematic reviews did not affect readers’ interpretation of the abstract results. Other studies are needed to confirm the results and explore the impact of a limitations section on a less expert panel of participants. Trial registration: ClinicalTrial.gov (NCT01848782). Trial registration: ClinicalTrial.gov (NCT01848782). Trial registration: ClinicalTrial.gov (NCT01848782). Keywords: Meta-analysis, Systematic review, Bias, Limits, Limitation, Interpretation, Interpretation bias, Misinterpretation, Abstract, Results Keywords: Meta-analysis, Systematic review, Bias, Limits, Limitation, Interpretation, Interpretation bias, Misinterpretation, Abstract, Results * Correspondence: ayavchitz@gmail.com 1Centre de Recherche Épidémiologies et Statistiques INSERM U1153, Paris, France 2Centre of Clinical Epidemiology, Assistance Publique des Hôpitaux de Paris, Hôtel Dieu Hospital, Place du Parvis Notre-Dame, 75181 Paris, Cedex 4, France Full list of author information is available at the end of the article © 2014 Yavchitz et al.; licensee BioMed Central Ltd. Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Open Access * Correspondence: ayavchitz@gmail.com 1Centre de Recherche Épidémiologies et Statistiques INSERM U1153, Paris, France 2Centre of Clinical Epidemiology, Assistance Publique des Hôpitaux de Paris, Hôtel Dieu Hospital, Place du Parvis Notre-Dame, 75181 Paris, Cedex 4, France Full list of author information is available at the end of the article Construction of the limitations section Construction of the limitations section According to the PRISMA statement for abstracts, the limitations section should address the following: 1) risk of bias common to many or all studies, such as lack of blinding for subjective outcomes or unavailability of data; 2) inconsistency of effect or association, as dem- onstrated by high heterogeneity; 3) imprecision due to few events or small sample size, for example; 4) indir- ectness of the evidence, such as use of an intermediate or short-term outcome; and 5) likelihood of publica- tion bias [12,16]. These limitations are the factors used to evaluate the level of evidence according to the GRADE approach [17]. Clinical decision makers and healthcare practitioners frequently rely on abstracts to decide the value of a study [6,7]. In some cases, healthcare practitioners have access to only the abstract and make healthcare decisions based solely on the information in the abstract [8]. To improve the transparency of abstracts [9,10], methodologists, re- searchers and editors have established recommendations for the presentation of systematic reviews in the PRISMA statements [11] with an extension for reporting abstracts [12]. These recommendations indicate that the limitations of the evidence should be described in the abstracts of systematic reviews [12]. This recommendation is rarely implemented [13], although some journals request that authors report a structured abstract with a limitations section [14]. However, the impact of adding this section to abstracts on readers’ interpretation is unknown. One author (AY) systematically searched for and ex- tracted from the full-text systematic review the follow- ing: 1) whether the systematic review had one or several of the limitations described above and 2) the limitations were reported by the authors of the systematic review in the full-text article or in the abstract. When limitations were outcome-specific, the limitations reported referred to the outcomes highlighted in the abstracts. We aimed to evaluate the impact of adding a limitations section, written according to the PRISMA statement for abstracts of systematic reviews, on readers’ interpretation of the abstract results. Then, for each selected abstract, one of the authors (AY) wrote a limitations section. The section focused on the limitations identified and was written in a standard- ized way, beginning with “This review is limited by….”, with a maximum of 2 sentences. When the original ab- stract was structured, we preserved the structured form and added the limitations section before the conclusions section with a heading “Limitations”. Abstract This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Page 2 of 9 Study design We planned a randomized controlled trial to compare the interpretation of systematic review abstracts reported with and without a limitations section. We selected 30 abstracts of systematic reviews, and then developed 2 formats of the selected abstracts: reported with and with- out a limitations section written according to the PRISMA statement for abstracts. Then, we randomized participants to read and interpret one abstract with or without a limi- tations section. The study reporting follows the 2010 CONSORT statement [15]. We voluntarily did not use the same terminology as PRISMA because it may not be well understood by the readers (e.g., imprecision, inconsistency of effect or asso- ciation and indirectness of the evidence are complex concepts). We kept the wording similar to that used by the authors of the systematic reviews, for example, when reporting the limitations in the discussion section. An- other author (IB) read the entire modified abstract to de- termine whether the limitations section was written according to our specific guidance, that is, whether the limitations section 1) focused on the limitations previously identified in the report of the systematic review, 2) was no longer than 2 sentences, and 3) focused on limitations as described in the PRISMA for abstracts. When the second Construction of the limitations section When the original abstract was not structured, we added the limitations section, without a heading, after the results and before the conclusion sentences. Background intervention and reported the risk of bias of primary studies in the full-text article, thus allowing selection of a relatively homogeneous sample. Abstracts from the cohort of 100 systematic reviews were screened by one author (AY) and reviewed by a second author (IB) to confirm eligibility. Any disagreements were resolved by consensus. Systematic reviews are the cornerstone of therapeutic evaluation [1]. Clinicians, decision makers and researchers use them to keep up-to-date with current medical litera- ture, develop clinical practice guidelines and sometimes plan future research [2-4]. However, systematic reviews may differ in quality depending on the conduct of the sys- tematic review as well as the availability and quality of the primary studies [5]. Consequently, readers should carefully examine the methodological quality of reviews to assess their confidence in the results and conclusions. Selection of abstracts of systematic reviews The abstracts were selected from a cohort of 100 system- atic reviews assessing the effects of healthcare interven- tions, published between January and March 2012, and indexed in the Database of Reviews of Effects [13]. The search strategy and eligibility criteria for this cohort were described elsewhere [13]. From this cohort, we selected the first 30 systematic reviews whose abstract conclu- sion favored the beneficial effect of the experimental Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Page 3 of 9 Page 3 of 9 2013 and indexed in PubMed Core Clinical Journals, with an email address available on PubMed. author found that the limitation section was not written according our specific guidance, the limitations section was re-written and discussed to reach consensus. Potential participants were invited by e-mail to partici- pate in an online survey on the interpretation of abstracts of systematic reviews. As an incentive, they were told that they would be entered into a draw along with all other participants for a chance to win an Apple iPad mini. If they agreed to participate, they logged onto a secure web- site and answered a screening question asking if they were a clinician; non-clinicians were excluded. Invitation emails were sent in waves until the planned number of clinicians had logged on and completed the assessment. A max- imum of 2 reminders were sent to participants. The email invitation and details of the questionnaire are in the Additional file 1. Randomization A t A computer-generated randomization list was generated for allocating participants to abstracts with or without a limitations section in a 1:1 ratio. Allocation was con- cealed by use of a web-based randomization procedure whereby participants logged onto the system and were randomized to evaluate an abstract with or without a limitations section. Clinicians who logged onto the sys- tem but did not evaluate an abstract were excluded and the abstract was automatically allocated to another clinician. An example of an abstract with and without a limita- tions section is given in the Table 1 and all abstracts with a limitations section are available in the Additional file 1. Participants Eligible participants were the corresponding authors of clinical trials published between January 2010 and June Construction of abstracts with and without a limitations section For each of the 30 selected abstracts, we obtained 1 ab- stract without a limitations section (i.e., the original ab- stract) and 1 with a limitations section (i.e., the original abstract plus the constructed limitations section). If the original abstract reported the limitations of the systematic review, this was deleted from the abstract without a limi- tations section and kept in the abstract with a limitations section. This situation occurred for 9 (30%) abstracts. All abstracts were standardized, with the treatment name (s), authors and journal masked. The experimental treat- ment name was replaced by “intervention A”. When the control treatment was an active treatment, “comparator B” replaced the treatment name. Acronyms and abbreviations were also deleted. BACKGROUND: BACKGROUND: Intervention A is a standard treatment of metastatic urothelial carcinoma (UC), though comparator B is frequently substituted due to improved tolerability. Because comparative effectiveness in clinical outcomes of intervention A - versus comparator B chemotherapy is lacking, a meta-analysis was carried out. BACKGROUND: Intervention A is a standard treatment of metastatic urothelial carcinoma (UC), though comparator B is frequently substituted due to improved tolerability. Because comparative effectiveness in clinical outcomes of intervention A - versus comparator B chemotherapy is lacking, a meta-analysis was carried out. METHODS: PubMed was searched for articles published from 1966 to 2010. Eligible studies included prospective randomized trials evaluating intervention A - versus comparator B regimens in patients with metastatic UC. Individual patient data were not available and survival data were inconsistently reported. Therefore, the analysis focused on overall response (OR) and complete response (CR) rates. The Mantel-Haenszel method was used for combining trials and calculating pooled risk ratios (RRs). METHODS: PubMed was searched for articles published from 1966 to 2010. Eligible studies included prospective randomized trials evaluating intervention A - versus comparator B regimens in patients with metastatic UC. Individual patient data were not available and survival data were inconsistently reported. Therefore, the analysis focused on overall response (OR) and complete response (CR) rates. The Mantel-Haenszel method was used for combining trials and calculating pooled risk ratios (RRs). RESULTS: A total of 286 patients with metastatic UC from four randomized trials were included. Intervention A was associated with a significantly higher likelihood of achieving a CR [RR =3.54; 95% confidence interval (CI) 1.48-8.49; P =0.005] and OR (RR =1.34; 95% CI 1.04-1.71; P =0.02). Survival end points could not be adequately assessed due to inconsistent reporting among trials. RESULTS: A total of 286 patients with metastatic UC from four randomized trials were included. Intervention A was associated with a significantly higher likelihood of achieving a CR [RR =3.54; 95% confidence interval (CI) 1.48-8.49; P =0.005] and OR (RR =1.34; 95% CI 1.04-1.71; P =0.02). Survival end points could not be adequately assessed due to inconsistent reporting among trials. RESULTS: A total of 286 patients with metastatic UC from four randomized trials were included. Intervention A was associated with a significantly higher likelihood of achieving a CR [RR =3.54; 95% confidence interval (CI) 1.48-8.49; P =0.005] and OR (RR =1.34; 95% CI 1.04-1.71; P =0.02). Abstract with limitations section TITLE: Comparative effectiveness of intervention A and comparator B for treatment of advanced urothelial carcinoma. TITLE: Comparative effectiveness of intervention A and comparator B for treatment of advanced urothelial carcinoma. TITLE: Comparative effectiveness of intervention A and comparator B for treatment of advanced urothelial carcinoma. TITLE: Comparative effectiveness of intervention A and comparator B for treatment of advanced urothelial carcinoma. BACKGROUND: Intervention A is a standard treatment of metastatic urothelial carcinoma (UC), though comparator B is frequently substituted due to improved tolerability. Because comparative effectiveness in clinica outcomes of intervention A - versus comparator B chemotherapy is lacking, a meta-analysis was carried out. Abstract without limitations section Abstract with limitations section Sample size h Each participant read 1 abstract with or 1 abstract with- out a limitations section. The unit of analysis was the abstract. A sample of 266 participants was theoretically needed to be able to detect an effect size of 0.4 with the primary outcome (with power of 90% and alpha risk 5%). An effect size of 0.4 is equivalent to a decrease in the primary outcome of 1 point (the minimum expected difference between groups on a 0–10 scale) with an SD of 2.5. Theoretically, each abstract must be read the same number of times according to the randomization group. Knowing that each participant would read only one abstract (with or without a limitations section), we chose to include 300 participants. Each abstract with and without a limitations section was assessed 5 times. Characteristics of participants Among the 4,807 potential participants who were invited by e-mail to participate in the survey between May 1 and June 30, 2013, 394 logged onto the study site; 89 were excluded because they were not clinicians and 5 did not complete the survey. From the 300 participants, 150 were randomized to the intervention group (i.e., ab- stracts with a limitations section), and 150 to the control group (i.e., abstracts without a limitations section). In total, 150 participants per group were included in the final ana- lysis (Figure 1). The median [Q1-Q3] participant age was 45 [range 38–54] years; 72% were male. Participants were mainly located in the European Union (49%) and in the United States (33%). Most had medical experience (74.7% had been clinicians for more than 10 years) and more than half (59%) regularly read reports of sys- tematic reviews. More than half (53%) had been in- volved in a systematic review, but 46% had never peer- reviewed a systematic review for a biomedical journal (Table 2). Characteristics of systematic reviews, abstracts and limitations sections The general characteristics of the included systematic reviews and the quality of the reporting of the abstracts (according to the PRSIMA statement for abstracts [12]) are in Table 3. Overall, 23 abstracts were structured abstracts, and we created a specific heading for the limitations section, whereas 7 abstracts were not structured, and a sen- tence reporting the limitations was added before the conclusions. Some limitations were reported in 9 ori- ginal abstracts. These limitations were deleted and rewritten according to the PRISMA guidelines. The quality assessment of the risk of bias in the selected systematic reviews was assessed with different tools. Authors of the included systematic reviews used the Cochrane or a modified Cochrane Collaboration tool in 10 abstracts (33%), the Jadad scale or modified Jadad scale in 5 (17%), the PEDro scale or modified PEDro scale in 3 (10%), and other tools in 4 (13%); the scale was not specified in 6 (20%). Blinding Participants were blinded to the study’s hypothesis. They were informed that they were participating in a study on the interpretation of abstracts of systematic reviews, but they were not aware that they were randomized to assess an abstract with or without a limitations section. BACKGROUND: Survival end points could not be adequately assessed due to inconsistent reporting among trials. LIMITATIONS: This review is limited by the small sample sizes and methodological quality of the included studies. None of the included studies was blinded or placebo controlled; two studies closed early. LIMITATIONS: This review is limited by the small sample sizes and methodological quality of the included studies. None of the included studies was blinded or placebo controlled; two studies closed early. CONCLUSIONS: Intervention A, as compared with comparator B, significantly increases the likelihood of both OR and CR in patients with metastatic UC. The impact of improved response proportions on survival end points could not be assessed. Conclusions: Intervention A, as compared with comparator B, significantly increases the likelihood of both OR and CR in patients with metastatic UC. The impact of improved response proportions on survival end points could not be assessed. Conclusions: Intervention A, as compared with comparator B, significantly increases the likelihood of both OR and CR in patients with metastatic UC. The impact of improved response proportions on survival end points could not be assessed. Page 4 of 9 Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 with and without a limitations section. P <0.05 was considered statistically significant. Ethical considerations The institutional review board from the University of Paris Descartes approved the protocol No. CL178200001. The study is registered in ClinicalTrials.gov (no. NCT01848782). Results The primary outcome was readers’ confidence in the results of the systematic review as stated in the abstract (i.e., How confident are you in the results of the systematic review?) assessed on a Likert scale, from 0 “not at all confident” to 10 “very confident”. The secondary out- comes were the confidence in the validity of the con- clusions (i.e., How confident are you in the validity of the conclusion of this study?), the beneficial effect of the experimental intervention (i.e., How confident are you that intervention “A” could be of benefit to patients?), the influence of the results on clinical practice (i.e., How confident are you that the results of this study could influ- ence your clinical practice?) and the rigor of the systematic review (i.e., Do you think that this systematic review was conducted rigorously?) assessed on an 11-point Likert scale. Statistical analysis Statistical analysis involved use of SAS v9.3 (SAS Inc., Cary, NC). All outcomes were quantitative; differences between groups were analyzed by a linear mixed model with a fixed factor (group) and random abstracts and ab- stract × group interaction effects. A random-effects model allowed for taking into account 2 levels of clustering: by abstract (each abstract was assessed 5 times in each group) and interclustering (pairing between the abstracts used in the 2 arms of the trial). Inference was based on restricted maximum likelihood. For primary outcome and secondary outcomes, we estimated the difference between means (with 95% confidence intervals [95% CIs]) for abstracts The limitations sections we created focused on risk of bias in 22 abstracts (73%), heterogeneity in 13 (43%), Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Page 5 of 9 Figure 1 Flow chart of participants in the trial. Figure 1 Flow chart of participants in the trial. Discussion publication bias in 15 (50%), imprecision of results in 12 (40%), and indirectness of the evidence in 2 (7%) abstracts. The median [Q1-Q3] word count for limitations sections was 27 [23–31]. The median number of limits described in the limitations sections was 2 (range 1–4). This study evaluated, in a randomized controlled trial, the impact of adding a limitations section to an abstract on the interpretation of abstracts of systematic reviews. This randomized controlled trial involved a large inter- national sample of clinicians and a sample of “real life” abstracts of systematic reviews (i.e., abstracts of pub- lished systematic reviews indexed in DARE). Despite the selection of a sample of systematic reviews of good qual- ity, the mean confidence of readers was low, and adding a limitations section had no impact on the interpretation of abstract results by expert-readers. Clinicians’ interpretation of abstracts Readers’ assessment of abstracts with and without a lim- itations section did not differ in the primary outcome – confidence in the results (mean [SD] =4.4 [2.3] and 4.6 [2.5], respectively; mean difference [95% CI] 0.2 [−0.4 to 0.7], p = 0.5). For the secondary outcomes, the assess- ment of abstracts with and without a limitations section did not differ for confidence in the validity of the conclu- sions (mean difference 0.07 [−0.5 to 0.6], p = 0.8); benefit of the experimental intervention to patients (mean differ- ence 0.1 [−0.4 to 0.7], p = 0.6); influence of the results on clinical practice (mean difference −0.08 [−0.6 to 0.5], p = 0.8) and rigor of the systematic review (mean difference −0.4 [−1.0 to 0.2], p = 0.2) (Table 4). Because abstracts are the first and sometimes the only source of information for readers, editors are attentive to their quality and their capacity to provide all the ne- cessary and important information on the research per- formed. In the 1960s, abstracts were usually reported on the last page of research articles and were moved to the beginning of research articles [18]. Since then, many edi- torial policies have been implemented to try to improve the content and the format of abstracts. These policies Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Page 6 of 9 Table 2 Characteristics of participants Table 2 Characteristics of participants Abstract without limitations n (%) Abstract with limitations n (%) Total n (%) N = 150 N = 150 N = 300 Qualification MD 78 (52.0) 78 (52.0) 156 (52.0) PhD 58 (38.7) 64 (42.7) 122 (40.7) Other 14 (9.3) 8 (5.3) 22 (7.3) Clinical experience <5 years 6 (4.0) 8 (5.3) 14 (4.7) 5 to 10 years 34 (22.7) 28 (18.7) 62 (20.7) >10 years 110 (73.3) 114 (76.6) 224 (74.7) Reading reports of systematic reviews Rarely or sometimes 61 (40.7) 62 (41.6) 123 (41.1) Regularly 89 (59.3) 87 (58.4) 176 (58.9) No. of randomized trials involved in 0 16 (10.7) 11 (7.3) 27 (9.0) 1–3 48 (32.0) 48 (32.0) 96 (32.0) 4–9 45 (30) 54 (36.0) 99 (33.0) >10 41 (27.3) 37 (24.7) 78 (26.0) Authored at least one systematic review 77 (51.3) 82 (55.0) 159 (53.2) No. Table 2 Characteristics of participants to believe the study findings [25]. Similarly, interpret- ation of study results is affected by the reporting of out- comes as absolute risk, relative risk or number needed to treat [26,27]. have involved the development and implementation of structured abstracts [18,19], reporting guidelines such as the CONSORT for abstracts of randomized controlled trials and the PRISMA statement for abstracts of sys- tematic reviews [12,20]. Such policies can improve the quality of reporting of abstracts [21] and should in the- ory improve the interpretation by readers. Our results did not show any impact of the abstract limitations section on expert-readers’ interpretation. Fur- thermore, our results highlight that confidence in results was low in both arms. The high level of expertise of the participants could explain these results. In fact, half of the clinicians included in this randomized controlled trial had some experience in the conduct and reviewing of systematic reviews. This level of expertise could in- crease their awareness of the common limitations of sys- tematic reviews such as the risk of publication bias or the limited quality of primary studies. Furthermore, the limitations section reported factual information and in a neutral form, and the conclusion of the systematic re- views’ abstract was not modified. Also, assessing the confidence in the results of a systematic review is com- plex because it depends both on how the systematic re- view is conducted and the quality of the primary studies included. However, despite these initiatives, the quality of reporting of abstracts remains questionable [9,10,22-24]. A recent study showed that despite systematic reviews including pri- mary studies with high risk of bias, just over half included a risk of bias assessment in the interpretation of results in the abstract [13]. Consequently, adding a limitations sec- tion could be useful to enhance readers’ awareness and improve their interpretation. However, a limitations sec- tion in the abstract is recommended by only a few journals and for systematic reviews in the PRISMA statement for abstracts [12]. For example, Annals of Internal Medicine has required authors to include a limitations section in the abstract of scientific articles since 2004 [14]. To our knowledge, the impact of adding a limitations section in abstracts of systematic reviews has never been evaluated. Previous studies have evaluated the impact of different reporting on the interpretation of the study by readers. Clinicians’ interpretation of abstracts of systematic reviews peer-reviewed 0 75 (50.0) 64 (43.0) 139 (46.5) 1–3 55 (36.7) 69 (46.3) 124 (41.5) >3 20 (13.3) 16 (10.7) 36 (12.0) Training in clinical epidemiology 101 (67.3) 76 (50.7) 177 (59.0) Training in methods of randomized trials 86 (57.3) 71 (47.3) 157 (52.3) Table 2 Characteristics of participants These studies mainly involved use of a sin- gle abstract of a fictional trial. For example, industry sponsorship can negatively influence the perception of the methodological quality of a study and the willingness Our study has some limitations. First, the readers did not access the full-text article to fully appraise the study results; they only assessed an abstract with or without a limitations section. However abstracts of systematic re- views are very important, because some readers cannot access full-text articles because of the fee requirement, Page 7 of 9 Page 7 of 9 Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Table 3 Characteristics of systematic reviews included Characteristics N = 30 Field of research, n (%) Medicine 27 (90) Surgery 3 (10) Impact factor, median; [Q1-Q3]; (min-max) 4.60; [3.18–7.51]; (0.00-18.0 No. of trials included, median; [Q1-Q3]; (min-max) 13; [6–23]; (4–54) Sample size, median; [Q1-Q3]; (min-max) 2872; [801–11005]; (257–782 Experimental treatment, n (%) Drugs 16 (53) Complex intervention 11 (37) Surgery 2 (7) Device 1 (3) Meta-analysis included in the systematic review, n (%) 26 (87) Funding source, n (%) Non-profit 11 (37) For-profit 2 (7) None 10 (33) Not specified 7 ((23) Content of the original abstract, n (%) Eligibility criteria 17 (57%) Key data bases and date of search 9 (30%) Method to assess the risk of bias 6 (20%) No. and type of the studies included in the systematic review 22 (73%) Summary measure and confidence interval for the main outcome results* 20/26 (77%) Strength and limitation 13 (43%) *This result applies on the 26 systematic reviews that included a meta-analysis. low Internet download capacity, or the full-text article being available only in a language not understood by the reader. Second, the participants were corresponding au- thors of articles of randomized trials and systematic re- views, who may be considered “reader-experts” and not representative of all readers of systematic reviews. Con- sequently, we cannot exclude that a limitations section may be useful for a less expert readership. Finally, we ex- plored only the impact of a limitations section added to abstracts reporting a systematic review and we cannot extrapolate our findings to limitations sections in abstracts reporting other types of studies such as randomized con- trolled trials and observational studies. However, this study has important implications. 95% CI, 95% confidence interval. Conclusions 6. Saint S, Christakis DA, Saha S, Elmore JG, Welsh DE, Baker P, Koepsell TD: Journal reading habits of internists. J Gen Intern Med 2000, 15:881–884. 6. Saint S, Christakis DA, Saha S, Elmore JG, Welsh DE, Baker P, Koepsell TD: Journal reading habits of internists. J Gen Intern Med 2000, 15:881–884. In conclusion, adding a limitations section in abstracts of systematic review may not affect expert-readers’ inter- pretation of abstract results and conclusions. Future studies are needed to confirm these results and explore the impact of a limitations section on a less expert panel of participants. 7. Scherer RW, Langenberg P, von Elm E: Full publication of results initially presented in abstracts. Cochrane Database Syst Rev 2007, (2):MR000005. 8. The PLoS Medicine Editors: The impact of open access upon public health. PLoS Med 2006, 3:e252. doi:10.1371/journal.pmed.0030252. 9. Hopewell S, Clarke M, Askie L: Reporting of trials presented in conference abstracts needs to be improved J Clin Epidemiol 2006 59:681–684 7. Scherer RW, Langenberg P, von Elm E: Full publication of results initially presented in abstracts. Cochrane Database Syst Rev 2007, (2):MR000005. 8. The PLoS Medicine Editors: The impact of open access upon public health. PLoS Med 2006, 3:e252. doi:10.1371/journal.pmed.0030252. 7. Scherer RW, Langenberg P, von Elm E: Full publication of results initially presented in abstracts. Cochrane Database Syst Rev 2007, (2):MR000005. 8. The PLoS Medicine Editors: The impact of open access upon public health. PLoS Med 2006, 3:e252. doi:10.1371/journal.pmed.0030252. 9. Hopewell S, Clarke M, Askie L: Reporting of trials presented in conference abstracts needs to be improved. J Clin Epidemiol 2006, 59:681–684. 9. Hopewell S, Clarke M, Askie L: Reporting of trials presented in conference abstracts needs to be improved. J Clin Epidemiol 2006, 59:681–684. 9. Hopewell S, Clarke M, Askie L: Reporting of trials presented in conference abstracts needs to be improved. J Clin Epidemiol 2006, 59:681–684. 10. Beller EM, Glasziou PP, Hopewell S, Altman DG: Reporting of effect direction and size in abstracts of systematic reviews. JAMA 2011, 306:1981–1982. 10. Beller EM, Glasziou PP, Hopewell S, Altman DG: Reporting of effect direction and size in abstracts of systematic reviews. JAMA 2011, 306:1981–1982. References 1. Sackett DL, Rosenberg WM, Gray JA, Haynes RB, Richardson WS: Evidence based medicine: what it is and what it isn’t. BMJ 1996, 312:71–72. 2. Bastian H, Glasziou P, Chalmers I: Seventy-five trials and eleven systematic reviews a day: how will we ever keep up? PLoS Med 2010, 7:e1000326. 3. Cook DJ, Mulrow CD, Haynes RB: Systematic reviews: synthesis of best evidence for clinical decisions. Ann Intern Med 1997, 126:376–380. 4. Bero LA, Jadad AR: How consumers and policymakers can use systematic reviews for decision making. Ann Intern Med 1997, 127:37–42. 1. Sackett DL, Rosenberg WM, Gray JA, Haynes RB, Richardson WS: Evidence based medicine: what it is and what it isn’t. BMJ 1996, 312:71–72. 2. Bastian H, Glasziou P, Chalmers I: Seventy-five trials and eleven systematic reviews a day: how will we ever keep up? PLoS Med 2010, 7:e1000326. 3. Cook DJ, Mulrow CD, Haynes RB: Systematic reviews: synthesis of best evidence for clinical decisions. Ann Intern Med 1997, 126:376–380. 4. Bero LA, Jadad AR: How consumers and policymakers can use systematic reviews for decision making. Ann Intern Med 1997, 127:37–42. 5. Shea BJ, Grimshaw JM, Wells GA, Boers M, Andersson N, Hamel C, Porter AC, Tugwell P, Moher D, Bouter LM: Development of AMSTAR: a measurement tool to assess the methodological quality of systematic reviews. BMC Med Res Methodol 2007, 7:10. g 5. Shea BJ, Grimshaw JM, Wells GA, Boers M, Andersson N, Hamel C, Porter AC, Tugwell P, Moher D, Bouter LM: Development of AMSTAR: a measurement tool to assess the methodological quality of systematic reviews. BMC Med Res Methodol 2007, 7:10. 5. Shea BJ, Grimshaw JM, Wells GA, Boers M, Andersson N, Hamel C, Porter AC, Tugwell P, Moher D, Bouter LM: Development of AMSTAR: a measurement tool to assess the methodological quality of systematic reviews. BMC Med Res Methodol 2007, 7:10. Authors’ contributions Conceived, designed and experiments: AY, IB, SH, PR, Performed the experiments: AY, IB, Analyzed the data: GB, Wrote the first draft: AY IB, Contributed to the writing of the manuscript: AY, IB, SH, GB, PR. All authors read and approved the final manuscript. 13. Hopewell S, Boutron I, Altman DG, Ravaud P: Incorporation of assessments of risk of bias of primary studies in systematic reviews of randomised trials: a cross-sectional study. BMJ Open 2013, 3:e003342. 13. Hopewell S, Boutron I, Altman DG, Ravaud P: Incorporation of assessments of risk of bias of primary studies in systematic reviews of randomised trials: a cross-sectional study. BMJ Open 2013, 3:e003342. 14. The Editors: Addressing the limitations of structured abstracts. Ann Intern Med 2004, 140:480–481. 14. The Editors: Addressing the limitations of structured abstracts. Ann Intern Med 2004, 140:480–481. Competing interests The authors declare that they have no competing interests. 12. Beller EM, Glasziou PP, Altman DG, Hopewell S, Bastian H, Chalmers I, Gøtzsche PC, Lasserson T, Tovey D: PRISMA for abstracts: reporting systematic reviews in journal and conference abstracts. PLoS Med 2013, 10:e1001419. 12. Beller EM, Glasziou PP, Altman DG, Hopewell S, Bastian H, Chalmers I, Gøtzsche PC, Lasserson T, Tovey D: PRISMA for abstracts: reporting systematic reviews in journal and conference abstracts. PLoS Med 2013, 10:e1001419. Received: 29 July 2014 Accepted: 13 November 2014 Published: 24 November 2014 this topic, the trial should be replicated, and other trials including a less expert readership or with different back- ground (e.g. authors of “clinical practice guidelines”) should be performed. Second, qualitative studies would probably be useful to help define how limitations sections should be reported to have a real impact on readers. Third, we recommend exploring the impact of a limita- tions section in abstracts of other study designs such as randomized controlled trials and observational studies. Overall, more research is needed on the interpretation of research results from abstracts because abstracts are widely disseminated. Financial support 18. Soffer A: Abstracts of clinical investigations. A new and standardized format. Chest 1987, 92:389–390. AY was funded by a grant from the Fondation pour la Recherche Médicale. AY was funded by a grant from the Fondation pour la Recherche Médicale. 19. Huth EJ: Structured abstracts for papers reporting clinical trials. Ann Intern Med 1987, 106:626. Transparency declaration 20. Hopewell S, Clarke M, Moher D, Wager E, Middleton P, Altman DG, Schulz KF: CONSORT for reporting randomized controlled trials in journal and conference abstracts: explanation and elaboration. PLoS Med 2008, 5:e20. AY affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained. AY and IB had access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. y y AY and IB had access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. 21. Hopewell S, Ravaud P, Baron G, Boutron I: Effect of editors’ implementation of CONSORT guidelines on the reporting of abstracts in high impact medical journals: interrupted time series analysis. BMJ 2012, 344:e4178. Additional file Additional file 1: The 30 Abstracts with limitations section. The invitation e-mail for the participants. The survey. 11. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gøtzsche PC, Ioannidis JPA, Clarke M, Devereaux PJ, Kleijnen J, Moher D: The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration. BMJ 2009, 339:b2700. 11. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gøtzsche PC, Ioannidis JPA, Clarke M, Devereaux PJ, Kleijnen J, Moher D: The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration. BMJ 2009, 339:b2700. Table 2 Characteristics of participants At this stage, we cannot make any recommendations for practice and we should probably not change guidelines and editorial policies related to the reporting of a limi- tations section in abstracts of systematic reviews. How- ever, this study highlights an important topic for future research. First, because our study is the first study on Table 4 Results of primary and secondary outcomes Abstract without limitations With Limitations Mean difference (95% CI) p-value Mean (SD) n = 150 Mean (SD) n = 150 How confident are you in the results of this study (0–10)? 4.6 (2.5) 4.4 (2.3) 0.2 (−0.4 to 0.7) p =0.5 How confident are you in the validity of the conclusion of this study (0–10)? 4.1 (2.5) 4.0 (2.3) 0.07 (−0.5 to 0.6) p =0.8 How confident are you that the intervention A could be benefit to patients (0–10)? 4.4 (2.6) 4.3 (2.3) 0.1 (−0.4 to 0.7) p =0.6 How confident are you that the results of this study could influence your clinical practice (0–10)? 3.8 (2.6) 3.8 (2.3) −0.08 (−0.6 to 0.5) p =0.8 Do you think that this systematic review was conducted rigorously (0–10)? 4.1 (2.7) 4.4 (2.6) −0.4 (−1.0 to 0.2) p =0.2 95% CI 95% confidence interval Table 4 Results of primary and secondary outcomes Page 8 of 9 Page 8 of 9 Page 8 of 9 Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 Received: 29 July 2014 Accepted: 13 November 2014 Published: 24 November 2014 Authors’ information On-line survey design and data management: Isabelle Pane, senior computer engineer and data manager, and Joan Denis, junior computer engineer and data manager, Centre de recherche Épidémiologies et Biostatistiques, INSERM U1153, Paris, France. 15. Schulz KF, Altman DG, Moher D: CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. J Clin Epidemiol 2010, 63:834–840. 15. Schulz KF, Altman DG, Moher D: CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. J Clin Epidemiol 2010, 63:834–840. 16. Guyatt GH: GrADe: what is “quality of evidence” and why is it important to clinicians? BMJ 2008, 336:995–998. 16. Guyatt GH: GrADe: what is “quality of evidence” and why is it important to clinicians? BMJ 2008, 336:995–998. Acknowledgements We thank all clinicians who participated to the study. 17. Guyatt GH, Oxman AD, Vist GE, Kunz R, Falck-Ytter Y, Alonso-Coello P, Sch\ünemann HJ: GRADE: an emerging consensus on rating quality of evidence and strength of recommendations. BMJ 2008, 336:924. We thank all clinicians who participated to the study. Additional file 1: The 30 Abstracts with limitations section. The invitation e-mail for the participants. The survey. Author details 1 1Centre de Recherche Épidémiologies et Statistiques INSERM U1153, Paris, France. 2Centre of Clinical Epidemiology, Assistance Publique des Hôpitaux de Paris, Hôtel Dieu Hospital, Place du Parvis Notre-Dame, 75181 Paris, Cedex 4, France. 3Faculté de Médecine, French Cochrane Center, Paris Descartes University, Sorbonne Paris Cité, Paris, France. 4Department of Anesthesiology and Critical Care, Beaujon University Hospital, Clichy, France. 5Department of Epidemiology, Columbia University Mailman School of Public Health, New York, NY, USA. 6Centre for Statistics in Medicine, University of Oxford, Wolfson College, Oxford OX2 6UD, UK. 22. Ioannidis JP, Lau J: Completeness of safety reporting in randomized trials: an evaluation of 7 medical areas. JAMA 2001, 285:437–443. 23. Yank V, Rennie D, Bero LA: Financial ties and concordance between results and conclusions in meta-analyses: retrospective cohort study. BMJ 2007, 335:1202–1205. 24. Boutron I, Dutton S, Ravaud P, Altman DG: Reporting and interpretation of randomized controlled trials with statistically nonsignificant results for primary outcomes. JAMA 2010, 303:2058–2064. Page 9 of 9 Page 9 of 9 Yavchitz et al. BMC Medical Research Methodology 2014, 14:123 http://www.biomedcentral.com/1471-2288/14/123 25. Kesselheim AS, Robertson CT, Myers JA, Rose SL, Gillet V, Ross KM, Glynn RJ, Joffe S, Avorn J: A randomized study of How physicians interpret research funding disclosures. N Engl J Med 2012, 367:1119–1127. 26. Bucher HC, Weinbacher M, Gyr K: Influence of method of reporting study results on decision of physicians to prescribe drugs to lower cholesterol concentration. BMJ 1994, 309:761–764. 27. Forrow L, Taylor WC, Arnold RM: Absolutely relative: how research results are summarized can affect treatment decisions. 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https://openalex.org/W2534559424	https://link.springer.com/content/pdf/10.1007%2Fs10459-016-9720-7.pdf	English	null	Scoring method of a Situational Judgment Test: influence on internal consistency reliability, adverse impact and correlation with personality?	Advances in health sciences education	2,016	cc-by	11,317	Adv in Health Sci Educ (2017) 22:243–265 DOI 10.1007/s10459-016-9720-7 1 Institute of Medical Education Research Rotterdam (iMERR), Erasmus MC, Room AE-239, PO Box 2040, 3000 CA Rotterdam, The Netherlands 2 Medical School, University of Buckingham, Buckingham, UK 3 School of Medicine, University of Dundee, Dundee, UK 4 Department of Psychology, Erasmus University Rotterdam, Rotterdam, The Netherlands 5 Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands Background Selection into medical school has been dominated by cognitive-based measures which are predictive for academic performance, but are less predictive for clinical performance (Ferguson et al. 2002; Salvatori 2001). Adding non-cognitive-based measures to cognitive- based measures may improve the predictive quality of a selection procedure (Kulatunga- Moruzi and Norman 2002; Lucieer et al. 2015; Powis 2015). Non-cognitive-based selec- tion instruments with good validity and reliability are essential for this purpose, because selection into medical school is highly competitive, with the number of applicants greatly exceeding the number of available places. An upcoming non-cognitive-based measure for selection into medical school is the Situational Judgment Test (SJT). An SJT presents applicants with several situations that they may encounter during the job (or at medical school), followed by a number of possible responses to that situation. Respondents are instructed to judge the appropriateness of these responses by stating what they would or should do in the described situation (Motowidlo et al. 1990; Weekley and Ployhart 2013). Administering SJTs in work-related selection procedures has several beneﬁcial characteristics: (1) good predictive validity with regard to job performance (McDaniel et al. 2001), (2) incremental validity over and above cognitive ability and personality (Clevenger et al. 2001), (3) less adverse impact than cognitive measures (McDaniel and Nguyen 2001), (4) higher favorability ratings by candidates than in cognitive tests (Lievens 2013) and (5) more efﬁcient administration to large groups of applicants than other non-cognitive-based instruments (e.g., assessment centers) (Mo- towidlo et al. 1990). Previous studies on the use of SJTs for selection into medical school have shown that these beneﬁcial characteristics of SJTs also apply in a medical school context (Koczwara et al. 2012; Lievens 2013; Lievens et al. 2005; Lievens and Sackett 2012; Patterson et al. 2009, 2011, 2015). Despite the good qualities mentioned above, some aspects of SJTs require more research. One of these aspects is the scoring method (Whetzel and McDaniel 2009). Scoring an SJT is more complicated than scoring a traditional knowledge test because there are no objectively correct answers, since SJTs consist of dilemmas with no clear-cut solutions (Bergman et al. 2006). Different researchers and practitioners have used different methods to convert the judgments on an SJT to a score, which has led to a large variety of scoring methods. Scoring method of a Situational Judgment Test: inﬂuence on internal consistency reliability, adverse impact and correlation with personality? W. E. De Leng1 • K. M. Stegers-Jager1 • A. Husbands2 • J. S. Dowell3 • M. Ph. Born4 • A. P. N. Themmen1,5 Received: 12 February 2016 / Accepted: 6 October 2016 / Published online: 18 October 2016 The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Situational Judgment Tests (SJTs) are increasingly used for medical school selection. Scoring an SJT is more complicated than scoring a knowledge test, because there are no objectively correct answers. The scoring method of an SJT may inﬂuence the construct and concurrent validity and the adverse impact with respect to non-traditional students. Previous research has compared only a small number of scoring methods and has not studied the effect of scoring method on internal consistency reliability. This study compared 28 different scoring methods for a rating SJT on internal consistency reliability, adverse impact and correlation with personality. The scoring methods varied on four aspects: the way of controlling for systematic error, and the type of reference group, distance and central tendency statistic. All scoring methods were applied to a previously validated integrity-based SJT, administered to 931 medical school applicants. Internal consistency reliability varied between .33 and .73, which is likely explained by the dependence of coefﬁcient alpha on the total score variance. All scoring methods led to signiﬁcantly higher scores for the ethnic majority than for the non-Western minorities, with effect sizes ranging from 0.48 to 0.66. Eighteen scoring methods showed a signiﬁcant small positive correlation with agreeableness. Four scoring methods showed a signiﬁcant small positive correlation with conscientiousness. The way of controlling for systematic error was the most inﬂuential scoring method aspect. These results suggest that the increased use of SJTs for selection into medical school must be accompanied by a thorough examination of the scoring method to be used. 123 244 W. E. De Leng et al. Keywords Situational Judgment Test Scoring method Medical school selection Internal consistency reliability Adverse impact Integrity Big Five Keywords Situational Judgment Test Scoring method Medical school selection Internal consistency reliability Adverse impact Integrity Big Five Aspect 1: controlling for systematic error SJTs with a rational scoring key and a Likert scale response format can be scored using raw, standardized, and dichotomous consensus (McDaniel et al. 2011). Raw consensus computes the distance between the applicant’s rating and the mean rating of the reference group using the raw data. Standardized consensus calculates the distance after conducting a within-person z standardization such that each applicant has a mean of zero and a standard deviation of one across the SJT items. Dichotomous consensus divides the Likert scale in the middle. Points are awarded when an applicant’s position on the Likert scale is on the same side as the reference group. Some dichotomous scoring methods increase the scoring range by applying a negative correction by subtracting points when applicants are on the other side of the Likert scale. By standardizing or dichotomizing the data, McDaniel et al. (2011) attempted to control for systematic error. Systematic error in an SJT score may be caused by response ten- dencies or coaching in strategies on how to use the Likert scale, for example only opt for the extremes or only opt for the middle of the scale (McDaniel et al. 2011). Moreover, response tendencies are inﬂuenced by ethnic differences. For example, Black and Hispanic Americans are more inclined to use the extremes of a Likert scale than White Americans (Bachman and O’Malley 1984; Hui and Triandis 1989). By standardizing or dichotomizing the data, these cultural differences in the use of a Likert scale no longer inﬂuence the SJT score. Raw consensus does not control for systematic error. McDaniel et al. (2011) examined the effect of these three scoring methods on the concurrent validity in two studies, using scores on a biodata scale measuring quitting tendencies and supervisory ratings of job performance as criterion. Higher concurrent validity was found for the standardized consensus and dichotomous consensus scales than for the raw consensus scale, which they explained by the removal of systematic error from the SJT score. In addition, the standardized and dichotomous consensus scales resulted in substantially smaller differences between White and Black respondents than the raw consensus scale, which they attributed to the removal of ethnic differences in the use of a Likert scale. Similarly, Legree et al. (2010) found a higher concurrent validity for a standardized scale than a raw scale. Background This study will investigate the effect of these various scoring methods on three psychometric qualities (i.e., internal consistency reliability, adverse impact and correlation with personality). For this purpose, we used a previously validated integrity- based SJT (Husbands et al. 2015) for the selection of medical school applicants at a Dutch medical school. Choice of scoring method depends on the type of scoring key and response format of an SJT. This study will focus on scoring methods for SJTs that use a rational scoring key and a Likert scale response format. A rational scoring key uses the judgments of a reference 123 123 Scoring method of a Situational Judgment Test: inﬂuence on… 245 group of Subject Matter Experts (SMEs) to determine the ‘‘correct’’ answer. SMEs are individuals highly experienced in the relevant domain (Bergman et al. 2006). The Likert scale response format instructs the respondents to rate the appropriateness of each response option on a rating scale (Weekley et al. 2013). group of Subject Matter Experts (SMEs) to determine the ‘‘correct’’ answer. SMEs are individuals highly experienced in the relevant domain (Bergman et al. 2006). The Likert scale response format instructs the respondents to rate the appropriateness of each response option on a rating scale (Weekley et al. 2013). Scoring methods The scoring methods in this study differ on four aspects: the way of controlling for systematic error, the type of reference group, the type of distance and the type of central tendency statistic. Aspect 2: reference group A second aspect on which scoring methods may differ is the reference group. As stated above, a rational scoring key uses the judgments of a group of SMEs to determine the ‘‘correct’’ answer on an SJT. Most SJT scoring methods use SMEs because it is expected that they have knowledge about what behavior is effective and ineffective in their ﬁeld (Motowidlo and Beier 2010). However, a number of SJT studies have used the group of respondents itself as a reference, a procedure called Consensus Based Measurement (CBM). Legree et al. (2005) argued that this procedure may be more appropriate for constructs for which no clear SMEs can be identiﬁed. A study on an SJT used for the US Airforce found that the mean ratings of the SMEs strongly correlated with the mean ratings of the group of respondents (Legree 1995; Legree and Grafton 1995). Similar results were found for an SJT measuring Tacit Knowledge of Military Leadership comparing lieu- tenants (i.e., SMEs) with cadets (Hedlund et al. 2003). Comparison of two SJT scoring keys based on either novices’ or experts’ mean effectiveness ratings found a correlation of .75 between the two keys (Motowidlo and Beier 2010). In addition, both scoring keys resulted in scores that had similar criterion-related validity coefﬁcients. These results were explained by novices’ possession of a different, more general type of knowledge outside the speciﬁc job context. Furthermore, Lineberry et al. (2014) stated that for script con- cordance tests used for assessing clinical reasoning skills, having experience does not indicate that someone is an infallible expert and that residents (i.e., novices) can outper- form most panelists (i.e., SMEs). We are not aware of any previous research on the effect of using a less experienced reference group in a medical selection context. Aspect 1: controlling for systematic error Next to using raw, standardized and dichotomous consensus, a score on an SJT with a rational scoring key and Likert scale response format can also be calculated using percent agreement (Legree et al. 2005). Percent agreement uses the endorsement ratios among the SMEs to determine the score corresponding to each rating. Percent agreement, like raw consensus, does not control for systematic error. An example of a scoring method using percent agreement assigns two points to the Likert scale point endorsed by 50 % or more of the SMEs and one point to the scale point 12 123 246 W. E. De Leng et al. endorsed by 25–50 % of the SMEs (Chan and Schmitt 1997). Another example assigns a score to each Likert scale point depending on the proportion of the reference group that endorsed that rating point (Lievens et al. 2015). endorsed by 25–50 % of the SMEs (Chan and Schmitt 1997). Another example assigns a score to each Likert scale point depending on the proportion of the reference group that endorsed that rating point (Lievens et al. 2015). Aspect 3: distance A third aspect on which scoring methods may differ is the type of distance that is cal- culated between an applicant’s rating and the overall rating of the reference group (SMEs or respondents). Some SJT studies have used the squared distance (McDaniel et al. 2011), whereas others have used the absolute distance (Legree 1995). Squaring the distance gives more weight to ratings that deviate more from the reference group (Legree et al. 2005). Present study The ﬁrst goal of this study was to investigate the effect of scoring method on the internal consistency reliability of an SJT score. The appropriateness of internal consistency as a reliability estimate for SJT scores is often called into question (Catano et al. 2012). Internal consistency reliability estimates, such as coefﬁcient alpha, are based on the assumption that all items measure the same latent trait on the same scale, i.e., that the same latent trait equally contributes to all item scores (Yang and Green 2011). The multidimensional nature of SJTs violates this strict assumption resulting in an inaccurate estimate of reliability (Graham 2006). However, the integrity-based SJT used in this study was designed to measure one dimension, which might lead to a less serious violation of the assumption of unidimensionality. This is supported by a meta-analysis of Campion et al. (2014) that reported a mean alpha of .57 across 129 coefﬁcients (range 0–.92). In addition, it was shown that coefﬁcient alpha was signiﬁcantly higher for SJTs that had a larger focus on one dimension. The focus of the current integrity-based SJT on one dimension may support the use of internal consistency reliability. So, given the anticipated unidimensionality of the SJT used in this study and because coefﬁcient alpha is still commonly reported in the SJT literature, we chose it as a measure of comparison between scoring methods. To the best of our knowledge, this will be the ﬁrst study to investigate the effect of different scoring methods on the internal consistency reliability. The second goal of this study was to examine the effect of scoring method on adverse impact, by analyzing the differences between Dutch and non-Western minority applicants. Adverse impact will be examined because SJTs may play an important role in promoting fairness in medical school selection, since SJT scores potentially demonstrate lower ethnic subgroup differences than cognitive ability test scores. On cognitive ability tests, White test takers have been shown to score approximately one standard deviation higher than non-White test takers (De Soete et al. 2013). A meta-analysis on ethnic subgroup differ- ences across 32 SJTs—mainly originating from the US—showed that White test takers score approximately 0.38 standard deviation higher than Black test takers, 0.24 standard deviation higher than Hispanic test takers and 0.29 standard deviation higher than Asian test takers (Whetzel et al. 2008). Aspect 4: central tendency statistic A fourth aspect on which SJT scoring methods may differ is the manner of how the judgments of the reference group are summarized (i.e., central tendency statistic). Most SJT scoring methods have used the mean as a central tendency statistic, whereas some studies have used the mode (De Meijer et al. 2010; Lievens et al. 2015). Scoring methods using the mode assign points to the Likert scale point that most of the people in the reference group endorse. Besides the mean and mode, another widely used central ten- dency statistic is the median, which reﬂects the number at the central point when the data are ranked in numerical order (McCluskey and Lalkhen 2007). To our knowledge, the median has so far never been used for scoring SJTs. For the sake of completeness, this study will include all three central tendency statistics. 12 Scoring method of a Situational Judgment Test: inﬂuence on… 247 Procedure The SJT was administered during the selection procedure for the Erasmus MC Medical School in 2014 and 2015 (N = 1025). The administration was solely for research purposes and participation was voluntarily. The Erasmus MC Medical School selects students on their participation in extracurricular activities, their performance on ﬁve cognitive tests during three on-site testing days (Urlings-Strop et al. 2009) and their pre-university Grade Point Average (GPA). The administration of the SJT was conducted during the on-site testing days, using paper-and-pencil. An additional questionnaire was administered regarding applicants’ demographic characteristics. A personality questionnaire was administered online when applicants registered for the selection procedure. The applicants were informed that the SJT and questionnaires were administered solely for research purposes and that their answers would not inﬂuence the outcome of the selection proce- dure. Participation was voluntarily. Present study 2010; McDaniel et al. 2011). We are unaware of any previous studies that have investigated the effect of type reference group, distance and central tendency statistic on the correlation of an SJT score with personality. (i.e., organized and persistent) (Costa and MacCrae 1992). The correlation with the Big Five was examined because three of the ﬁve dimensions (i.e., conscientiousness, emotional sta- bility and agreeableness) have been shown to moderately and positively correlate with SJT scores (McDaniel et al. 2007) and integrity test scores (Marcus et al. 2007). Moreover, the validity and reliability of thescores on the Big Five measure used inthis study [i.e.,NEO-PI-R (Costa and MacCrae 1992)] has repeatedly been demonstrated (Costa and McCrae 2008), including in samples of adolescents (De Fruyt et al. 2000). It is therefore expected that the integrity-based SJT will becorrelated to these three BigFive dimensionsand that theresulting correlation coefﬁcients will provide a good measure of comparison between the scoring methods. We hypothesize that scoring methods that control for systematic error will lead to higher correlation coefﬁcients, because the inﬂuence of response tendencies regarding the use of Likert scales is removed from the SJT score (Legree et al. 2010; McDaniel et al. 2011). We are unaware of any previous studies that have investigated the effect of type reference group, distance and central tendency statistic on the correlation of an SJT score with personality. Measures Integrity-based Situational Judgment Test Present study A Dutch study also found that the ethnic subgroup difference in an integrity SJT score (d = 0.38) was lower than in a cognitive ability test score (d = 0.48) (De Meijer et al. 2010). Selection on only cognitive ability test scores might lead to the rejection of more ethnic minority applicants than ethnic majority applicants, whereas selection on SJT scores may increase the admission rate among ethnic minorities, resulting in a more culturally diverse medical student population. To promote the expected positive inﬂuence of an SJT on fairness, it is crucial to investigate the potential inﬂuence of scoring method on adverse impact. In line with the ﬁndings of McDaniel et al. (2011), we expect that scoring methods controlling for systematic error (i.e., standardized and dichotomous consensus) will lead to smaller ethnic differences than scoring methods that do not (i.e., raw consensus and percent agreement). The other scoring method aspects (i.e., type of reference group, distance and central tendency statistic) have not been studied in combination with adverse impact before. The third goal of this study was to investigate the effect of scoring method on the corre- lation between the SJT score and three of the Big Five personality traits. The Big Five describes someone’s personality using ﬁve broad dimensions: neuroticism (i.e., emotional instability), extraversion (i.e., outgoing and energetic), openness to experience (i.e., intel- lectual curiosity), agreeableness (i.e., altruistic and compassionate) and conscientiousness 123 123 248 W. E. De Leng et al. (i.e., organized and persistent) (Costa and MacCrae 1992). The correlation with the Big Five was examined because three of the ﬁve dimensions (i.e., conscientiousness, emotional sta- bility and agreeableness) have been shown to moderately and positively correlate with SJT scores (McDaniel et al. 2007) and integrity test scores (Marcus et al. 2007). Moreover, the validity and reliability of thescores on the Big Five measure used inthis study [i.e.,NEO-PI-R (Costa and MacCrae 1992)] has repeatedly been demonstrated (Costa and McCrae 2008), including in samples of adolescents (De Fruyt et al. 2000). It is therefore expected that the integrity-based SJT will becorrelated to these three BigFive dimensionsand that theresulting correlation coefﬁcients will provide a good measure of comparison between the scoring methods. We hypothesize that scoring methods that control for systematic error will lead to higher correlation coefﬁcients, because the inﬂuence of response tendencies regarding the use of Likert scales is removed from the SJT score (Legree et al. 123 Personality questionnaire In 2014, the Dutch version of the NEO-PI-R was administered to assess the applicants’ standing on the Big Five personality traits (Costa and MacCrae 1992; Hoekstra et al. 1996). The questionnaire consisted of 240 statements that applicants had to judge on a ﬁve-point Likert scale (1 Strongly disagree–5 Strongly agree). The ﬁve personality subscales demonstrated good internal consistency reliabilities (coefﬁcient alpha): .92 for neuroti- cism, .87 for extraversion, .85 for openness, .87 for agreeableness and .88 for conscien- tiousness. Due to the length of the questionnaire, the NEO-PI-R was not administered in 2015. Demographics An applicant was considered a non-Western minority when one of his/her parents was born outside Europe or North-America (Statistics Netherlands; www.cbs.nl). An applicant was considered a non-Western minority when one of his/her parents was born outside Europe or North-America (Statistics Netherlands; www.cbs.nl). The socio-economic status of an applicant was determined by the level of education of his/her parents. A division was made between ﬁrst-generation and non-ﬁrst-generation university students. First-generation university students were deﬁned as students whose parents did not attend university (either a research university or a university of applied science). Integrity-based Situational Judgment Test The integrity-based SJT used in this study was developed in the United Kingdom (UK) (Husbands et al. 2015). The authors translated this SJT to Dutch. This translation was validated using the back translation procedure described by Brislin (1970). The back translation was conducted by an independent commercial translation ofﬁce. The authors discussed and made appropriate changes to the translated version. The SJT consisted of ten scenarios describing problematic situations that could occur during medical school. Each scenario was followed by ﬁve response options. The respondents had to judge the appropriateness of each response option on a four-point Likert scale (1 Very inappropriate–4 Very appropriate) in terms of what should be done given the situation [i.e., knowledge-based instructions (Ployhart and Ehrhart 2003)]. An example of an SJT item is presented in Appendix 1. 123 123 249 Scoring method of a Situational Judgment Test: inﬂuence on… A rational scoring key for this SJT was developed based on the judgments of 16 SMEs (75 % female). The mean age of this group was 40.8 years (SD = 11.1). The SMEs were individuals involved in teaching professionalism in the medical curriculum. Two of the SMEs were medical doctors. The mean number of years of experience with profession- alism in the medical curriculum of this group was 6.4 (SD = 5.9). All SMEs were native Dutch. The intraclass correlation coefﬁcient (ICC) among the SMEs was .65, indicating a moderate agreement (two-way mixed model, absolute agreement). Scoring methods In preparation for this study we combined the four aspects on which scoring methods can differ; this yielded 28 scoring methods to be tested (Fig. 1). These scoring methods fol- lowed the categorization into raw, standardized and dichotomous consensus scoring methods as proposed by McDaniel et al. (2011). Within each of the raw and standardized scoring methods, the distance (absolute or squared) was calculated between the applicant’s rating and the overall rating of the ref- erence group on the Likert scale. The reference group was either made up of the 16 SMEs or of the group of respondents itself. The overall rating of this reference group was reﬂected by either the mean, median or mode. In addition to the raw and standardized consensus scoring methods, the dichotomous consensus scoring method was applied. The reference group consisted of either the SMEs or the group of respondents itself. Another variation was applied by either assigning zero points to or subtracting one point from applicants whose rating was located on the opposite side of the Likert scale than the reference group. The 24 scoring methods based on either raw, standardized or dichotomous consensus were complemented with four scoring methods based on percent agreement (Legree et al. 2005). These scoring methods used either the 25–50 % endorsement rule used by Chan and 123 123 250 W. E. De Leng et al. Fig. 1 Schematic representation of the 28 scoring methods. SMEs Subject Matter Experts Fig. 1 Schematic representation of the 28 scoring methods. SMEs Subject Matter Experts Schmitt (1997) or assigned a score to each Likert scale point corresponding to the pro- portion of subjects in the reference group who endorsed that point (Lievens et al. 2015). The reference group consisted of either the SMEs or the respondents. The correlations between the 28 scoring methods are presented in Appendix 2. Although some correlation coefﬁcients indicated a large overlap between the scoring methods (i.e., within the raw consensus scoring method set), other scoring methods showed less overlap (i.e., between the raw and dichotomous scoring method sets). Scoring methods To our knowledge, of half of these scoring methods no results have been published in the context of application to an SJT (i.e., scoring methods using the median, scoring methods calculating the distance from the group mode, dichotomous scoring methods using the SMEs, percent agreement scoring methods using the endorsement rate of the group and the proportions of the SMEs). 123 Participants Nine-hundred thirty-one medical school applicants responded (response rate = 90.8 %). The demographic characteristics of this sample are depicted in Table 1. The two cohorts (2014 and 2015) were similar with regard to gender, age and ethnicity. Cohort 2015 consisted of signiﬁcantly more ﬁrst-generation students than cohort 2014, but the size of this effect was small [X2(1) = 6.02, p = .014, u = .08]. Personality data were obtained from 73.3 % of the participants from cohort 2014. SJT scores did not signiﬁcantly differ between respondents and non-respondents to the personality questionnaire. Statistical analysis Both SPSS (IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.) and R (Version 3.1.0) were used to convert the judgments on the SJT to a score, using the different scoring methods. The raw and standardized consensus scoring methods that used the group of respondents itself as a reference were conducted using a leave-one-out method (Hastie et al. 2009). This method removes the applicant whose score needs to be calculated from the dataset, and calculates the summary statistic across the remaining group members. The distance between the applicant and the remaining group members composes the applicant’s score. Coefﬁcient alpha was used as an estimate of internal consistency reliability (Cronbach 1951). Independent t-tests were used to examine the 28 different SJT scores on disparities between ﬁrst-generation and non-ﬁrst-generation university applicants and between Dutch and non-Western minority applicants. The effect sizes of the social and ethnic disparities were reﬂected by Cohen’s d (Cohen 1988). A stricter alpha level (a = .001) was used because of the large number of comparisons. 123 123 251 Scoring method of a Situational Judgment Test: inﬂuence on… For each scoring method, Pearson correlations were used to determine the correlation between the SJT score and the three Big Five personality traits for which we expected a correlation. General linear models were used to examine which scoring method aspects signiﬁcantly inﬂuenced the outcome measures (i.e., coefﬁcient alpha, effect size and correlation coef- ﬁcient). For each outcome measure, four general linear models were tested, namely one model for each scoring method aspect. The four aspects were tested in separate models because the small number of data points (i.e., 28) did not allow entering all four aspects in one model. The effect sizes were corrected for the reliability of the scoring method by dividing Cohen’s d by coefﬁcient alpha, since low reliability may obscure subgroup dif- ferences (Lievens et al. 2008). Internal consistency reliability Coefﬁcient alpha varied from .33 to .73 depending on the scoring method (Table 2). The lowest coefﬁcient alpha was found for the scoring method that calculated the absolute distance from the mean of the group of respondents itself using standardized consensus. The highest coefﬁcient alpha was found for the scoring method that calculated the absolute distance from the mean of the group of respondents itself using raw consensus. For the general linear models with coefﬁcient alpha as dependent variable, the way of controlling for systematic error was the only signiﬁcant factor with a very large effect size, F(3, 24) = 40.05, p \ .001, g2 = .83. Raw consensus led to a signiﬁcantly higher coef- ﬁcient alpha than the other three methods of controlling for systematic error. In addition, Table 1 Demographic characteristics of the participants in this study for each cohort 2014 (N = 521) 2015 (N = 410) Gender (% female) 64.1 62.7 Age [mean (SD)] 19.1 (1.9) 19.2 (1.9) Ethnicity % Dutch 58.3 57.2 % non-Western minority 31.3 32.2 % Western minority 10.4 10.6 SES (% ﬁrst-generation university students) 24.0 31.6 SD standard deviation, SES socio-economic status Table 1 Demographic characteristics of the participants in this study for each cohort 252 W. E. De Leng et al. Table 2 Descriptive statistics and internal consistency reliability (alpha coefﬁcient) for the 28 rate-SJT scoring methods Scoring method M (SD) Min.–Max. Alpha Raw consensus 1. Absolute distance—SME mean 34.32 (6.02) 20.01–64.99 .67 2. Absolute distance—SME median 33.11 (6.61) 13.50–66.50 .56 3. Absolute distance—SME mode 32.95 (6.52) 14.50–65.50 .55 4. Squared distance—SME mean 36.25 (12.50) 11.48–107.72 .67 5. Squared distance—SME median 42.44 (13.27) 12.75–122.75 .61 6. Squared distance—SME mode 41.81 (13.18) 13.25–121.25 .60 7. Absolute distance—Group mean 31.26 (6.31) 16.32–63.09 .73 8. Absolute distance—Group median 28.93 (7.00) 11–63 .61 9. Absolute distance—Group mode 29.07 (6.99) 11–63 .59 10. Squared distance—Group mean 30.35 (11.56) 8.47–100.35 .73 11. Squared distance—Group median 35.67 (12.85) 11–113 .65 12. Squared distance—Group mode 36.28 (13.01) 11–115 .63 Standardized consensus 13. Absolute distance—SME mean 32.86 (4.63) 21.24–51.67 .44 14. Absolute distance—SME median 33.52 (4.68) 19.09–51.54 .41 15. Squared distance—SME mean 34.46 (9.57) 14.31–34.46 .49 16. Squared distance—SME median 36.29 (9.61) 13.47–79.99 .45 17. Absolute distance—Group mean 30.42 (3.91) 20.99–50.67 .33 18. Absolute distance—Group median 29.91 (4.57) 18.27–51.00 .43 19. Squared distance—Group mean 29.11 (7.77) 13.58–74.24 .45 20. Squared distance—Group median 30.08 (8.89) 12.63–79.44 .51 Dichotomous consensus 21. Internal consistency reliability SME as reference 34.34 (3.55) 21–44 .34 22. SME as reference—negative correction 18.78 (7.04) -8–38 .34 23. Group as reference 37.56 (3.59) 22–47 .34 24. Group as reference—negative correction 25.21 (7.11) -6–44 .34 Percent agreement 25. Endorsement rate—SME 54.23 (7.32) 29–74 .49 26. Endorsement rate—Group 47.53 (5.17) 26–60 .46 27. Proportions—SME 19.39 (2.16) 11.18–25.59 .54 28. Proportions—Group 18.84 (1.55) 11.34–22.63 .58 M mean, SD standard deviation, SME Subject Matter Expert, Min. minimum, Max. maximum standardized consensus and percent agreement yielded a signiﬁcantly higher coefﬁcient alpha than dichotomous consensus. standardized consensus and percent agreement yielded a signiﬁcantly higher coefﬁcient alpha than dichotomous consensus. Adverse impact All scoring methods led to signiﬁcantly higher scores for the Dutch majority than for the non-Western minorities (Table 3). The effect sizes (d) of these differences ranged from 0.48 to 0.66 (medium effect). The largest differences were found for the scoring methods 123 Scoring method of a Situational Judgment Test: inﬂuence on… 253 Table 3 Results of the independent t tests for Dutch versus non-Western differences in SJT scores gen- erated by the 28 different scoring methods Scoringmethod Dutch (N = 490) Non-Western (N = 269) d Raw consensus 1. Absolute distance—SME mean 32.88 (5.38) 36.51 (6.50) 0.61 2. Absolute distance—SME median 31.47 (5.92) 35.58 (7.05) 0.63 3. Absolute distance—SME mode 31.34 (5.82) 35.37 (6.95) 0.63 4. Squared distance—SME mean 33.28 (10.86) 40.82 (13.98) 0.60 5. Squared distance—SME median 39.24 (11.50) 47.39 (14.84) 0.61 6. Squared distance—SME mode 38.70 (11.37) 46.67 (14.74) 0.61 7. Absolute distance—Group mean 29.95 (5.61) 33.16 (7.03) 0.50 8. Absolute distance—Group median 27.29 (6.27) 31.31 (7.49) 0.58 9. Absolute distance—Group mode 27.37 (6.21) 31.51 (7.48) 0.60 10. Squared distance—Group mean 27.94 (9.88) 33.96 (13.37) 0.51 11. Squared distance—Group median 32.66 (11.06) 40.02 (14.35) 0.57 12. Squared distance—Group mode 33.13 (11.10) 40.86 (14.59) 0.60 Standardized consensus 13. Absolute distance—SME mean 31.69 (4.23) 34.52 (4.43) 0.65 14. Absolute distance—SME median 32.30 (4.25) 35.22 (4.60) 0.66 15. Squared distance—SME mean 32.07 (8.52) 37.80 (9.36) 0.64 16. Squared distance—SME median 33.88 (8.51) 39.69 (9.56) 0.64 17. Absolute distance—Group mean 29.53 (3.63) 31.55 (3.72) 0.55 18. Absolute distance—Group median 28.83 (4.25) 31.30 (4.34) 0.58 19. Squared distance—Group mean 27.47 (7.14) 31.13 (7.40) 0.50 20. Squared distance—Group median 28.10 (8.11) 32.52 (8.58) 0.53 Dichotomous consensus 21. SME as reference 35.07 (3.32) 33.43 (3.46) 0.48 22. SME as reference—negative correction 20.22 (6.59) 16.98 (6.86) 0.48 23. Group as reference 38.31 (3.37) 36.69 (3.44) 0.48 24. Group as reference—negative correction 26.70 (6.66) 23.49 (6.79) 0.48 Percent agreement 25. Endorsement rate—SME 56.04 (6.76) 51.72 (7.35) 0.61 26. Endorsement rate—Group 48.74 (4.71) 45.78 (5.11) 0.60 27. Proportions—SME 19.93 (1.99) 18.66 (2.16) 0.61 28. Proportions—Group 19.20 (1.37) 18.32 (1.63) 0.58 All differences were signiﬁcant (p \ .001) SME Subject Matter Expert, d Cohen’s d (effect size) that calculated the absolute distance from the SME median using standardized consensus. The smallest ethnic difference was observed for all scoring methods that used dichotomous consensus. that calculated the absolute distance from the SME median using standardized consensus. Table 4 Pearson correlation coefﬁcients between the SJT score and the three Big Five personality dimensions for which we expect a correlation with the integrity-based SJT assessed by the NEO-PI-R in cohort 2014 only (N = 382) Adverse impact The smallest ethnic difference was observed for all scoring methods that used dichotomous consensus. For the general linear models with the corrected effect size as dependent variable, the way of controlling for systematic error was again the only signiﬁcant factor with a very large effect size, F(3,24) = 15.54, p \ .001, g2 = .66. Raw consensus led to smaller corrected effect sizes than standardized and dichotomous consensus, but not percent agreement. 123 254 W. E. De Leng et al. None of the scoring methods led to signiﬁcant differences between ﬁrst-generation university applicants and non-ﬁrst-generation university applicants (data available upon request). Due to the lack of signiﬁcant differences, no general linear models were tested. Correlation with personality Eighteen scoring methods resulted in an SJT score that had a signiﬁcant but small positive correlation with agreeableness (Table 4). The largest correlation coefﬁcients were found for scoring methods calculating the distance from the SME mean using standardized consensus. In addition, four scoring methods resulted in an SJT score that had a signiﬁcant but small positive correlation with conscientiousness. The largest correlation coefﬁcients Scoringmethod N A C Raw consensus 1. Absolute distance—SME mean -.03 -.11 -.04 2. Absolute distance—SME median .01 -.11 -.07 3. Absolute distance—SME mode 0 -.11 -.06 4. Squared distance—SME mean -.03 -.12 -.04 5. Squared distance—SME median -.01 -.12 -.06 6. Squared distance—SME mode -.01 -.12 -.05 7. Absolute distance—Group mean -.06 -.07 .02 8. Absolute distance—Group median -.06 -.08 0 9. Absolute distance—Group mode -.03 -.08 0 10. Squared distance—Group mean -.06 -.09 0 11. Squared distance—Group median -.06 -.11 0 12. Squared distance—Group mode -.05 -.11 -.01 Standardized consensus 13. Absolute distance—SME mean 0 -.15 -.12 14. Absolute distance—SME median -.01 -.12 -.12 15. Squared distance—SME mean 0 -.15 -.10 16. Squared distance—SME median 0 -.13 -.11 17. Absolute distance—Group mean .01 -.10 -.07 18. Absolute distance—Group median 0 -.11 -.06 19. Squared distance—Group mean .02 -.10 -.06 20. Squared distance—Group median .01 -.11 -.06 Dichotomous consensus 21. SME as reference -.07 .07 .10 22. SME as reference—negative correction -.07 .07 .10 23. Group as reference .02 .14 .05 24. Group as reference—negative correction .02 .14 .05 Percent agreement 25. Endorsement rate—SME 0 .10 .05 26. Endorsement rate—Group .04 .06 .01 27. Proportions—SME .03 .11 .05 28. Proportions—Group .04 .08 .01 Bold coefﬁcients reﬂect a signiﬁcant relationship. For the scoring methods using distance metrics (number 1 to 20), a negative correlation coefﬁcient reﬂects a positive relationship and vice versa N neuroticism, A agreeableness, C conscientiousness, SME Subject Matter Expert 12 3 Scoring method of a Situational Judgment Test: inﬂuence on… 255 were found for scoring methods calculating the absolute distance from the SME mean and median both using standardized consensus. Due to the low effect sizes and the small range of signiﬁcant correlation coefﬁcients, no general linear models were tested. were found for scoring methods calculating the absolute distance from the SME mean and median both using standardized consensus. Due to the low effect sizes and the small range of signiﬁcant correlation coefﬁcients, no general linear models were tested. Discussion This study shows that the psychometric quality of an SJT greatly depends on the choice of scoring method, speciﬁcally in the way the scoring method controls for systematic error. Firstly, the way of controlling for systematic error strongly affects the internal consistency reliability of an SJT score, with higher reliability estimates for scoring methods that use raw consensus. Secondly, the way of controlling for systematic error inﬂuences the adverse impact of the SJT score, with a lower adverse impact for scoring methods that use raw consensus compared to dichotomous and standardized consensus. Lastly, the different scoring methods had a minor inﬂuence on the correlation with agreeableness and con- scientiousness, but the practical signiﬁcance of these correlations was negligible. Internal consistency reliability Our ﬁrst ﬁnding was that the way a scoring method controls for systematic error strongly inﬂuences the internal consistency reliability. This strengthens the concerns about the use of coefﬁcient alpha as a reliability estimate for an SJT score. Changing only the scoring method could alter the acceptability of the resulting reliability estimate from poor to sufﬁcient, even for an SJT that was speciﬁcally constructed to measure one dimension. This large variety in internal consistency reliability is likely explained by the dependence of coefﬁcient alpha on the total score variance (Streiner 2003). Standardized and dichotomous consensus and percent agreement were associated with a reduction in total score variance, which is demonstrated by the lower standard deviations in Table 2. This reduction in total score variance will most likely lead to a lower coefﬁcient alpha. This line of reasoning implies that coefﬁcients alpha reported in previous studies on SJTs may be strongly inﬂuenced by irrelevant aspects, such as the total score variance generated by the scoring method used. Assuming that most studies on SJTs arbitrarily choose one scoring method rather than another, choice of scoring method contributes to the limited usefulness of coefﬁcient alpha as a reliability estimate for SJTs. Future studies should investigate whether the large variation in coefﬁcient alpha caused by different scoring methods also occurs in other reliability estimates (e.g., alternate forms reliability) to ﬁnd out whether this large variation is an artifact of coefﬁcient alpha only. A more accurate reliability estimate might be obtained by a combination of a more thoroughly construct-based SJT development (Christian et al. 2010) and a reliability estimate that takes into account the imposed factor structure of the SJT, for example a structural equation modeling (SEM) reliability estimate (Yang and Green 2011) or strat- iﬁed alpha (Catano et al. 2012). Future research is required on the application of construct- based development methods and alternative internal consistency estimates for SJTs. Adverse impact Although all scoring methods led to signiﬁcant ethnic differences in SJT score, the way a scoring method controlled for systematic error inﬂuenced the size of these effects. 123 12 256 W. E. De Leng et al. Speciﬁcally, the effect size decreased when using raw consensus instead of standardized or dichotomous consensus. This result is not in line with the ﬁndings of McDaniel et al. (2011) who found lower ethnic subgroup differences for scoring methods that controlled for systematic error (i.e., standardized and dichotomous consensus), which they explained by the removal of ethnicity related response tendencies in the use of Likert scales. However, the uncorrected effect sizes do show some support for this line of reasoning with the lowest effect sizes reported for the scoring methods using dichotomous consensus. The absence of lower effect sizes for standardized consensus might be caused by the low number of scale points (i.e., four) on the Likert scale that was used. Narrow Likert scales may not be as strongly affected by response tendencies as Likert scales with more scale points (Flaskerud 1988), resulting in no differences when controlling for the response tendencies. A study on script concordance tests recommended a reduction of the Likert scale from ﬁve to three points in order to decrease the inﬂuence of construct-irrelevant factors such as examinee response styles (Lineberry et al. 2013). Dichotomizing the Likert scale does seem to have some effect on adverse impact, but at the cost of low internal consistency reliability, leading to a similar issue as the diversity–validity dilemma (De Soete et al. 2013). Another noteworthy ﬁnding is that adverse impact was similar for both reference groups (SMEs and respondents). Previous studies which compared different reference groups found similar validity coefﬁcients for the scores of both groups (Legree et al. 2005; Motowidlo and Beier 2010), but did not study the effect of the reference group on adverse impact. Most SJTs use SMEs as a reference group under the assumption that they have considerable experience in a relevant setting and therefore know what kind of behaviors are appropriate in the described situations. Our results suggest that the use of a reference group of inexperienced respondents (i.e., secondary school students) does not affect the adverse impact of an SJT. A possible explanation for this comparable adverse impact is the better representa- tiveness of the group of respondents with respect to ethnicity. Adverse impact All our SMEs in this study were native Dutch, while only 57 % of the applicants were native Dutch. Little is known about the cultural susceptibility of integrity. However, medical professionalism has been found to depend on cultural context (Chandratilake et al. 2012; Jha et al. 2015) and since integrity is an important aspect of medical professionalism, it too might depend on cultural context (Arnold and Stern 2006). A reference group that is more representative of the demographic characteristics of the applicant group may lead to a more accurate mea- surement of the targeted construct and may therefore result in equal or less adverse impact. Future research should investigate the effect of the demographic composition of the ref- erence group on the psychometric quality of an SJT. Another explanation for the equal adverse impact for both type of reference groups might be that there were too few SMEs to be able to achieve proper consensus on the difﬁcult dilemmas described in the scenarios. This was reﬂected by the non-perfect agreement in the SMEs’ evaluation of the response options (ICC = .65). A group of 931 individuals might result in more meaningful consensus. This contention is supported by Legree et al. (2005), who stated that in light of equal validity coefﬁcients, an examinee- based scoring standard gives more reliable values than an expert-based scoring standard, due to the larger number of examinees. 123 123 12 257 Scoring method of a Situational Judgment Test: inﬂuence on… Correlation with personality Our last ﬁnding was that 18 scoring methods showed a correlation with agreeableness and four scoring methods showed a correlation with conscientiousness, which was in line with previous research (Marcus et al. 2007; McDaniel et al. 2007). However, these correlations must be interpreted with caution, since all correlation coefﬁcients represent small effects and it is likely that the large sample size has contributed to the statistical signiﬁcance of these small effects. The larger number of signiﬁcant correlations among scoring methods using standardized consensus is in line with the ﬁndings of McDaniel et al. (2011) and might be explained by the removal of systematic error from the SJT score. However, the small effect size of these correlations between the integrity-based SJT score and the three Big Five personality traits precludes any conclusive statements about the effect of scoring method on the correlation with personality. The small number of signiﬁcant correlations between the SJT score and the Big Five personality traits is in consonance with a previously reported non-association between the Big Five personality traits and the score on a multiple mini interview (MMI), another widely used selection instrument for medical school (Kulasegaram et al. 2010). This non- association might be explained by the fact that personality tests assess non-cognitive traits, whereas MMIs and SJTs assess non-cognitive behaviors. Non-cognitive behaviors are more dependent on situational factors than personality traits (Eva 2005). This is in line with a previous study which demonstrated that a contextualized personality measure had higher criterion validity for academic performance and counterproductive academic behavior than a generic personality measure (Holtrop et al. 2014). The lack of contextu- alization of the NEO-PI-R limits the usefulness of personality tests in medical school selection and may be an explanation for the absence of any meaningful correlations between the SJT score and personality. Scoring method aspects revisited Four scoring method aspects were examined. Differences in internal consistency reliability and adverse impact were found for only one aspect: the way of controlling for systematic error, with raw consensus leading to scores with the highest coefﬁcient alpha and the smallest ethnic subgroup differences. As mentioned above, these differences might be explained by the effect of this scoring method aspect on the total score variance and the negligible effect of response tendencies due to the narrow Likert scale used in this study. No differences were found for the other three aspects (i.e., reference group, distance and central tendency statistic). As stated before, the absence of differences for reference group might be caused by the larger size and better representativeness of the group of respondents itself, which might remove the beneﬁts of using a highly experienced but small group of SMEs. Another potential reason is that integrity-related issues in the beginning stage of medical school do not require speciﬁc knowledge but more general knowledge which can be possessed by both reference groups, which is reﬂected by a correlation of .90 between the group of SMEs and group of respondents itself in their average rating. The absence of differences for the scoring method aspect of distance (absolute vs. squared) may be explained by the low number of scale points on the Likert scale (i.e., four), which means that the maximum distance between an applicant’s rating and the overall rating can never exceed three. This may not be sufﬁcient to get a signiﬁcant 3 258 W. E. De Leng et al. difference in the outcome measure when squaring the distance between both ratings. Future research should examine the scoring method aspect of distance for SJTs using Likert scales with more scale points. difference in the outcome measure when squaring the distance between both ratings. Future research should examine the scoring method aspect of distance for SJTs using Likert scales with more scale points. Lastly, the similar results for the three different central tendency statistics may be explained by the distribution of the ratings across the Likert scale. Data with a symmetric distribution are best summarized using the mean. Since the mean is strongly inﬂuenced by extreme scores (Field 2013), asymmetrically distributed data are better summarized using the median or mode. Practical implications The most important practical implication of this study is that it creates awareness about the importance of carefully considering the immense number of possibilities for con- verting the judgments on an SJT to a score. Instead of arbitrarily choosing one of the many existing methods, researchers and practitioners should accompany the development of an SJT with a thorough examination of the scoring method to be used. In addition, this study demonstrated that the results when using the group of respondents itself are similar to those obtained when using a group of SMEs as reference. Using the group of respondents has practical and economic advantages, since the collection of data from SMEs can be difﬁcult. Unfortunately, this study does not allow any conclusive statements about which scoring method is best, because the ﬁndings are highly dependent on this particular SJT measuring this particular construct in this particular setting. Firstly, this study was conducted in the Netherlands, where medical school applicants are relatively young (17–18 years). The use of more mature applicants may lead to different results for scoring methods that use the group of respondents itself as a reference. Secondly, the cultural context may inﬂuence the way the reference group judges integrity-related dilemmas (Chandratilake et al. 2012; Jha et al. 2015). Finally, SJTs measuring other constructs than integrity might be differentially inﬂuenced by changing the scoring method. Future research should replicate this study with other SJTs measuring different constructs in other settings to investigate the gener- alizability of these ﬁndings and to provide clarity on which scoring method is best for which situation. Scoring method aspects revisited A four-point Likert scale precludes extreme scores leading to similar values for the mean, median and mode and likely causes the comparable results for this scoring method aspect. Conclusion In conclusion, although the SJT scoring method is often chosen arbitrarily, this study shows that changing the scoring method strongly inﬂuences the internal consistency reli- ability and adverse impact of an SJT score. The most inﬂuential characteristic of a scoring method is the way of controlling for systematic error. Given the increasing use of SJTs for selection into medical school, it is crucial to thoroughly examine which scoring method is best to use. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Inter- national 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. Strengths and limitations To our knowledge, this is the ﬁrst study to compare such a large number of scoring methods, varying not only the way of controlling for systematic error and the type of reference group, but also the type of distance and central tendency statistic. Next to the large number of scoring methods examined, this study also contributes to previous research by the examining the effect of scoring method on internal consistency reliability. Embedding the administration of the SJT into the selection procedure led to a very high response rate, ensuring that our results were not inﬂuenced by a volunteer bias. The credibility of our results is further supported by a relatively small restriction of range. Unlike many other selection procedures, the current selection procedure was not preceded by a pre-selection on cognitive competencies. 123 123 12 259 Scoring method of a Situational Judgment Test: inﬂuence on… Although this study compared a large number of scoring methods, we do not claim that this list is exhaustive. Examples of other approaches for scoring SJTs are the squared Mahalanobis distance (Barbot et al. 2012) and the use of paired comparisons (Gold and Holodynski 2015). It seems that the possibilities are endless and future studies should investigate these other scoring methods. For practical reasons, the number of scoring methods in this study was limited to 28. Appendix 1: example scenario Michael questions Sarah, a fellow medical student about extreme and provocative com- ments about individuals’ sexual preferences on her Facebook page. Sarah argues she should be free to express her personal views. She also insists that her personal views have no bearing on her performance as a medical student or patient care. How appropriate are each of the following responses by Michael in this situation? 1. Advise Sarah to remove all controversial comments from her Facebook page 2. Alert Facebook that Sarah’s page contains potentially inappropriate content as they could remove it 3. Ask Sarah to ensure her privacy settings are restricted so her page is inaccessible to patients or the general public 4. Inform a member of staff about Sarah’s Facebook comments 4. Inform a member of staff about Sarah’s Facebook comments 5. Withhold advice to Sarah as her views do not affect patient care or performance as a medical student Appendix 2 See Table 5. 123 260 W. E. De Leng et al. .95 .94 .99 .98 .93 .92 .95 .96 .95 .97 .95 .95 .96 .97 .99 .91 .83 .84 .89 .85 .86 .86 .83 .84 .85 .84 .84 .93 .86 .85 .86 .85 .84 .85 .92 .98 .91 .84 .85 .92 .88 .89 .98 .92 .91 .87 .84 .84 .89 .87 .88 .92 .96 .95 .95 .87 .85 .86 .89 .88 .89 .91 .95 .96 .95 .99 .67 .72 .74 .66 .69 .70 .50 .60 .63 .54 .63 .64 .70 .78 .79 .70 .73 .74 .50 .59 .62 .54 .61 .64 .98 .65 .70 .72 .68 .70 .72 .48 .58 .61 .55 .64 .66 .96 .94 .65 .72 .74 .68 .72 .74 .47 .57 .60 .54 .62 .64 .95 .95 .61 .64 .68 .61 .63 .66 .61 .70 .72 .63 .71 .72 .88 .84 .60 .64 .67 .60 .62 .65 .60 .75 .76 .62 .74 .74 .87 .82 .57 .62 .65 .60 .62 .65 .53 .64 .65 .61 .70 .70 .87 .84 .58 .62 .66 .60 .62 .65 .53 .67 .69 .60 .73 .73 .87 .83 -.58 -.62 -.61 -.57 -.58 -.58 -.31 -.37 -.39 -.34 -.39 -.41 -.74 -.82 -.59 -.63 -.61 -.57 -.58 -.58 -.31 -.37 -.39 -.34 -.39 -.41 -.75 -.82 -.51 -.52 -.56 -.51 -.51 -.54 -.49 -.63 -.63 -.49 -.61 -.61 -.75 -.71 -.52 -.53 -.56 -.51 -.51 -.54 -.50 -.63 -.63 -.50 -.61 -.61 -.75 -.71 -.88 -.91 -.93 -.84 -.84 -.85 -.75 -.77 -.80 -.74 -.76 -.77 -.75 -.78 -.73 -.74 -.77 -.72 -.72 -.75 -.73 -.80 -.80 -.75 -.79 -.79 -.75 -.74 -.90 -.91 -.92 -.86 -.86 -.87 -.78 -.80 -.82 -.77 -.79 -.80 -.77 -.80 -.82 -.81 -.83 -.82 -.81 -.83 -.87 -.91 -.91 -.87 -.90 -.89 -.71 -.70 Scoring method of a Situational Judgment Test: inﬂuence on… 261 Table 5 continued Method 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. .99 17. .87 .85 18. .85 .83 .97 19. .91 .89 .96 .93 20. .91 .88 .95 .95 .99 21. -.74 -.77 -.58 -.57 -.60 -.60 22. -.75 -.77 -.58 -.57 -.60 -.60 1 23. -.73 -.70 -.81 -.85 -.76 -.80 .61 .60 24. -.74 -.71 -.81 -.86 -.76 -.80 .60 .60 1 25. Appendix 2 -.71 -.71 -.67 -.67 -.64 -.65 .67 .67 .59 .58 1 3 Method 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. .99 17. .87 .85 18. .85 .83 .97 19. .91 .89 .96 .93 20. .91 .88 .95 .95 .99 21. -.74 -.77 -.58 -.57 -.60 -.60 22. -.75 -.77 -.58 -.57 -.60 -.60 1 23. -.73 -.70 -.81 -.85 -.76 -.80 .61 .60 24. -.74 -.71 -.81 -.86 -.76 -.80 .60 .60 1 25. -.71 -.71 -.67 -.67 -.64 -.65 .67 .67 .59 .58 12 W. E. De Leng et al. 262 Table 5 continued Method 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 26. -.76 -.71 -.85 -.85 -.82 -.81 .56 .55 .69 .68 .77 27. -.74 -.74 -.71 -.71 -.67 -.68 .68 .68 .64 .63 .97 .81 28. -.71 -.70 -.83 -.83 -.78 -.79 .49 .47 .69 .68 .82 .94 .85 All correlations are signiﬁcant. The numbers in the table correspond to the scoring methods in Tables 2, 3 and 4 Scoring method of a Situational Judgment Test: inﬂuence on… 263 References Odessa: Psychological Assessment Resources Inc. 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https://openalex.org/W3093131214	https://www.researchsquare.com/article/rs-11897/latest.pdf	English	null	The Effect of Multimorbidity Patterns and the Impact of Comorbid Anxiety and Depression on Primary Health Service Use: The Men Androgen Inflammation Lifestyle Environment and Stress (MAILES) Study	American journal of men's health	2,020	cc-by	8,973	The effect of multimorbidity patterns, and the impact of comorbid anxiety and depression, on primary health service use: the Men Androgen In§ammation Lifestyle Environment and Stress (MAILES) Study Shu Kay Ng (  s.ng@gri¨th.edu.au ) Gri¨th University https://orcid.org/0000-0002-6 Sean A Martin The University of Adelaide Robert J Adams The University of Adelaide Peter O'Loughlin SA Pathology Gary A Wittert The University of Adelaide Shu Kay Ng (  s.ng@gri¨th.edu.au ) Gri¨th University https://orcid.org/0000-0002-6865-9384 Sean A Martin The University of Adelaide Robert J Adams The University of Adelaide Peter O'Loughlin SA Pathology Gary A Wittert Research article Posted Date: January 20th, 2020 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at American Journal of Men's Health on September 1st, 2020. See the published version at https://doi.org/10.1177/1557988320959993. The effect of multimorbidity patterns, and the impact of comorbid anxiety and depression, on primary health service use: the Men Androgen Inflammation Lifestyle Environment and Stress (MAILES) Study Shu Kay Ng, PhD1, Sean A. Martin, PhD2, Robert J. Adams, MD, PhD3, Peter O’ Loughlin, PhD4, and Gary A. Wittert, MD, PhD2 1 School of Medicine and Menzies Health Institute Queensland, Griffith University, Nathan, Q4111, Australia 1 School of Medicine and Menzies Health Institute Queensland, Griffith University, Nathan, Q4111, Australia 2 Freemasons Foundation Centre for Men’s Health, University of Adelaide, Adelaide, SA5005, Australia 2 Freemasons Foundation Centre for Men’s Health, University of Adelaide, Adelaide, SA5005, Australia 3 The Health Observatory, Discipline of Medicine, University of Adelaide, The Queen Elizabeth Hospital Campus, Woodville, SA5011, Australia 3 The Health Observatory, Discipline of Medicine, University of Adelaide, The Queen Elizabeth Hospital Campus, Woodville, SA5011, Australia 4 Chemical Pathology, SA Pathology, Adelaide, SA5000, Australia Corresponding author. School of Medicine, Griffith University, Room 2.41, N78, 170 Kessels Road, Nathan, Q4111, Australia. Phone: +617 37359131; E-mail: s.ng@griffith.edu.au Phone: +617 37359131; E-mail: s.ng@griffith.edu.au 1 1 ABSTRACT Background: Multimorbidity has been identified as a serious challenge on global health system, closely associated with lower quality of life, poorer health outcomes, and higher utilisation of health services. However, there are major gaps in our knowledge around multimorbidity, especially its effect on primary care services and the burden of comorbid mental health conditions on multimorbidity patterns. This study sought to determine patterns of multimorbidity and quantify their impact on use of primary health services in the presence and absence of anxiety and depression among a cohort of urban community-dwelling men. Methods: This was a prospective cohort study with Australian population. The study population consisted of 2,039 men aged ≥40, who were enrolled either in the Florey Adelaide Male Ageing Study (FAMAS) Stage 2 between 2007-2010 or in the North-West Adelaide Health Study (NWAHS) Stage 3 between 2008-2010. Data have been collected on the prevalence of 8 chronic conditions and linked Medicare data about individual health service utilization information on annual GP visits. Multinomial logistic regression was adopted to quantify the impact of anxiety and depression on the frequencies of GP visits, with adjustment for participant’s demographic and lifestyle characteristics. Results: Obesity and cardiovascular disease (CVD) were associated with the highest number of comorbid conditions. Two non-random multimorbidity “clusters” emerged: (CVD, Obesity, Diabetes) and (CVD, Obesity, Osteoarthritis). Participants with conditions comorbid with CVD were more likely to have 10 or more annual GP visits, compared to multimorbidity involving other conditions. Comparing to participants without CVD, the presence of CVD increased the chance of having 10 or more annual GP visits (adjusted risk ratio: 3.7; 95% CI: 2.8-4.8). When CVD was comorbid with anxiety and depression having 10 or more annual GP visits was more common (adjusted risk ratio: 1.8; 95% CI: 1.2-2.5). 2 2 Conclusions: In Australian, community-dwelling men, multimorbidity is associated with a high use of GP services; especially for multimorbidity that includes CVD with comorbid anxiety and depression. Multimorbidity patterns involving CVD should be considered in developing clinical trials to better inform medical decision making and care for patients with CVD and comorbid conditions. Keywords: Multimorbidity, Primary health services use, Anxiety, Depression, Cardiovascular disease, Cohort studies, Men’s health 3 3 Setting and population MAILES study was established in 2009 to investigate the associations of sex steroids, The MAILES study was established in 2009 to investigate the associations of sex steroids, inflammation, environmental and psychosocial factors with cardio-metabolic disease risk in men. The study population consists of 2,568 men from two cohort studies: all participants of the Florey Adelaide Male Ageing Study (FAMAS) and age-matched male participants of the North-West Adelaide Health Study (NWAHS). Data have been collected on a number of chronic conditions, as well as linked Medicare data about individual health service claims and utilization information. All protocols were approved by the Royal Adelaide Hospital and the Queen Elizabeth Hospital Research Ethics Committees, with written, informed consent obtained from all participants. Detailed information on recruitment and follow-up process was reported elsewhere [19]. The data in MAILES Stage 2 study contain 2,039 men (FAMAS Stage 2: 2007–2010; NWAHS Stage 3: 2008– 2010), representing data collected at clinics approximately 5 years after baseline visits in the two studies. The MAILES study was established in 2009 to investigate the associations of sex steroids, inflammation, environmental and psychosocial factors with cardio-metabolic disease risk in men. The study population consists of 2,568 men from two cohort studies: all participants of the Florey Adelaide Male Ageing Study (FAMAS) and age-matched male participants of the North-West Adelaide Health Study (NWAHS). Data have been collected on a number of chronic conditions, as well as linked Medicare data about individual health service claims and utilization information. All protocols were approved by the Royal Adelaide Hospital and the Queen Elizabeth Hospital Research Ethics Committees, with written, informed consent obtained from all participants. Detailed information on recruitment and follow-up process was reported elsewhere [19] The data in Health service usage Health service use was obtained from a self-reported, piloted, health service utilization questionnaire. The number of general practitioner (GP) visits in one year was categorized into four categories (zero, 1-4, 5-9, and 10 or more). Information was also obtained regarding a participant’s main reason for visiting the GP, their overall rating of the visit, whether other health issues were raised, and the use of other health service providers. Background Multimorbidity has recently been identified as one of the greatest challenges facing the global health system [1]. Estimates from the WHO demonstrate that between 40-60% of the adult population in developed countries have two or more chronic conditions [2]. The presence of multimorbidity has been associated with lower quality of life [3], increase mortality [4], and higher utilisation of hospital and primary care services [5-8]. Despite this, the study of multimorbidity, as distinct from earlier studies focussed on comorbidity [9], is only relatively new. Multimorbidity is particularly applicable to the primary care setting, where the focus of the general practitioner tends to focus on the whole care of the patient rather than one particular condition [10]. Recent editorials [11-13] have detailed major gaps in our knowledge around multimorbidity, and especially its effect on primary care services. For instance, while the prevalence of multimorbidity is known to increase with age [14] the higher number of absolute cases of multimorbidity in those aged under 65 and the limited opportunities for intervention in elderly patients has motivated a life course approach to multimorbidity in primary care [15]. Despite this, there remains an absence of studies that examine multimorbidity in younger to middle-aged cohorts [12]. The burden of comorbid mental health conditions on multimorbidity patterns, particularly anxiety and depression, has also been highlighted as requiring further study, with around one in three patients with multimorbidity having a concomitant mental health disorder [1, 16]. The recent UK Academy of Medical Sciences report [17] has recommended as a research priority the need to better understand the biopsychosocial determinants of multimorbidity clusters. However to date most evidence on multimorbidity is derived from large disease surveillance systems that have limited capacity to understand multimorbidity as “a non-random series of predictable clusters” [1], and identify modifiable targets for intervention in primary care [18]. Given this, the objectives of our study are to examine: (a) the patterns of multimorbidity of eight chronic conditions (anxiety, asthma, CVD, depression, diabetes, obesity, osteoarthritis, 4 rheumatoid arthritis), and (b) the impact of comorbid anxiety and depression, on the utilization of general practitioner services for urban-dwelling, middle-aged to elderly men. rheumatoid arthritis), and (b) the impact of comorbid anxiety and depression, on the utilization of general practitioner services for urban-dwelling, middle-aged to elderly men. Chronic Conditions Data on chronic disease status (CVD, diabetes, arthritis, depression, anxiety, asthma), were collected through self-report to the question ‘Have you ever been told by a doctor that you have any 5 of the following conditions?’ Classification of diabetes was by self-report and biomedical measures (fasting blood glucose ≥7.0mmol/L and/or HbA1c ≥6.5) [19]. Obesity is indicated by a waist circumference ≥100cm as measured at the clinic visit. Depressive symptoms were assessed using the Beck Depression Inventory (BDI-Ia) [20] for FAMAS men and the Center for Epidemiologic Studies - Depression Scale (CES-D) [21] for NWAHS men. Cut-off scores of 10 and 16 for the BDI-1a & CES-D, respectively, were employed to classify into depression categories (Yes/No). Both the BDI-1a and CES-D show comparable specificities from the classification of major depression in residential, older men [22]. Anxiety symptoms were assessed using the Generalised Anxiety Disorder 7 item (GAD-7) scale [23]. A cut-off score of 10 was used to categorise into anxiety categories (Yes/No). The GAD-7 shows good overall specificity for generalised anxiety disorder in comparable men [23]. Demographic and lifestyle factors Age, marital status, household income, education and qualifications, work status, smoking, alcohol consumption, and physical activity were utilized as collected by self-reported questionnaires at MAILES Stage 2. Information about country of birth was from baseline. Statistical analysis Analysis of chronic conditions was conducted based on the clustering method of pairwise concordance statistics [24], which adopts the asymmetric Somers’ D statistic to quantify the degree of multimorbidity beyond chance [25-26]. Identification of significant (non-random) multimorbidity between conditions, also known as “associative multimorbidity” [27], is more informative to view disease patterns for a potential sharing of risk factors of the diseases [25, 28-30]. The clustering method adopts the Benjamini-Hochberg procedure to control for the false discovery rate at α=0.05 [31]. [31]. Chi-square analysis (for categorical variables) or ANOVA (for quantitative variables) were used to test for significant differences in participant’s characteristics and multimorbidity patterns between the four groups according to the number of GP visits in one year. Multinomial logistic 6 regression was adopted to assess the impact of depression and anxiety on the frequencies of GP visits via additive interaction terms, separately for obesity, CVD, and diabetes, with adjustment for participant’s demographic and lifestyle characteristics. Adjusted relative-risk ratios (RRRs) of GP visits relative to the reference category of 1-4 GP visits were obtained, along with their 95% confidence intervals (CIs). Predicted probabilities of 10+ annual GP visits were calculated to illustrate the effects from either obesity, CVD, or diabetes alone as well as the impact of comorbid anxiety or depression. It is well recognized that any factor, which is caused in part by the exposure (incidence of chronic condition) and is associated with outcome of interest (frequency of GP visits), should not be treated as a confounder and should not be adjusted for in the regression analysis [32]. Bias can result from adjusting for this “intermediate factor” as the estimated exposure-related risk will be markedly reduced. On the basis of literature and clinical evidence, medication was hypothesized to be part of the causal pathway between multimorbidity and GP visits; i.e., multimorbidity is associated with more medications [33-34] and in turn more medications have been shown to increase the health service utilization [35-36]. Therefore, medication was not adjusted for in the multinomial logistic regression analysis. regression was adopted to assess the impact of depression and anxiety on the frequencies of GP visits via additive interaction terms, separately for obesity, CVD, and diabetes, with adjustment for participant’s demographic and lifestyle characteristics. Adjusted relative-risk ratios (RRRs) of GP visits relative to the reference category of 1-4 GP visits were obtained, along with their 95% confidence intervals (CIs). Statistical analysis Predicted probabilities of 10+ annual GP visits were calculated to illustrate the effects from either obesity, CVD, or diabetes alone as well as the impact of comorbid anxiety or depression. It is well recognized that any factor, which is caused in part by the exposure (incidence of chronic condition) and is associated with outcome of interest (frequency of GP visits), should not be treated as a confounder and should not be adjusted for in the regression analysis [32]. Bias can result from adjusting for this “intermediate factor” as the estimated exposure-related risk will be markedly reduced. On the basis of literature and clinical evidence, medication was hypothesized to be part of the causal pathway between multimorbidity and GP visits; i.e., multimorbidity is associated with more medications [33-34] and in turn more medications have been shown to increase the health service utilization [35-36]. Therefore, medication was not adjusted for in the multinomial logistic regression analysis. Comparison analysis and logistic regression were performed using STATA (SE 13.1; StataCorp, College Station, Texas) on the basis of 1,904 participants (93.4% of 2,039) with complete information on the annual frequency of GP visits. Sensitivity analyses were conducted regarding the definitions of anxiety and depression based on either formal clinical diagnosis (GAD7 for anxiety; BDI-1a/CES-D for depression) or medications for anxiety and/or depression, compared to self-reported questionnaire. Multimorbidity of chronic conditions Prevalence rates in decreasing order of the eight chronic conditions among the MAILES Stage 2 cohort were: Obesity (49.9%), Diabetes (19.8%), Asthma (13.1%), CVD (11.8%), Osteoarthritis 7 (11.1%), Depression (9.2%), Anxiety (7.2%), and Rheumatoid arthritis (3.7%). Fig. 1 displays the multimorbidity patterns among these chronic conditions, along with seven pairs of conditions with significant non-random multimorbidity. From Fig. 1, obesity and CVD have the highest number of associated comorbid conditions. Two non-random multimorbidity “clusters” were identified: (CVD, Obesity, and Diabetes) and (CVD, Obesity, and Osteoarthritis). Primary health service use The demographic and lifestyle characteristics of all participants (N=2,039) and separately for participants with completed or missing information on the annual frequency of GP visits are shown in Supplementary Table S1. Overall, the mean age of participants was 59.8, 77.8% married, 67.0% born in Australia, and 14.8% with a degree. About 55.4% of participants were employed, with 36.9% retired. Majority of participants were non-smokers (85.0%) and consumed <2 standard alcoholic drinks per day (79.7%). Participants who had complete information on GP visits were more likely younger (mean age of 59.6 versus 62.6, p=0.005), born in Australia (p=0.016), and employed (p=0.045). Other demographic and lifestyle characteristics were not different significantly between participants with completed or missing information on GP visits. Among 1,904 participants with GP service utilization information, 156 (8.2%) did not visit a GP in last 12 months, 1,084 (57.0%) visited 1-4 times, 416 (21.8%) visited 5-9 times, and 248 (13.0%) participants had 10 or more GP visits. The median range of GP visits was 3-4 times in a year. Participant’s characteristics among the four categories of GP visits are provided in Table 1. Those attending their GP more frequently tended to be older (there is a trend of increasing mean ages from 51.7 to 66.6 years, p<0.001). Related to this, the category of 10+ GP visits has significantly higher proportions of people being separated/widowed (p=0.006), of lower income (p<0.001), and retired (p<0.001). From Table 1, it is also observed that the category of 10+ GP visits contains significantly less Australian born people (p=0.021) and less smokers (p=0.002). As described in the statistical analysis section, Table 1 shows the trend of increasing mean number of medications from 0.11 to 3.17 (p<0.001), indicating a positive association between medication and 8 the frequency of GP visits. There are no differences between the four categories of GP visits in education qualification and alcohol consumption. Impact of multimorbidity on primary health service use Table 2 displays the differences in nine types of comorbid conditions (identified from Fig. 1) among the four categories of GP visits. From Table 2, participants with comorbid conditions have generally more GP visits compared to those without any comorbid conditions (namely, decrease in Table 2 displays the differences in nine types of comorbid conditions (identified from Fig. 1) among the four categories of GP visits. From Table 2, participants with comorbid conditions have generally more GP visits compared to those without any comorbid conditions (namely, decrease in proportions of zero GP visit and 1-4 GP visits but increase in proportions of 5-9 or 10+ GP visits); see also Supplementary Fig. S1. Men with comorbid conditions that include CVD were more likely to have 10 or more annual GP visits. For those participants without neither anxiety nor depression, 54.1% of participants with CVD, obesity, and diabetes, 50.0% of participants with CVD and osteoarthritis, 43.8% of participants with CVD, obesity, and osteoarthritis, 42.9% of participants with CVD and diabetes, and 36.1% of participants with CVD and obesity had 10 or more annual GP visits. Table 2 also shows the increased proportions of participants with 10+ GP visits when symptoms of anxiety or depression was also present (for example, from 54.1% to 85.7% and from 50.0% to 85.7% for participants, respectively, with (CVD, Obesity, and Diabetes) and with (CVD and Osteoarthritis). The results of multinomial logistic regression models assessing the impact of anxiety and/or depression (defined by self-reported questionnaire) on the frequencies of GP visits are provided in Table 3, separately for Conditions A (obesity), B (CVD), and C (diabetes). Besides age, household income and work status, other demographic and lifestyle characteristics were not significant. The presence of obesity increased the frequency of GP visits (adjusted RRRs: 1.4 for 5-9 GP visits and 2.3 for 10+ GP visits over 1-4 GP visits) for participants without anxiety and depression. For participants with obesity, the presence of anxiety or depression further increased the frequency of GP visits (adjusted RRRs: 2.2 for 5-9 GP visits and 3.8 for 10+ GP visits relative to 1-4 GP visits). The predicted probabilities of 10+ GP visits, comparing men without obesity, anxiety, or depression to those with obesity but no anxiety or depression; and those with obesity and also 9 anxiety, depression or both, are displayed in Fig. 2(a). Impact of multimorbidity on primary health service use The corresponding predicted probabilities of 10+ GP visits for these 3 groups were 4.5%, 9.1%, and 22.3%, respectively. The adjusted risk ratios of 10+ GP visits for obesity alone was 2.0 (95% CI: 1.5-2.8), whereas those attributed to anxiety or depression was 2.4 (95% CI: 1.9-3.2). The presence of CVD increased the frequency of GP visits (adjusted RRRs: 1.7 for 5-9 GP visits and 4.8 for 10+ GP visits) for participants without anxiety and depression. For participants with CVD, the presence of anxiety or depression further increased the frequency of GP visits (adjusted RRRs: 4.2 for 5-9 GP visits and 5.0 for 10+ GP visits relative to 1-4 GP visits). The predicted probabilities of 10+ GP visits were 5.7%, 21.0%, and 36.8% for the groups without CVD anxiety or depression, CVD without anxiety or depression, CVD with anxiety and/or depression, respectively (Fig. 2b). The adjusted risk ratios of 10+ GP visits for CVD alone was 3.7 (95% CI: 2.8-4.8), whereas those attributed to anxiety or depression was 1.8 (95% CI: 1.2-2.5). The presence of diabetes increased the frequency of GP visits (adjusted RRRs: 1.9 for 5-9 GP visits and 3.1 for 10+ GP visits) for participants without anxiety and depression. For participants with Diabetes, the presence of anxiety or depression further increased the chance of 10+ GP visits over 1-4 GP visits, with adjusted RRR of 3.0. The predicted probabilities of 10+ GP visits were 5.5%, 13.5%, and 30.1% for the groups without diabetes, anxiety or depression, diabetes without anxiety and depression, and diabetes with anxiety and/or depression, respectively. The adjusted risk ratios of 10+ GP visits for diabetes alone was 2.4 (95% CI: 1.9-3.2), whereas those attributed to anxiety or depression was 2.2 (95% CI: 1.6-3.1) (Fig. 2(c)). Additional results of the sensitivity analyses on the definition of anxiety and depression based on formal clinical diagnosis (GAD7 for anxiety; BDI-1a/CES-D for depression) or medications for anxiety and/or depression were provided in Supplementary Tables S2-S3, which indicated the same conclusion as above that the presence of clinically-diagnosed or medication- based anxiety and/or depression further increased significantly the chance of 10+ GP visits. 10 Discussion Without accounting for gender difference, it has been documented that comorbid depression in persons with diabetes is associated with increased healthcare utilization for a Hungarian population aged >18 and a sample of African-American patients age ≥40 [43-44]. Our study revealed for the first time the synergistic effect of comorbid anxiety and/or depression with either obesity, CVD, or diabetes on primary health service use for older aged men. The likelihood of having 10 or more annual GP visits were increased by 140%, 80%, and 120%, respectively, for comorbid obesity, CVD, or diabetes due to anxiety and/or depression. But the overall effect is still the largest (the predicted probability of 10+ GP visits was 36.8%) for anxiety and/or depression with comorbid CVD compared to obesity (22.3%) and diabetes (30.1%). The significance of comorbid depression and anxiety is not only a matter of increased use of primary care services [45- 47]. Nabi et al. [48] reported that depression symptoms are associated with an increased risk of all- cause death for middle-aged men and women with comorbid coronary heart disease and depression (the British Whitehall-II study) and May et al. [49] found using a large cohort of patients underwent angiography (aged ≥18) that a depression diagnosis at any time following coronary artery disease diagnosis was the strongest predictor of all-cause death, emphasising the need for continual screening of depression among patients with heart diseases. The major strength of the MAILES study is its value-added benefit of combining selected approximately 27.5% of men with mental disorders made use of any services for mental health problems in a year, compared to 8.8% of the general Australian men population [42]. Our study found that 33.8% of men with anxiety and depression had 10 or more annual GP visits, compared to 10.4% of men without anxiety and depression. Primary health service use particularly in community dwelling men with anxiety and/or depression in addition to other chronic health conditions was less researched. Without accounting for gender difference, it has been documented that comorbid The major strength of the MAILES study is its value-added benefit of combining selected participants from two high-quality cohort studies to provide a wealth of measured and self-reported information about multiple chronic conditions, together with the data collected on a wide range of biomedical and socio-demographic variables as well as linked information about primary health- service utilization from Medicare data. Discussion Based on a national survey (aged 16-85), 11 approximately 27.5% of men with mental disorders made use of any services for mental health problems in a year, compared to 8.8% of the general Australian men population [42]. Our study found that 33.8% of men with anxiety and depression had 10 or more annual GP visits, compared to 10.4% of men without anxiety and depression. Primary health service use particularly in community dwelling men with anxiety and/or depression in addition to other chronic health conditions was less researched. Without accounting for gender difference, it has been documented that comorbid depression in persons with diabetes is associated with increased healthcare utilization for a Hungarian population aged >18 and a sample of African-American patients age ≥40 [43-44]. Our study revealed for the first time the synergistic effect of comorbid anxiety and/or depression with either obesity, CVD, or diabetes on primary health service use for older aged men. The likelihood of having 10 or more annual GP visits were increased by 140%, 80%, and 120%, respectively, for comorbid obesity, CVD, or diabetes due to anxiety and/or depression. But the overall effect is still the largest (the predicted probability of 10+ GP visits was 36.8%) for anxiety and/or depression with comorbid CVD compared to obesity (22.3%) and diabetes (30.1%). The significance of comorbid depression and anxiety is not only a matter of increased use of primary care services [45- 47]. Nabi et al. [48] reported that depression symptoms are associated with an increased risk of all- cause death for middle-aged men and women with comorbid coronary heart disease and depression (the British Whitehall-II study) and May et al. [49] found using a large cohort of patients underwent angiography (aged ≥18) that a depression diagnosis at any time following coronary artery disease diagnosis was the strongest predictor of all-cause death, emphasising the need for continual screening of depression among patients with heart diseases. approximately 27.5% of men with mental disorders made use of any services for mental health problems in a year, compared to 8.8% of the general Australian men population [42]. Our study found that 33.8% of men with anxiety and depression had 10 or more annual GP visits, compared to 10.4% of men without anxiety and depression. Primary health service use particularly in community dwelling men with anxiety and/or depression in addition to other chronic health conditions was less researched. Discussion This study presents findings about the utilization of GP services by older-aged community-dwelling men (≥40 years) in relation to their patterns of multimorbidity, where two non-random multimorbidity “clusters” of (CVD, Obesity, Diabetes) and (CVD, Obesity, Osteoarthritis) were identified, and the impact of comorbid anxiety and depression. There is a common misperception that “men don’t go to the doctors”. In 2014-15, an estimated 7.4 million males aged ≥15 years (78%) in Australia had seen a GP at least once in the previous year; the proportion increased with age (from 80.4% at age of 45-54 to 96.3% at age ≥65). In 2013-14, expenditure on primary healthcare and hospital services was 38% and 40% of total health funding respectively in Australia [37]. We have previously reported that >90% of men in our cohort attended their GP at least once in the preceding year [38]. The current study shows higher proportions of at least one GP visit in previous year for men with comorbid conditions (e.g. 96% for men with diabetes and obesity; 100% for men with diabetes, obesity, and CVD). Specifically, this study revealed that men with chronic conditions comorbid with CVD are more likely to have 10 or more annual GP visits, compared to multimorbidity involving other conditions such as diabetes, obesity, arthritis or depression and anxiety, in the absence of CVD. Primary health service use has previously been shown to be frequent in men with multimorbidity involving heart failure in older men [39] or congenital heart disease in younger men (mean age 28.1 years) [40]. Our study quantified the impact of multimorbidity involving obesity, CVD, or diabetes on the relative risks of higher frequencies in annual GP visits, showing again higher impact from CVD compared to multimorbidity involving obesity or diabetes without CVD, after adjustment for demographic factors. Furthermore there is a two to three-fold increase in the chance of having 10 or more GP services annually for Australian men with CVD alone. Another significant driver of primary health care service use among men is mental health Another significant driver of primary health care service use among men is mental health disorders. Data from the FAMAS cohort (men aged 35-80) showed an adjusted OR of 3.9 of 10+ annual GP visits versus none for depressed men [41]. Discussion The MAILES study has a sound epidemiological base, a 12 comparatively large sample of randomly selected community-dwelling men and a high overall response rate, allowing its findings to be generalized to the broader population [19]. As with most cohort studies, the key limitation of the MAILES study is its reliance on self-reported information for some lifestyle and medical factors, such as rheumatic diseases [50]. However, De-Loyde et al. [51] reported that the use of patient self-reported questionnaires to ascertain comorbid conditions remains a valid method for health services research, as shown in the sensitivity analyses which indicated the same conclusion for using clinically diagnosed or medication-based anxiety and depression. Moreover, self-reports of cardiac and stroke events have been reported to be accurate [52-53]. Another limitation of the study was the lack of information on the severity of the disease and type of treatment. Although the study participants were representative of its target population, they were also predominantly Caucasian, aged 35–80 years (at recruitment) and community- dwelling [19]. Conclusions Our study strengthens the evidence-based information about the nature of multimorbidity in men and its impact on primary health services use, which is critical to inform guidelines and health management for effective and efficient care of men with multimorbidity and comorbid anxiety and/or depression. Coexisting conditions may also influence the effectiveness of therapies or modify patients' priorities concerning their health care [54]. Effective management of this patient group thus requires effective management of other comorbid conditions as well [55-56], bearing on different pattern in men’s health service use [57]. More importantly, multimorbidity patterns involving CVD should be considered in the development of clinical trials and guidelines to better inform medical decision-making and provide comprehensive or collaborative care for patients with CVD and comorbid conditions including anxiety and/or depression [54, 58-60]. Additional file Abbreviations ANOVA: Analysis of variance; CI: confidence interval; CVD: Cardiovascular disease; FAMAS: Florey Adelaide Male Ageing Study; GP: General practitioner; ANOVA: Analysis of variance; CI: confidence interval; CVD: Cardiovascular disease; FAMAS: Florey Adelaide Male Ageing Study; GP: General practitioner; MAILES: Men Androgen Inflammation Lifestyle Environment and Stress; NWAHS: North-West Adelaide Health Study; OR: odds ratio; RRR: Relative risk ratio MAILES: Men Androgen Inflammation Lifestyle Environment and Stress; NWAHS: North-West Adelaide Health Study; OR: odds ratio; RRR: Relative risk ratio Consent for publication Not applicable. Availability of data and material The data that support the findings of this study are available from the MAILES cohort study team but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of the MAILES cohort study team. Additional file 1 contains supplementary material including Tables S1-S3 and Fig. S1. Additional file 1 contains supplementary material including Tables S1-S3 and Fig. S1. Ethics approval and consent to participate All protocols were approved by the Royal Adelaide Hospital and the Queen Elizabeth Hospital Research Ethics Committees, with written, informed consent obtained from all participants. Additional file Additional file Additional file 13 Funding This work was funded through the Australian National Health and Medical Research Council (Project Grant #627227). The funding sources had no role in the design and conduct of the study; collection, management, analysis, or interpretation of the data; or preparation, review, or approval of the manuscript. Authors’ contributions SN, SM, RA, GW conceptualised and designed the research project. SN was responsible for the statistical analysis. SN, SM, GW interpreted the results and drafted the first version of the manuscript. All authors (SN, SM, RA, PO, GW) critically revised, read and approved the manuscript. Competing interests GW reports grants from Bayer Schering, grants from Eli Lilly, grants from Lawley GW reports grants from Bayer Schering, grants from Eli Lilly, grants from Lawley Pharmaceuticals, non-financial support from Eli Lilly, non-financial support from Novo Nordisk, personal fees from Bayer Schering, personal fees from Eli Lilly, personal fees from Sanofi, personal Pharmaceuticals, non-financial support from Eli Lilly, non-financial support from Novo Nordisk, personal fees from Bayer Schering, personal fees from Eli Lilly, personal fees from Sanofi, personal uticals, non-financial support from Eli Lilly, non-financial support from Novo Nordisk, personal fees from Bayer Schering, personal fees from Eli Lilly, personal fees from Sanofi, personal 14 fees from Novo Nordisk, personal fees from AstraZeneca, personal fees from I-Nova, personal fees from Elsevier, outside the submitted work. All other authors have no declarations. Acknowledgements The authors are most grateful for the generosity of the cohort participants in giving their time and effort to the study. The study team also is very appreciative of the work of the clinic, recruiting, and research support staff for their substantial contribution to the success of the study. 15 References 1. Pearson-Stuttard J, Ezzati M, Gregg EW. Multimorbidity – a defining challenge for health systems. Lancet Public Health. 2019;4:e599-e600. 2. World Health Organization. Multimorbidity: Technical Series on Safer Primary Care. Geneva: World Health Organization, 2016. Licence: CC BY-NC-SA 3.0 IGO. 2. World Health Organization. Multimorbidity: Technical Series on Safer Primary Care. Geneva: World Health Organization, 2016. Licence: CC BY-NC-SA 3.0 IGO. 3. Fortin M, Lapointe L, Hudon C, Vanasse A, Ntetu AL, Maltais D. Multimorbidity and quality of life in primary care: a systematic review. Health Qual Life Outcomes. 2004;2:51. 4. 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BMC Fam Pract. 2009;10:46. 21 Fig. 1 Multimorbidity analysis: (a) Significant non-random multimorbidity between 8 chronic conditions (nodal size is proportional to the number of conditions that are significantly comorbid with the condition; bolded lines link the “closest” pairs of conditions, with which the pairwise Somers’ D statistic is maximum and significant); (b) Significant comorbid chronic conditions (higher Somers’ D statistic (maximum is 1.0) represents a higher degree of non-random multimorbidity, where the strength of multimorbidity is measured through the number of concordant pairs indicating the presence of both conditions [24]. Fig. 1 Multimorbidity analysis: (a) Significant non-random multimorbidity between 8 chronic conditions (nodal size is proportional to the number of conditions that are significantly comorbid with the condition; bolded lines link the “closest” pairs of conditions, with which the pairwise Somers’ D statistic is maximum and significant); (b) Significant comorbid chronic conditions (higher Somers’ D statistic (maximum is 1.0) represents a higher degree of non-random multimorbidity, where the strength of multimorbidity is measured through the number of concordant pairs indicating the presence of both conditions [24]. 22 23 Fig. 2 Adjusted predictions of 10 or more annual GP visits with 95% CIs for Conditions (a) Obesity; (b) CVD; and (c) Diabetes. Fig. 2 Adjusted predictions of 10 or more annual GP visits with 95% CIs for Conditions (a) Obesity; (b) CVD; and (c) Diabetes. differences among the four categories of GP visits (p<0.05). a Test for differences in frequencies among the four categories using chi-square tests; test for differences in means using ANOVA ANOVA. * Significant differences among the four categories of GP visits (p<0.05). quencies among the four categories using chi-square tests; test for differences in means using diff h f i f GP i i ( 0 05) References 23 Table 1 Demographic and lifestyle characteristics of participants in the four categories of GP visits (N = 1,904) Characteristicsa Frequency (%) or Mean (SD) Total (n=1,904) Zero GP visit (n=156) (8.2%) 1-4 GP visits (n=1084) (57.0%) 5-9 GP visits (n=416) (21.8%) 10+ GP visits (n=248) (13.0%) Age* 51.7 (8.3) 57.8 (10.9) 63.2 (11.2) 66.6 (10.6) 59.6 (11.5) Marital status* Married Separated/widowed Never married missing 118 (76.6%) 23 (14.9%) 13 (8.4%) 2 860 (80.2%) 148 (13.8%) 65 (6.1%) 11 315 (76.6%) 74 (18.0%) 22 (5.4%) 5 172 (69.6%) 58 (23.5%) 17 (6.9%) 1 1465 (77.7%) 303 (16.1%) 117 (6.2%) 19 Country of birth* Australia UK/Ireland Europe Asia/Other missing 104 (66.7%) 27 (17.3%) 18 (11.5%) 7 (4.5%) 0 754 (69.6%) 196 (18.1%) 91 (8.4%) 43 (4.0%) 0 284 (68.3%) 83 (20.0%) 38 (9.1%) 11 (2.6%) 0 146 (59.1%) 52 (21.1%) 40 (16.2%) 9 (3.6%) 1 1288 (67.7%) 358 (18.8%) 187 (9.8%) 70 (3.7%) 1 Household income* Up to $20K $20 - $60K $60 - $80K >$80K missing 8 (5.5%) 47 (32.2%) 26 (17.8%) 65 (44.5%) 10 99 (9.6%) 431 (41.6%) 154 (14.9%) 353 (34.0%) 47 74 (18.9%) 198 (50.5%) 56 (14.3%) 64 (16.3%) 24 72 (31.7%) 121 (53.3%) 19 (8.4%) 15 (6.6%) 21 253 (14.0%) 797 (44.2%) 255 (14.2%) 497 (27.6%) 102 Education Qualification High school Trade Cert./Diploma Degree missing 39 (25.2%) 39 (25.2%) 51 (32.9%) 26 (16.8%) 1 279 (26.0%) 254 (23.6%) 369 (34.3%) 173 (16.1%) 9 132 (31.8%) 98 (23.6%) 125 (30.1%) 60 (14.5%) 1 79 (32.2%) 61 (24.9%) 84 (34.3%) 21 (8.6%) 3 529 (28.0%) 452 (23.9%) 629 (33.3%) 280 (14.8%) 14 Work status* Employed Unemployed Retired Other missing 128 (83.7%) 5 (3.3%) 15 (9.8%) 5 (3.3%) 3 702 (65.1%) 20 (1.9%) 311 (28.8%) 45 (4.2%) 6 165 (40.1%) 6 (1.5%) 218 (53.0%) 22 (5.4%) 5 56 (22.6%) 4 (1.6%) 147 (59.3%) 41 (16.5%) 0 1051 (55.6%) 35 (1.9%) 691 (36.6%) 113 (6.0%) 14 Smoking* Yes/Occasionally No missing 32 (21.1%) 120 (78.9%) 4 167 (16.0%) 876 (84.0%) 41 37 (9.3%) 359 (90.7%) 20 34 (14.4%) 203 (85.7%) 11 270 (14.8%) 1558 (85.2%) 76 Alcohol <2 drinks 3-4 drinks 5-8 drinks >8 drinks missing 118 (77.1%) 18 (11.8%) 10 (6.5%) 7 (4.6%) 3 810 (78.4%) 115 (11.1%) 73 (7.1%) 35 (3.4%) 51 315 (80.4%) 45 (11.5%) 18 (4.6%) 14 (3.6%) 24 191 (84.5%) 19 (8.4%) 10 (4.4%) 6 (2.7%) 22 1434 (79.5%) 197 (10.9%) 111 (6.2%) 62 (3.4%) 100 Number of medications* 0.11 (0.6) 0.76 (1.6) 2.04 (3.0) 3.17 (4.2) 1.30 (2.6) a Test for differences in frequencies among the four categories using chi-square tests; test for differences in means using ANOVA mographic and lifestyle characteristics of participants in the four categories of GP 904) 24 Table 2 Frequency of GP visits for nine types of comorbid conditions Table 2 Frequency of GP visits for nine types of comorbid conditions Multimorbidity Count (row %) Total Zero GP visit n=156 (8.2%) 1-4 GP visits n=1084 (57.0%) 5-9 GP visits n=416 (21.8%) 10+ GP visits n=248 (13.0%) Anxiety & Depression Nil 1 (1.4%) 136 (8.7%) 27 (38.0%) 933 (59.7%) 19 (26.8%) 330 (21.1%) 24 (33.8%) 163 (10.4%) 71 1562 Diabetes & Obesity (anx, dep or both) Diabetes & Obesity (no anx nor dep) Nil 1 (2.9%) 8 (4.2%) 92 (11.7%) 9 (25.7%) 84 (43.5%) 505 (64.0%) 8 (22.9%) 55 (28.5%) 131 (16.6%) 17 (48.6%) 46 (23.8%) 61 (7.7%) 35 193 789 Diabetes & CVD (anx, dep or both) Diabetes & CVD (no anx nor dep) Nil 0 (0%) 0 (0%) 138 (10.2%) 1 (9.1%) 12 (21.4%) 849 (62.9%) 3 (27.3%) 20 (35.7%) 259 (19.2%) 7 (63.6%) 24 (42.9%) 103 (7.6%) 11 56 1349 CVD & Obesity (anx, dep or both) CVD & Obesity (no anx nor dep) Nil 1 (5.6%) 0 (0%) 96 (11.8%) 1 (5.6%) 36 (37.1%) 515 (63.3%) 6 (33.3%) 26 (26.8%) 146 (18.0%) 10 (55.6%) 35 (36.1%) 56 (6.9%) 18 97 813 Rheumatoid & Obesity (anx, dep or both) Rheumatoid & Obesity (no anx nor dep) Nil 0 (0%) 2 (4.9%) 96 (11.2%) 3 (33.3%) 14 (45.2%) 531 (61.9%) 2 (22.2%) 9 (29.0%) 158 (18.4%) 4 (44.4%) 6 (19.4%) 73 (8.5%) 9 31 858 Osteoarthritis & CVD (anx, dep or both) Osteoarthritis & CVD (no anx nor dep) Nil 0 (0%) 0 (0%) 148 (9.7%) 0 (0%) 9 (30.0%) 932 (61.3%) 1 (14.3%) 6 (20.0%) 305 (20.1%) 6 (85.7%) 15 (50.0%) 135 (8.9%) 7 30 1520 Osteoarthritis & Obesity (anx, dep or both) Osteoarthritis & Obesity (no anx nor dep) Nil 1 (6.3%) 1 (1.2%) 94 (11.7%) 1 (6.3%) 40 (46.0%) 503 (62.5%) 7 (43.8%) 25 (28.7%) 149 (18.5%) 7 (43.8%) 21 (24.1%) 59 (7.3%) 16 87 805 Diab & CVD & Obesity (anx, dep or both) Diab & CVD & Obesity (no anx nor dep) Nil 0 (0%) 0 (0%) 91 (12.3%) 0 (0%) 9 (24.3%) 482 (65.2%) 1 (14.3%) 8 (21.6%) 122 (16.5%) 6 (85.7%) 20 (54.1%) 44 (6.0%) 7 37 739 Arth & CVD & Obesity (anx, dep or both) Arth & CVD & Obesity (no anx nor dep) Nil 0 (0%) 0 (0%) 93 (12.4%) 0 (0%) 6 (37.5%) 480 (64.0%) 1 (33.3%) 3 (18.8%) 130 (17.3%) 2 (66.7%) 7 (43.8%) 47 (6.3%) 3 16 750 Data are counts (row percentages) in each category of GP visits. ( p g ) g y Comparison is between participants with the specific type of comorbid conditions (split by those with comorbid anxiety and/or depression (anx, dep or both) and those with no anxiety nor depression (no anx nor dep)) relative to those participants without any of the specific comorbid conditions (denoted as Nil); participants with either of the specific comorbid conditions were excluded from the comparisons). p ) The highest-frequency category of GP visits is highlighted for each pattern of comorbid conditions. Data are counts (row percentages) in each category of GP visits. References Comparison is between participants with the specific type of comorbid conditions (split by those with comorbid anxiety Total p ) The highest-frequency category of GP visits is highlighted for each pattern of comorbid conditions. 25 26 Table 3 Multinomial logistic regression on the four categories of GP visits Characteristics Zero GP visit (n=138) versus 1-4 GP visits (n=952) 5-9 GP visits (n=363) versus 1-4 GP visits (n=952) 10+ GP visits (n=200) versus 1-4 GP visits (n=952) Condition A (for Obesity)a Obesity effect for participants without anx or dep Extra effect from anx, dep, or both for participants with obesity Age Household income Up to $20K $20 - $60K $60 - $80K >$80K Work status Employed Unemployed Retired Other 0.7 (0.5-1.1) 0.9 (0.3-2.7) 0.9* (0.9-1.0) Reference 0.9 (0.3-2.3) 0.9 (0.3-2.7) 1.0 (0.4-2.7) Reference 1.6 (0.5-5.2) 0.7 (0.3-1.6) 0.8 (0.3-2.2) 1.4* (1.1-1.9) 2.2* (1.3-3.7) 1.0 (1.0-1.0) Reference 0.8 (0.6-1.2) 1.1 (0.6-1.8) 0.6 (0.4-1.0) Reference 0.9 (0.3-2.5) 2.2* (1.5-3.4) 1.5 (0.8-2.8) 2.3* (1.5-3.5) 3.8* (2.2-6.6) 1.1* (1.0-1.1) Reference 0.7 (0.5-1.1) 0.6 (0.3-1.3) 0.3* (0.1-0.6) Reference 1.0 (0.3-3.9) 1.6 (0.9-2.9) 6.3* (3.4-11.6) Condition B (for CVD) a CVD effect for participants without anx or dep Extra effect from anx, dep, or both for participants with CVD Age Household income Up to $20K $20 - $60K $60 - $80K >$80K Work status Employed Unemployed Retired Other N/A 1.7* (1.1-2.6) 4.2* (1.0-16.8) 1.0 (1.0-1.0) Reference 0.8 (0.6-1.2) 1.0 (0.6-1.6) 0.5* (0.3-0.9) Reference 0.8 (0.3-2.3) 2.2* (1.5-3.3) 1.5 (0.8-2.7) 4.8* (3.0-7.6) 5.0* (1.3-19.7) 1.0* (1.0-1.1) Reference 0.6* (0.4-1.0) 0.5* (0.3-1.0) 0.2* (0.1-0.5) Reference 1.0 (0.3-3.7) 1.6 (0.9-2.7) 5.8* (3.2-10.5) Condition C (for Diabetes) a Diabetes effect for participants without anx or dep Extra effect from anx, dep, or both for participants with diabetes Age Household income Up to $20K $20 - $60K $60 - $80K >$80K Work status Employed Unemployed Retired Other 0.8 (0.4-1.6) 1.7 (0.3-8.9) 0.9* (0.9-1.0) Reference 0.8 (0.3-2.0) 0.8 (0.3-2.3) 0.9 (0.3-2.2) Reference 1.9 (0.6-5.5) 0.7 (0.3-1.6) 0.8 (0.3-2.1) 1.9* (1.4-2.7) 1.2 (0.5-2.8) 1.0 (1.0-1.0) Reference 0.9 (0.6-1.3) 1.0 (0.6-1.8) 0.6* (0.4-1.0) Reference 0.8 (0.3-2.3) 2.1* (1.4-3.2) 1.5 (0.8-2.8) 3.1* (2.1-4.8) 3.0* (1.3-6.8) 1.1* (1.0-1.1) Reference 0.7 (0.5-1.1) 0.6 (0.3-1.3) 0.3* (0.1-0.6) Reference 0.9 (0.2-3.5) 1.5 (0.9-2.6) 5.8* (3.2-10.7) Data are adjusted relative risk ratios (RRRs) with 95% CI (significant results marked with an *). B because there are no participants with comorbid conditions involving CVD in the category of zero GP p p medications was not adjusted in the models; see text for details. References N/A for Model B because there are no participants with comorbid conditions involving CVD in the category of zero GP visit. Other work status includes student and people with home duties. a Number of medications was not adjusted in the models; see text for details. Table 3 Multinomial logistic regression on the four categories of GP visits 26 Figures Figure 1 Multimorbidity analysis: (a) Signi¦cant non-random multimorbidity between 8 chronic conditions (nodal size is proportional to the number of conditions that are signi¦cantly comorbid with the condition; bolded lines link the “closest” pairs of conditions, with which the pairwise Somers’ D statistic is maximum and signi¦cant); (b) Signi¦cant comorbid chronic conditions (higher Somers’ D statistic (maximum is 1.0) represents a higher degree of non-random multimorbidity, where the strength of multimorbidity is measured through the number of concordant pairs indicating the presence of both conditions [24]. Figure 2 Adjusted predictions of 10 or more annual GP visits with 95% CIs for Conditions (a) Obesity; (b) CVD; and (c) Diabetes. Adjusted predictions of 10 or more annual GP visits with 95% CIs for Conditions (a) Obesity; (b) CVD; and (c) Diabetes. Figure 1 Multimorbidity analysis: (a) Signi¦cant non-random multimorbidity between 8 chronic conditions (nodal size is proportional to the number of conditions that are signi¦cantly comorbid with the condition; bolded lines link the “closest” pairs of conditions, with which the pairwise Somers’ D statistic is maximum and signi¦cant); (b) Signi¦cant comorbid chronic conditions (higher Somers’ D statistic (maximum is 1.0) represents a higher degree of non-random multimorbidity, where the strength of multimorbidity is measured through the number of concordant pairs indicating the presence of both conditions [24]. Figure 2 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. BMCHSRadditional¦le1revised¦nal.pdf
https://openalex.org/W3197798210	https://pubs.rsc.org/en/content/articlepdf/2021/na/d1na00420d	English	null	Role of cooperative factors in the photocatalytic activity of Ba and Mn doped BiFeO<sub>3</sub> nanoparticles	Nanoscale advances	2,021	cc-by	9,743	Introduction wastewater treatment.7,8 Single phase multiferroic BiFeO3 (BFO),9 is a p-type semiconductor,10 and features a narrow band gap, non-toxicity and chemical inertness making it a promising candidate for photocatalytic applications.11,12 The recycling of wastewater contaminated by organic pollutants is one of the major priorities in our ecosystem. In this area, research is getting promoted on processes that improve the oxidative degradation of organic pollutants. These processes include photocatalysis,1 Fenton oxidation,2 and ozonation.3 In the advanced oxidation processes like heterogeneous photo- catalysis, water purication happens on the surface of a photo- catalyst under the irradiation of photons resulting in total mineralization of the dyes. The eﬃciency of the method is based on the formation of the hydroxyl radical $OH, which acts as an oxidation agent and is responsible for the photo- degradation of the organic pollutants. BFO and doped BFO nanoparticles (NPs) are ferroelectric materials.13,14 It has been shown that in photoferroelectric materials15 the spatial separation of electrons (e) and holes (h+) and their transfer from bulk to the surface is assisted by the presence of intrinsic spontaneous polarization induce local electric elds known as depolarization elds.16 The role of this intrinsic electric eld for charge carrier dynamics in the ferro- electric photocatalyst for the photocatalytic redox reactions is fascinating, but the mechanisms are still not understood. Aer poling, a signicant enhancement in the visible-light photo- catalytic activity of BFO NPs was reported recently.17 BFO has been studied for the decomposition of organic dyes under UV + visible light irradiation.18 However, the utilisation of BFO is limited due to rapid charge carrier recombination, and the formation of detrimental defect and trap states in the electronic band structure due to oxygen vacancies. Subsequently, diﬀerent strategies have been developed to improve its photocatalytic eﬃciency, e.g., by altering the morphology,19–21 synthesizing composites,22–25 exploring nanohybrids,26 and tailoring its properties via doping.27 In the eld of photocatalysis, semiconductors have contrib- uted immensely.4 The state-of-the-art photocatalytic materials like TiO2, ZnO et al. can only degrade the pollutants under UV light, which excludes the visible light range of the solar spec- trum. aInstitute for Materials Science and Center for Nanointegration Duisburg-Essen (CENIDE), University of Duisburg-Essen, 45141, Essen, Germany. E-mail: astita. dubey@uni-due.de bWerkstoﬀe der Elektrotechnik & CENIDE, Universit¨at Duisburg-Essen, Bismarckstraße 81, 47057 Duisburg, Germany † Electronic supplementary information (ESI) available: Supportive images and table. See doi: 10.1039/d1na00420d Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5: This article is licensed under a Creative Commons Attribution 3.0 Un The escalated photocatalytic (PC) eﬃciency of the visible light absorber Ba-doped BiFe0.95Mn0.05O3 (BFM) nanoparticles (NPs) as compared to BiFeO3 (BFO) NPs is reported for the degradation of the organic pollutants rhodamine B and methyl orange. 1 mol% Ba-doped-BFM NPs degrade both dyes within 60 and 25 minutes under UV + visible illumination, respectively. The Ba and Mn co-doping up to 5 mol% in BFO NPs increases the speciﬁc surface area, energy of d–d transitions, and PC eﬃciency of the BFO NPs. The maximum PC eﬃciency found in 1 mol% Ba doped BFM NPs is attributed to a cooperative eﬀect of factors like its increased light absorption ability, large surface area, active surface, reduced recombination of charge carriers, and spontaneous polarization to induce charge carrier separation. The 1 mol% Ba and 5 mol% Mn co-incorporation is found to be the optimum dopant concentration for photocatalytic applications. These properties of co-doped BFO NPs can, e.g., be exploited in the ﬁeld of water splitting. Received 3rd June 2021 Accepted 25th August 2021 DOI: 10.1039/d1na00420d rsc.li/nanoscale-advances Received 3rd June 2021 Accepted 25th August 2021 Nanoscale Advances Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. © 2021 The Author(s). Published by the Royal Society of Chemistry Role of cooperative factors in the photocatalytic activity of Ba and Mn doped BiFeO3 nanoparticles† Astita Dubey, *a Alexander Schmitz,b Vladimir V. Shvartsman, a Gerd Bacher,b Doru C. Lupascua and Marianela Escobar Castilloa Cite this: Nanoscale Adv., 2021, 3, 5830 Introduction Single doped BFO NPs like, Sc-doped BFO NPs and nanobers were utilised for the photodegradation of MB dye.37 Mn-doped BFO NPs were able to degrade acid red dye, and Ba doped BFO NPs was eﬀectual to degrade MO dye.38,39 The photodegradation of CR dye using Ba and Mn co-doped and Ca doped BFO nanobers was reported recently.40,41 In spite of its calibre in photocatalysis, nanober production is diﬃcult and not worthwhile for large-scale application. To overcome this, an economical synthesis route is required with low-cost dopants without compromising the advantageous properties of BFO NPs. Ba and Mn as dopants are inexpensive and amply available elements on earth. It has been shown that these elements are easy to incorporate, and their doping bestows the properties of BFO.14,42 The versatile oxidation state and similar ionic radius of Mn makes it one of the best transition metals to substitute Fe. Nevertheless, to our knowledge there is no study on Ba and Mn co-doped BFO NPs for photocatalysis. To obtain such NPs we have implemented a modied sol–gel method, which is an easy, cost-eﬀective, and quite reproducible route. We have incorporated from 1 to 5 mol% of Ba into BiFe0.95Mn0.05O3 (BFM) NPs. We study the role of these dopants in the overall increase of photocatalytic eﬃciency of the BFO NPs. There are several factors which aﬀect the rate of degradation of dyes including surface area, light absorption, charge carrier separa- tion, and their recombination rate [Fig. S1†]. We nd that 1 mol% Ba doped BFM NPs show the best photocatalytic activity among all co-doped NPs examined in this study and can completely degrade MO and RhB dyes within 25 and 60 minutes, respectively, for 105 M dye concentration. We found that an optimum dopant concentration is the key factor to control the photocatalytic parameters. Sample preparation Ba and Mn co-doped BFO NPs (Bi1xBaxFe0.95Mn0.05O3: x ¼ 0.00, 0.01, 0.03, 0.05) were synthesized by a modied wet chemical ‘sol–gel route’ assisted by a calcination step at 773 K.14 Bismuth nitrate Bi(NO3)3$5H2O ($98%), iron nitrate Fe(NO3)3- $9H2O ($98%), manganese acetate Mn(CH3COO)2$4H2O ($99%), barium hydroxide Ba(OH)2$H2O (99.995%), tartaric acid C4H6O6 ($99%), and nitric acid (HNO3) were purchased from Sigma Aldrich and were used as precursors without further X-ray diﬀraction (XRD) The phase and lattice parameters of all NPs were investigated by powder XRD using a Panalytical Empyrean (Cu Ka radiation) diﬀractometer over a 2-theta range of 10 to 80 with a step size of 0.026. The lattice parameters were obtained by Rietveld renement using the High Score Plus soware with consider- ation of nonstructural eﬀects, crystallite size, and strain. Experimental An ESEM Quanta 400 FEG, FEI instrument was used to study the shape and morphology of the NPs. The atomic composition of the NPs was estimated by energy dispersive X-ray spectroscopy (EDXS) using an analytical SEM device (EDS, energy resolution <132 eV for Mn Ka, detector area 10 mm2). Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Studies on photodegradation of dyes by alkaline and tran- sition metal doped BFO NPs are also found to be eﬀective. Single doped BFO NPs like, Sc-doped BFO NPs and nanobers were utilised for the photodegradation of MB dye.37 Mn-doped BFO NPs were able to degrade acid red dye, and Ba doped BFO NPs was eﬀectual to degrade MO dye.38,39 The photodegradation of CR dye using Ba and Mn co-doped and Ca doped BFO nanobers was reported recently.40,41 In spite of its calibre in photocatalysis, nanober production is diﬃcult and not worthwhile for large-scale application. To overcome this, an economical synthesis route is required with low-cost dopants without compromising the advantageous properties of BFO NPs. Ba and Mn as dopants are inexpensive and amply available elements on earth. It has been shown that these elements are easy to incorporate, and their doping bestows the properties of BFO.14,42 The versatile oxidation state and similar ionic radius of Mn makes it one of the best transition metals to substitute Fe. For the PC in acidic medium, the pH of the solution (dye + NPs) was adjusted up to 2.2 by adding a few drops of 2 N HNO3. To keep a constant temperature (298 K) during PC, a continuous water ow was maintained around the jacketed beaker to rule out any thermal catalytic eﬀect. To study the degradation of the dye, 1 ml solution was taken every 5 minutes and centrifuged using a Hettich Zentrifugen Universal 320 R device at 9000 RPM for 10 minutes in the dark to separate the NPs from the dye. Absorption measurements were then performed on the ob- tained solution. Nevertheless, to our knowledge there is no study on Ba and Mn co-doped BFO NPs for photocatalysis. To obtain such NPs we have implemented a modied sol–gel method, which is an easy, cost-eﬀective, and quite reproducible route. We have incorporated from 1 to 5 mol% of Ba into BiFe0.95Mn0.05O3 (BFM) NPs. We study the role of these dopants in the overall increase of photocatalytic eﬃciency of the BFO NPs. There are several factors which aﬀect the rate of degradation of dyes including surface area, light absorption, charge carrier separa- tion, and their recombination rate [Fig. S1†]. Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. We nd that 1 mol% Ba doped BFM NPs show the best photocatalytic activity among all co-doped NPs examined in this study and can completely degrade MO and RhB dyes within 25 and 60 minutes, respectively, for 105 M dye concentration. We found that an optimum dopant concentration is the key factor to control the photocatalytic parameters. Aer the complete degradation of the dye (colourless solu- tion), the NPs were conveniently separated with the help of magnets under the PC vessel and used for the next cycle of PC. Aer 3 successful PC cycles, the NPs were again structurally characterized to compare with previous results. Introduction Their application as antibacterial and photo- electrochemical agents are quite promising, like e.g., for ZnO and TiO2 respectively.5,6 Presently, there is a demand for pho- tocatalysts, which can not only utilise a maximum range of solar light, but are a low cost and sustainable technology for the A moderate amount of doping into BFO at both Bi- and Fe- sites aﬀects its ferroelectric, magnetic and optical proper- ties.14,28 It has been shown that cation doping can reduce the particle size and alter the band gap of pristine BFO.29 These fruitful changes increase the surface to volume ratio and enhance the visible light absorption of the NPs, which are © 2021 The Author(s). Published by the Royal Society of Chemistry 5830 \| Nanoscale Adv., 2021, 3, 5830–5840 5830 Nanoscale Advances View Article Online Nanoscale Advances View Article Online Nanoscale Advances View Article Online Paper modication. We use the following abbreviations in the manuscript BiFe0.95Mn0.05O3 ¼ BFM, Bi0.99Ba0.01Fe0.95Mn0.05O3 ¼ 1BBFM, Bi0.97Ba0.03Fe0.95Mn0.05O3 ¼ 3BBFM, Bi0.95Ba0.05- Fe0.95Mn0.05O3 ¼ 5BBFM. important parameters for photocatalytic application. In this context, rare-earth metal doping has been widely studied to improve the photocatalytic properties of BFO. Rhodamine B (RhB) dye was degraded using Gd-doped BFO NPs.30 The exploitation of Gd and Sn co-doped BFO NPs and La and Se co- doped BFO NPs was reported for the photodegradation of several dyes like methylene blue (MB), congo-red (CR) and methyl violet (MV).31,32 Like this, there are many other reports on Nd, Dy, La, and Sm-doped BFO materials based on compli- cated synthesis methods.33–36 From an economic and environ- mental point of view rare-earth metal doping is not justiable, especially for water cleaning purposes. p p y p p (RhB) dye was degraded using Gd-doped BFO NPs.30 The exploitation of Gd and Sn co-doped BFO NPs and La and Se co- doped BFO NPs was reported for the photodegradation of several dyes like methylene blue (MB), congo-red (CR) and methyl violet (MV).31,32 Like this, there are many other reports on Nd, Dy, La, and Sm-doped BFO materials based on compli- cated synthesis methods.33–36 From an economic and environ- mental point of view rare-earth metal doping is not justiable, especially for water cleaning purposes. Studies on photodegradation of dyes by alkaline and tran- sition metal doped BFO NPs are also found to be eﬀective. Photocatalysis (PC) procedure 105 M of methyl orange (MO) and rhodamine B (RhB) aqueous suspensions were prepared. To establish an adsorption– desorption equilibrium between the dye and the photocatalyst, 50 ml of organic dye with 0.025 g of NPs were placed into a jacketed beaker in the dark for 30 minutes. To achieve good dispersion of the ligand free NPs in the dye solution, 9 minutes ultrasonication was performed in the dark. The degradation of RhB and MO was carried out under the irradiation of a halogen lamp (LOT-Oriel) in a dark chamber to avoid additional light sources. During PC experiments, continuous magnetic stirring of the NPs was performed to avoid their settling. A UV cut-oﬀ lter (390 nm cut-oﬀwavelength) was used for the experi- ments under visible light only. Fluorescence spectrometry To measure the emission spectra, a Varian Cary Eclipse Fluo- rescence spectrophotometer was used. The 450 nm laser exci- tation source was used to collect emission spectra in the range from 550–650 nm. A stable dispersion of NPs was prepared in ethanol (optical grade) and a baseline was obtained by measuring pure ethanol. Fig. 1 XRD diﬀractograms of BFO, BFM, 1BBFM, 3BBFM, and 5BBFM NPs along with their rhombohedral (R3cH) crystal structure on the right. s Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5 This article is licensed under a Creative Commons Attribution 3.0 U These co-doped BFO NPs were used to perform photo- degradation of the organic dye RhB. The representative result of photodegradation of RhB using the 1BBFM sample is shown in Fig. 4a. The absorbance peak at 550 nm is a characteristic absorption feature of the RhB dye. A decrease in the magnitude of this peak with time indicates a decrease in the concentration of the dye in the solution due to photodegradation, since the absorbance is directly proportional to the concentration of the dye.43 Fig. 4b compares the photodegradation of the dye within 180 minutes for all NPs under visible light illumination only (halogen lamp, with 390 nm cut-oﬀlter) at pH ¼ 4.4 of the solution. The photodegradation eﬃciency of BFM is slightly better than that of BFO NPs. However, incorporating 1 mol% Ba into BFM increases the eﬃciency drastically. For larger barium content in the co-doped NPs, the degree of photodegradation slightly decreases, but the 5BBFM NPs still show better photo- catalytic properties than both BFM and BFO NPs. The photo- degradation trend under the UV + visible light source (halogen lamp, without 390 nm lter) at pH ¼ 4.4 is shown in Fig. 4c, where we observe the same trend as in Fig. 4b but with a slightly UV-Vis spectrometry The diﬀuse reectance spectra (DRS) of the NPs were collected by a UV-Vis spectrometer Shimadzu 2600 in the wavelength range 300–900 nm. The NP-powder was pressed and then measured inside an integrating sphere in reectance mode. Barium sulfate was used as a reference and the baseline was corrected before recording each spectrum. Transmission spectra of diluted NPs and NPs drop-cast onto quartz substrates were recorded using a Shimadzu UV-2550 spectrometer equip- ped with an integrating sphere in transmission geometry. Nanoscale Advances Nanoscale Advances Nitrogen physisorption measurements The specic surface area of the NPs was determined from nitrogen sorption measurements using a Coulter SA 3100 analyzer (Beckman Coulter). The samples were degassed under vacuum at room temperature for 24 h. The Brunauer– Emmett–Teller (BET) equation was applied to determine the specic surface areas of the NPs. The Barrett–Joyner–Halenda (BJH) method was applied to determine the pore volume distribution. Transmission electron microscopy (TEM) The size and crystallinity of the NPs were analyzed using a high- resolution TEM on a JEOL JEM-2200FS microscope using 200 kV acceleration voltage and a probe side aberration corrector. © 2021 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv., 2021, 3, 5830–5840 \| 5831 Paper View Article Online Paper View Article Online View Article Online Nanoscale Advances Piezoresponse force microscopy (PFM) To address the ferroelectric properties of the NPs, PFM measurements were performed using a commercial scanning probe microscope MFP-3D (Asylum Research). Pt/Cr coated cantilevers Multi 75E-G (Budget Sensors) with a spring constant of 3 N m1 were used. PFM measurements were conducted at a probing voltage of amplitude Uac ¼ 5 V and frequency f ¼ 50 kHz. The NPs were drop-cast onto conductive carbon tape for the PFM measurements. Structure and morphology of doped BFO NPs According to the XRD patterns, all NPs exhibit rhombohedral (R3c) crystal structure without any secondary phase above the XRD detection limit (Fig. 1). The unit cell volume and lattice parameters including microstrain of the same NPs have been reported in our earlier work.14 The Rietveld renement analyses show distortion in the lattice parameters and an increased microstrain and decreased crystallite size upon Ba doping into Mn doped BFO NPs also in this study.14 In accordance with SEM measurements, both the pristine BFO and co-doped NPs manifest nearly spherical shape with an almost homogeneous size distribution (Fig. 2a–d). We have observed a decrement in the particle size upon co-doping which matches with previously reported PFM and TEM data, where the particle size decreases with a slight alteration in the morphology of the NPs.14 In Fig. 3a, b representative TEM images of the 1BBFM NPs are shown, and an average particle size of 39 nm is calculated. The high crystallinity of the 1BBFM NPs can be seen in Fig. 3c. Photoluminescence (PL) spectroscopy Temperature-resolved PL measurements were conducted inside a CryoMech PT-403 closed-cycle cryostat under high vacuum conditions (<105 mbar). The samples were excited with the ltered 325 nm line of a Kimmon IK-series He–Cd laser. Luminescence spectra were collected using a fused- silica lens system and dispersed using a Horiba iHR320 grating monochromator before being recorded by a Horiba Symphony I CCD camera. Samples were dissolved in optical grade ethanol and treated in an ultrasonic bath for 30 minutes. 20 mL of the dispersion were then drop-cast onto a cleaned Si substrate and dried at room temperature. A reference sample was prepared by drop-casting 20 mL of ethanol onto an identical Si substrate. Fig. 1 XRD diﬀractograms of BFO, BFM, 1BBFM, 3BBFM, and 5BBFM NPs along with their rhombohedral (R3cH) crystal structure on the right. © 2021 The Author(s). Published by the Royal Society of Chemistry 5832 \| Nanoscale Adv., 2021, 3, 5830–5840 5832 Nanoscale Advances View Article Online Nanoscale Advances View Article Online Paper Fig. 2 SEM images of undoped (a) and Ba and Mn co-doped BFO NPs with Ba concentrations of 1 mol% (b), 3 mol% (c), and 5 mol% (d). ln(C0/Ct) for a photocatalytic measurement under visible light and UV + visible illumination up to only 180 min, respectively. From the line ts of ln(C0/Ct) in the time interval from 0 h to 5 h the slope (rate constant) values were obtained. The rate constant values for the doped and undoped NPs in the presence of UV and UV-visible light are plotted in Fig. 4d. For the 1BBFM NPs the rate constant is maximal and almost twice as high as for the pristine BFO NPs. However, further doping with Ba decreases the rate constant of the photocatalytic reaction under both illumination conditions. In short, doping increases the PC eﬃciency of the BFO NPs, and the best photocatalyst among all NPs found is 1BBFM under natural condition, pH ¼ 4.4. pen Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To further enhance the photocatalytic response of the 1BBFM NPs, we decreased the pH of the dye solution to 2.2 by adding a few drops of dilute HNO3. Photoluminescence (PL) spectroscopy For such pH conditions and under UV-visible illumination, 1BBFM completely degraded the dye within 60 minutes (Fig. 5a). We also performed photo- degradation of another organic dye, namely methyl orange under the same conditions. The 1BBFM NPs could completely degrade methyl orange within 25 minutes (Fig. 5b). From Fig. 5c, the photodegradation reaction follows rst order kinetics for both dyes. The rate constant for RhB photo- degradation increased by about 6 times at pH ¼ 2.2 as compared to the solution with pH ¼ 4.4. We observed that the nanopowders are better dispersed in RhB at pH ¼ 2.2 in comparison to the natural condition (pH ¼ 4.4). The zeta potential value of the dispersion increases from 17.2 mV at pH ¼ 4.4 to 39.4 mV at pH ¼ 2.2. This promotes more dye adsorption onto the NPs surface, and it could be one of the major causes for the increase in PC eﬃciency upon decreasing the pH of the solution. Fig. 2 SEM images of undoped (a) and Ba and Mn co-doped BFO NPs with Ba concentrations of 1 mol% (b), 3 mol% (c), and 5 mol% (d). Fig. 3 TEM images of 1 mol% Ba and 5 mol% Mn co-doped BFO (1BBFM) NPs at diﬀerent regions are shown in (a) and (b) with the particle size distribution histogram. Image (c) shows a high-resolution TEM image at 5 nm scale bar for 1BBFM. These PC reactions were run at least three times and were found to have similar eﬃciencies as the rst cycle. A represen- tative graph of cycles for 1BBFM is shown in Fig. S2.† The NPs were removed aer the PC with the help of a magnet attached to the PC jacketed beaker. For the photocatalyst it is important that it does not self-degrade and remains chemically inert with respect to the dye and solution during the PC reaction. To check the stability of the NPs we have performed XRD measurements for all NPs aer the PC and found that they are stable. In Fig. S3† the XRD patterns of BFO and 1BBFM NPs are shown aer PC at pH 2.2 and at pH 4.4 along with TEM images of 1 BBFM sample aer PC at pH 2.2. Photoluminescence (PL) spectroscopy (d) Variation of the rate constants (k) for the photocatalysts under visible and UV + visible light excitation at pH ¼ 4.4. Nanoscale Advances Paper Fig. 4 Absorption spectra of RhB with respect to time in the presence of 1BBFM NPs under visible light and at normal pH of the RhB solution (pH ¼ 4.4) show the degradation of the RhB dye in (a). The relative concentration of RhB versus time under visible light and UV + visible light (at pH ¼ 4.4) is shown in (b) and (c), respectively. The insets show the kinetics of the photodegradation. (d) Variation of the rate constants (k) for the photocatalysts under visible and UV + visible light excitation at pH ¼ 4.4. the data, we have rst compared the absorption spectra of the pure BFO NPs using three methods. Fig. S5† shows the absor- bance spectra obtained using the powder DRS method repre- sented by the black line (a), the transmittance of the NPs dispersed in ethanol (blue line) (b), and the transmittance of the drop-cast and dried NPs (red line) (c). The spectra show a weak absorption feature at around 650 nm (1.90 eV) followed by a steep absorption edge at 550 nm (2.25 eV), possibly con- taining contributions from two diﬀerent transitions. These results match well with earlier reports for polycrystalline bulk BFO and diﬀerent sized BFO NPs.47,48 We observed that the drop-cast (red curve) and powder DRS (black curve) methods give well resolved and nearly similar shapes of the spectra, but for the dispersion (blue curve) method we only see the most prominent features of the absorption spectrum, where other important peaks are hidden. Here, we learn the eﬀect of the chosen method on the absorption spectra of BFO NPs. Since these NPs exhibit strong scattering, the peak 650 nm dimin- ishes in the transmittance measurement of the dispersed NPs, generation of charge carriers (e and h+) upon light absorption by the photocatalyst (see also Fig. S1†). Aerwards, the charge carriers get separated to diﬀuse from the bulk to the surface of the NPs along with competition against their recombination or trapping. In the last step, redox reactions occur at the surface of the NPs driven by the photogenerated charge carriers. Photoluminescence (PL) spectroscopy From these results, we conclude that there is no formation of any secondary phase or degradation of the NPs, indicating that the NPs recover as a photocatalyst aer successful PC. Additionally, we have per- formed total organic count (TOC) measurements for 1 BBFM (Fig. S4†), using a Shimadzu-TOC L device. From the TOC results, we found that all of the RhB was successfully degraded and decayed into CO2 and H2O. Fig. 3 TEM images of 1 mol% Ba and 5 mol% Mn co-doped BFO (1BBFM) NPs at diﬀerent regions are shown in (a) and (b) with the particle size distribution histogram. Image (c) shows a high-resolution TEM image at 5 nm scale bar for 1BBFM. faster degradation for all samples. This shows an eﬀect of UV light together with visible light on the photocatalytic eﬃciency of the studied NPs, which is due to the increase in the number of photons in total. To study the kinetics of PC, Langmuir–Hinshelwood tting was used.44 If the degradation/reaction follows a rate law (eqn (1)) then the reaction has rst order kinetics.45 Based on these data, the 1BBFM NPs shows the best photo- degradation ability. What could be the reason of 1BBFM to be the best photocatalyst among the NPs under study? How does the overall doping into BFO NPs aﬀect the photocatalysis? To unveil the possible reasons, we conducted further experiments to study the various parameters crucial for eﬃcient photo- catalysis in these samples. Photocatalysis involves the kt ¼ ln(C0/Ct) (1) (1) here, k is the rate constant of the reaction, C0 is the initial concentration of the dye in the dark, and Ct is the concentration of the dye at time t during the PC reaction under light. Inserts in Fig. 4b and c show the representative time dependencies of Nanoscale Adv., 2021, 3, 5830–5840 \| 5833 © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online Nanoscale Advances Fig. 4 Absorption spectra of RhB with respect to time in the presence of 1BBFM NPs under visible light and at normal pH of the RhB solution (pH ¼ 4.4) show the degradation of the RhB dye in (a). The relative concentration of RhB versus time under visible light and UV + visible light (at pH ¼ 4.4) is shown in (b) and (c), respectively. The insets show the kinetics of the photodegradation. Photoluminescence (PL) spectroscopy Charge carriers not only promote dye reduction, but possibly also react with electron scavengers (e.g., O2) to form radical anions (e.g., O2 ), oxidize organic molecules, or react with OH/H2O to form $OH radicals.46 So in the next sections, we will discuss about the light absorption by NPs and charge carrier generation, charge carrier recombination and available surface area for the pho- tocatalysis process. © 2021 The Author(s). Published by the Royal Society of Chemistry Light absorption and charge carrier generation To study the charge carrier generation by light absorption, we have performed UV-visible absorption measurements. There are diﬀerent methods to collect absorption spectra of nano- powders. To avoid method-related ambiguities in the analysis of © 2021 The Author(s). Published by the Royal Society of Chemistry 5834 \| Nanoscale Adv., 2021, 3, 5830–5840 View Article Online Fig. 5 Absorbance plots with respect to time of RhB (a) and MO (b) i the presence of 1BBFM NPs at pH ¼ 2.2 under UV + visible illumination Photodegradation trend Ct/C0 as a function of time for both dyes (c where the inset shows the reaction kinetics and rate constant (k values. Paper p g This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. F(RN) ¼ K/S ¼ (1 RN)2/RN (2) RN ¼ Rsample/Rreference (3) here, RN is the reectance of an innitely thick sample. K and S are the absorption and scattering coeﬃcients. The band gap of BFO is mainly formed by strong hybridization of Fe 3d, O 2p and Bi 6p orbitals,50,51 and it exhibits a complex electronic structure caused by spin-charge-lattice couplings due to the convolution of charge transfer bands (interatomic transitions) and absorption bands (d–d transitions).47,52 In Fig. 6b, the spec- trum exhibits peaks at 1 41 eV (879 nm) 1 90 eV (652 nm) Fig. 5 Absorbance plots with respect to time of RhB (a) and MO (b) in the presence of 1BBFM NPs at pH ¼ 2.2 under UV + visible illumination. Photodegradation trend Ct/C0 as a function of time for both dyes (c), where the inset shows the reaction kinetics and rate constant (k) values. Fig. 6 Normalised diﬀuse reﬂectance spectra (a) and Kubelka–Munk transformed (F(R)) normalised absorption spectra (b) for all Ba, Mn co- doped BFO NPs. Paper Nanoscale Advances This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 6 Normalised diﬀuse reﬂectance spectra (a) and Kubelka–Munk transformed (F(R)) normalised absorption spectra (b) for all Ba, Mn co- doped BFO NPs. Fig. 6 Normalised diﬀuse reﬂectance spectra (a) and Kubelka–Munk transformed (F(R)) normalised absorption spectra (b) for all Ba, Mn co- doped BFO NPs. F(RN) ¼ K/S ¼ (1 RN)2/RN (2) RN ¼ Rsample/Rreference (3) F(RN) ¼ K/S ¼ (1 RN)2/RN (2) (2) (3) here, RN is the reectance of an innitely thick sample. K and S are the absorption and scattering coeﬃcients. Light absorption and charge carrier generation The band gap of BFO is mainly formed by strong hybridization of Fe 3d, O 2p and Bi 6p orbitals,50,51 and it exhibits a complex electronic structure caused by spin-charge-lattice couplings due to the convolution of charge transfer bands (interatomic transitions) and absorption bands (d–d transitions).47,52 In Fig. 6b, the spec- trum exhibits peaks at 1.41 eV (879 nm), 1.90 eV (652 nm), 2.50 eV (496 nm), and 3.34 eV (371 nm) in the limit of the measurement range. The peaks at lower energies, 1.41 eV and 1.90 eV, can be attributed to the crystal eld d–d transitions 6A1g / 4T1g and 6A1g / 4T2g respectively.48 The peak at 2.50 eV may be attributed to the band gap of BFO NPs,52 and a hump near 3.34 eV may correspond to the well-known p / d charge transfer band.53 Upon Mn incorporation, the global shape of the BFO absorption spectra changes and upon further Ba doping the shape is altered again, which shows the individual eﬀect of these Fig. 5 Absorbance plots with respect to time of RhB (a) and MO (b) in the presence of 1BBFM NPs at pH ¼ 2.2 under UV + visible illumination. Photodegradation trend Ct/C0 as a function of time for both dyes (c), where the inset shows the reaction kinetics and rate constant (k) values. which was conducted in front of an integrating sphere. The collection of reectance measurements in the solid form inside an integrating sphere to compensate scattering is a reliable way to observe the absorption features of these NPs. Based on the above experiment, we collected the powder DRS spectra of all synthesized nanopowders [Fig. 6a]. Fig. 6b shows the transformed absorption spectra of doped and undoped BFO NPs via the Kubelka–Munk function (F(RN)).49 © 2021 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv., 2021, 3, 5830–5840 \| 5835 © 2021 The Author(s). Published by the Royal Society of Chemistry 5835 View Article Online Fig. 7 Topography (a), vertical (b) and lateral (c) PFM images of 1BBFM NPs. The cross sections of vertical PFM (d) and lateral PFM (e) images (the cross-section location is marked by a red line in the corre- sponding images). The local piezoresponse phase (f) and amplitude (g) hysteresis loops. Paper View Article Online Paper Nanoscale Advances Paper dopants on the electronic structure of BFO NPs. Light absorption and charge carrier generation The 6A1g / 4T2g transition (1.90 eV) becomes more intense and broader for BFM, which shows a distortion in energy levels due to the Jahn–Teller eﬀect originating from the presence of Mn3+ ions as conrmed by XPS analysis in our earlier report.14 This peak is even more intense and shied to a higher energy for 1BBFM. Upon further Ba doping, the absorption peak at 1.98 eV slightly broadens and the intensity of the peak 2.51 eV increases. One can see that in the photon energy range from 1.6 to 2.50 eV the Ba and Mn co-doped NPs absorb more solar light than the BFM and undoped BFO NPs. Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In literature, BFO is oen considered as direct band gap semiconductor by both theoretical and experimental proofs54–56 whereas there are also reports claiming an indirect band gap.57–59 The band gap is considered to be 2.5 eV for BFO single crystals.55 For BFO NPs, the band gap is extracted to be 2.74 eV for 100 nm, 2.24 eV for 190 nm, 2.22 eV for 120 nm, 2.21 eV for 50 nm, and 2.18 eV for 30 nm NPs using the Tauc model from optical methods.48,60 However, the Tauc plot model is based on a three-dimensional density of states and thus does not include, e.g., d–d transitions. Therefore, we refrain from using the Tauc plot approach here for estimating the band gap for doped NPs. So far, we have observed the distortion in elec- tronic structure of BFO, and increased light absorption by doping the NPs. The utilization of maximum solar light for charge carrier generation is reected by the increased photo- catalytic activity of the doped NPs. Fig. 7 Topography (a), vertical (b) and lateral (c) PFM images of 1BBFM NPs. The cross sections of vertical PFM (d) and lateral PFM (e) images (the cross-section location is marked by a red line in the corre- sponding images). The local piezoresponse phase (f) and amplitude (g) hysteresis loops. Local ferroelectric properties Aer charge carrier generation, their eﬃcient separation also plays a crucial role during the photocatalysis process. In a previous report we have proven by PFM technique that BFO NPs and their co-doped fellows are ferroelectric.14 In ferroelec- tric materials, it has been shown that charge carriers get sepa- rated easily upon light illumination.61 In photoferroelectric materials a broad space charge region exists due to internal screening of spontaneous polarization by free charge carriers and defects within the material. The resulting band bending depends on the surface polarity and facilitates the separation of photogenerated charge carriers.62,63 One can expect that the larger spontaneous polarization of NPs will be benecial for charge carrier separation and photocatalytic activity. To address the polarization of our best photocatalyst i.e., 1BBFM NPs we applied PFM. The PFM signal depends on the local piezores- ponse, which is proportional to the polarization value. Fig. 7 shows the topography (a), vertical PFM (VPFM) and lateral PFM (LPFM) images. The intensities of the VPFM and LPFM signals depend on the out-of-plane and in-plane components of polarization, respectively. The 1BBFM NPs are mostly single domain as shown by red dotted circles, similar to the BFO and BFM NPs.14 The local PFM hysteresis loops (Fig. 7f, g) conrm the switching of the polarization direction by an external elec- tric eld conrming the ferroelectric state of the NPs. In our previous study we found that Mn doping slightly increases the PFM signal of the BFO NPs, but co-doping with 5 mol% Ba decreases it.14 Comparing the PFM response of the 1BBFM NPs with those data, one can see that the intensity of the PFM is about 30% larger than in the BFM NPs (Fig. S6†). As piezores- ponse of a material is proportional to the spontaneous polari- zation, we can conclude that 1BBFM has a larger spontaneous polarization than the BFO and BFM NPs. A larger polarization means a larger depolarization eld, which can facilitate photoinduced charge carrier separation and may best explain the PC eﬃciency of the 1BBFM NPs to be the best. Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Though surface area can be a major factor, other factors also aﬀect the degradation rate of doped NPs. As photocatalysis is a surface-mediated process, not only the available surface area of the NPs plays an important role for their eﬃciency, but also the surface activity. It has been demonstrated that for a variety of metal-oxides, visible uorescence is connected to the adsorption and modication of surface adsorbates such as oxygen or -OHx groups by the excitation light.68,69 The evolution of such uorescence under continuous excitation is hence connected to the surface activity. To support the benecial properties of our co-doped NPs as compared to pure BFO, we drop cast a dispersion of the NPs in ethanol onto a cleaned Si substrate and conducted PL measurements under UV excita- tion. The dried NPs were excited with a 325 nm (3.81 eV) laser source in vacuum to collect PL spectra at 290 K and at 5 K. We nd the PL spectra in the visible range from 400–700 nm. During continuous illumination, a strong variation of the PL intensity of all samples was observed as exemplied by the spectra of the doped 1BBFM NPs (Fig. 9a). No change can be detected in the shape of the spectra. Fig. 9 (a) The illumination time (t) dependent emission spectra of 1BBFM NPs at 290 K with 325 nm excitation. (b) The change in intensity versus illumination time in minutes at 290 K and 5 K for pristine BFO NPs (blue) and 1BBFM NPs (red), respectively. The occurrence of such broad spectra, as well as the observed change in intensity, has been described in literature and is most likely associated with the adsorption of OHx groups from the solvent (or atmosphere) onto the NPs surface and their modi- cation under the action of incident laser radiation.66,67 Although the experiments were conducted under high vacuum, the –OH groups are expected to still persist on the highly porous NPs surfaces and to be trapped between agglomerated particles. The observed brightening of the PL spectra in the course of illumination manifests light induced surface activity attributed to the modication of adsorbed OH-groups on the surface of the NPs. Comparing the emission intensity change of the pristine and 1BBFM NPs (Fig. 9b) reveals a much stronger brightening eﬀect for the 1BBFM sample at both room and cryogenic temperatures. Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This eﬀect indicates a higher optically induced surface activity of 1BBFM in terms of the adsorption and alteration of attached –OH groups as compared to the pristine BFO NPs. The improved adsorption of –OH groups will lead to increased $OH radical formation aer charge carrier genera- tion. This eﬀect is expected to improve the photocatalytic eﬃ- ciency of the doped sample in the presence of a dye. Fig. 8 The speciﬁc surface area trend (red) versus doping in BFO NPs is compared to the relative photodegradation (vertical bars) of RhB under UV-visible light (pH ¼ 4.4) up to 180 minutes. Charge carrier recombination Once the charge carriers are separated, their recombination should be inhibited for their diﬀusion from the bulk to the surface of the NPs. The PL spectroscopy can be used to identify not only the defect states but the recombination of charge carriers as well.64 Therefore, the PL spectra of all NPs were measured by excitation of the NPs by a 450 nm (2.75 eV) laser light source (Fig. S7†). For both, the pristine and doped BFO NPs, we found similar emission peaks, which indicates that no new types of in-band defect levels are formed at this dopant concentration. However, the peaks get slightly shied, but this eﬀect is not as signicant as the change in the total peak area (Fig. S7b†). The 1BBFM sample has the smallest peak area for both emission peaks among the NPs, which is associated with a lower charge carrier recombination,65 and a less absorption of 1BBFM at 500 nm (2.48 eV). Therefore, the larger PL peak area for the 5BBFM NPs than for the 1BBFM NPs may correlate with the lower PC eﬃciency of the 5BBFM sample. © 2021 The Author(s). Published by the Royal Society of Chemistry 5836 \| Nanoscale Adv., 2021, 3, 5830–5840 5836 View Article Online Paper Nanoscale Advances Fig. 9 (a) The illumination time (t) dependent emission spectra of 1BBFM NPs at 290 K with 325 nm excitation. (b) The change in intensity versus illumination time in minutes at 290 K and 5 K for pristine BFO NPs (blue) and 1BBFM NPs (red), respectively. Available surface area for photocatalysis Available surface area for photocatalysis The specic surface area of the co-doped BFO NPs was evaluated using BET measurements. The results are summarized in Table S1.† Doping with Mn alone does not lead to a signicant change in the surface area, but by adding 1% Ba the surface area increases considerably. For the samples with more Ba content, the surface area decreases slightly, but they still exhibit more surface area than the pure BFO NPs. The trend of the specic surface area compared to the photodegradation trend at pH ¼ 4.4 under UV-visible light for all NPs is shown in Fig. 8. Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:02:33 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Discussion The obtained results indicate that the increased photocatalytic eﬃciency of BFO NPs upon Ba and Mn doping can be explained by a combination of several factors, where the foremost is the Fig. 8 The speciﬁc surface area trend (red) versus doping in BFO NPs is compared to the relative photodegradation (vertical bars) of RhB under UV-visible light (pH ¼ 4.4) up to 180 minutes. © 2021 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv., 2021, 3, 5830–5840 \| 5837 View Article Online View Article Online Paper View Article Online Nanoscale Advances Paper degradation of organic dyes (rhodamine B and methyl orange) under UV-Visible light. Among them the NPs with 5 mol% Mn and 1 mol% Ba doping show the best results and can degrade MO and RhB dyes in 25 and 60 minutes, respectively, at pH ¼ 2.2. The surface area and pore volume increase for the doped NPs, while the 1BBFM NPs have the largest surface area and pore volume amidst the NPs under study. The electronic band structure of BFO gets altered due to doping. This increases the light absorption capability of doped NPs in the visible range, in particular between 500 and 800 nm. The larger spontaneous polarization and related depolarization eld make the charge carrier separation more eﬀective in the 1BBFM NPs than in the BFO and other doped NPs. The emission spectra show that for 1BBFM the charge carrier recombination is lower than for other NPs. A stronger increase of luminescence from adsorbed species on the co-doped NPs during continuous UV excitation compared to the pristine BFO NPs further indicates an enhanced surface activity. Overall, the maximum photocatalytic eﬃciency for the 1BBFM NPs is attributed to the cooperative eﬀect of increased light absorption, a larger surface area, more eﬀective charge separation, and less recombination of photo- generated charge carriers. The dopant concentration is a key factor in controlling the parameters of the photocatalytic process. In our case doping with 1 mol% Ba into BiFe0.95- Mn0.05O3 NPs is proven to be optimal for the photocatalysis eﬃciency. alteration in the lattice parameters and the change in size and morphology of the NPs. The spherical morphology of the doped BFO NPs under study implies a large surface area, which provides more sites for incoming charge carriers to participate in redox reactions at the surface. Author contributions Third, the spontaneous polarization of these ferroelectric NPs at room temperature causes band bending, which assists the separation of charge carriers. This eﬀect can also promote the photocatalysis process considerably. The PFM data indicate the larger polarization in the 1BBFM NPs as compared to other NPs under study, which correlates with their best photocatalytic performance. A D has synthesized the NPs and performed structural, morphological, reectance and emission characterization of NPs via XRD, TEM, SEM, UV-Vis, and PL methods. A D per- formed all photocatalysis experiments and wrote the manuscript. V V S performed the PFM measurements and analyzed with A D. A S has collected the PL measurements with respect to illumination time and analyzed the results with A D. M E C has initialized this research and was responsible for scientic project administration. V V S, G B and D C L have contributed equally to the scientic discussion. The fourth factor is the slowest charge carrier recombination for the 1BBFM among all the NPs, as evidenced by the decreased peak area of the PL emission spectra collected at 450 nm. The temporal evolution of the PL intensities collected at 325 nm provides a conclusive link to the catalytic activities of the NPs via adsorbing –OH groups onto their surface. The increasing rate of the PL intensity growth under continuous illumination observed in the 1BBFM sample as compared to the undoped BFO indicates stronger interaction of the nanoparticle surface with adsorbed OH groups. This nding also relates the increased photocatalytic eﬃciency of the 1BBFM NPs to the increased surface activity. Acknowledgements We are grateful to DFG (Deutsche Forschungsgemeinscha) for nancial support through the project LU 729/21-1 (project number 396469149). We would like to thanks Ms Claudia Schenk and Ms Anam Ashgar for help in BET and TOC measurements, respectively. Conﬂicts of interest The authors declare no conict of interest. Discussion The 1BBFM sample has the largest surface area of almost 1.6 times more than that of the pure BFO NPs (Fig. 8). The larger surface area and the higher pore volume are major factors in the photocatalytic properties of the co-doped BFO NPs. Second, the optical absorption edge in the electronic struc- ture of the BFO NPs is altered due to doping. In the Mn doped BFO NPs, the existence of manganese both as Mn3+ (Jahn–Teller cation) and Mn4+ in the crystalline lattice was conrmed by XPS and Raman analyses.14 The presence of a Jahn–Teller cation in the MnO6 octahedra can distort the electronic structure of BFO by splitting the energy levels further.68 Upon further co-doping Ba into BFM, the unit cell volume increases and the Mn3+ concentration reduces in favor of Mn4+. It is important to notice that this eﬀect is most signicant for the 1BBFM, which also has the largest unit cell volume.14 For co-doped BFO NPs, two main factors cause important changes in the electronic structure: increased unit cell volume due to bigger Ba2+, and presence of a Jahn–Teller cation (Mn3+). In total, such structural distortion leads to an alteration in Fe–O bond lengths and inuences the d–d crystal eld transition energy levels,68 and can alter the electronic transition of C3v crystal symmetry of BFO. As per Wei et al., an increase in crystal eld splitting due to the reduction in the unit cell volume via doping by rare-earth elements leads to a decrease in the energy edge of d–d transitions.69 In our case the increase in unit cell volume due to Ba doping into BFM leads to an increase of the 6A1g / 4T2g d–d transition energies (Fig. 6b). The increase of the light absorption associated with the 6A1g / 4T2g d–d transition due to the doping inuences the photocatalytic eﬃciency signicantly. Open Access Article. Published on 26 August 2021. Downloaded on 10/24/2024 5:0 This article is licensed under a Creative Commons Attribution 3.0 Unp 35 M. Sakar, S. Balakumar, P. Saravanan and S. Bharathkumar, Nanoscale, 2015, 7, 10667–10679. 9 S. Goswami, D. Bhattacharya, P. 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https://openalex.org/W4243997851	https://revistas2.uepg.br/index.php/olhardeprofessor/article/download/4035/3259, https://www.redalyc.org/pdf/684/68425573004.pdf	es		Profesorado principiante: entre montañas y veredas, aprendiendo a ser maestro en el sureste mexicano	Olhar de Professor	2,013	cc-by	6,926	DOI: 10.5212/OlharProfr.v.15i2.0003 PROFESORADO PRINCIPIANTE: ENTRE MONTAÑAS Y VEREDAS, APRENDIENDO A SER MAESTRO EN EL SURESTE MEXICANO BEGINNING TEACHERS: BETWEEN MOUNTAINS AND SIDEWALKS, LEARNING TO BE A TEACHER IN SOUTHEAST MEXICO Iván Alexis Pinto Diaz* Resumen: Resultado del trabajo de campo de la investigación “los primeros años de ejercicio docente en educación básica. Reconstrucción de prácticas y experiencias en contextos desfavorecidos”, se presenta evidencias de la situación que enfrentan los profesores principiantes en las comunidades rurales del estado de Chiapas, en el sureste mexicano. Se recupera las peripecias, angustias y los aprendizajes de los primeros días y meses que como profesores principiantes viven en el centro de trabajo y la comunidad. A partir de una serie de entrevistas in situ se documenta la experiencia, pero sobre todo se desentraña la distancia entre los procesos de formación que ofrecen las instituciones formadoras de profesores y la realidad que les toca vivir a los nuevos trabajadores de la educación; se destaca que no existe apoyo y acompañamiento para esos primeros momentos del ejercicio docente. Se hace evidente y como lo señalan los profesores, es la realidad lo que los forma y los compromete con la actividad profesional en la que se encuentran inmersos. El trabajo forma parte de una intención mayor, la de documentar la realidad y participar en el debate sobre la necesidad de establecer programas y apoyos para que los profesores principiantes se desarrollen y consoliden en la tarea docente. Como se notará, los profesores principiantes viven intensamente sus primeros contactos con su profesión (a pesar de las penurias y conﬂictos), es una experiencia reveladora y signiﬁcativamente formativa, la cuestión es sistematizarla y presentarla como un recurso que puede potencializar la formación continua. Palabras clave: Profesores principiantes. Diﬁcultades. Retos. Formación. Abstract: This article presents the rresults of the investigation called "The ﬁrst years of teaching in basic education: reconstruction of practices and experiences in disadvantaged contexts" which presents evidence of the situation faced by beginning teachers in rural communities in the state of Chiapas in the southeast of Mexico.The article recovers the adventures, anxieties and lessons from the ﬁrst days and months that beginning teachers experience in the workplace, and in the community. From a series of on-site interviews the article reports not only the experience, but also the gap between the training processes offered by teacher education institutions, and the reality lived by beginning teachers. The article emphasizes that there is not support, and assistance for the ﬁrst years of teaching. It also provides evidence based on teachers’ testimonies, that it is the reality they experience that turns them into teachers and, commits them to the profession in which they are immersed. The article has a broader purpose, which is to document the reality, and participate in the debate about the need to establish, and support programs for beginning teachers to develop and strengthen the teaching task. As it will be noticed, * Maestro en Educación Superior. Universidad Autónoma de Chiapas. México. Maestro en Educación con especialidad en Investigación Educativa. Universidad Autónoma de Chiapas. México. Candidato a Doctor en Educación. Universidad Pedagógica Nacional. México. Profesor de la Universidad Autónoma de Chiapas. E-mail: <iapidiaz70@hotmail.com>. * Master in Education. Autonomous University of Chiapas. Mexico. Master in Education with a degree in Educational Research. Autonomous University of Chiapas. Mexico. Doctoral student in Education. National Pedagogy University. Mexico. Professor at the Autonomous University of Chiapas. E-mail: <iapidiaz70@hotmail.com>. Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> 237 Profesorado principiante: entre montañas y veredas, aprendiendo a ser maestro en el sureste mexicano beginning teachers live intensively their ﬁrst teaching years, despite the difﬁculties and conﬂicts, it is a revealing and formative meaningful experience. The issue is systematized and presented as a resource that can enrich continuing education. Keywords: Beginning teachers. Difﬁculties. Challenges. Education. Introducción Este trabajo es producto de las consideraciones desarrolladas en el marco del proyecto de investigación no. 146031, denominado “Los primeros años de ejercicio docente en educación básica. Reconstrucción de prácticas y experiencias en contextos desfavorecidos” que se lleva a cabo por académicos y estudiantes de la Universidad Pedagógica Nacional –Unidad Ajusco-,1 en el marco de la convocatoria SEB-2009-1 del Consejo Nacional de Ciencia y Tecnología del gobierno mexicano. La investigación tiene como propósito “analizar cualitativamente las prácticas, diﬁcultades y logros de los maestros noveles de educación primaria y secundaria” en contextos desfavorecidos y rurales. Para el estudio se consideró a profesores de dos entidades mexicanas, una de ellas, y es a lo que reﬁere este trabajo, tiene que ver con profesores principiantes del estado de Chiapas. Poco se ha investigado en el contexto mexicano, la realidad que enfrentan los recién egresados de las instituciones formadoras de profesores, cuando viven sus primeras experiencias como trabajadores de la educación. Cómo y qué deﬁne la experiencia entre pasar de la tranquilidad de ser estudiante normalista y el proceso de hacerse maestro. El estudio observa con 1 La investigación es coordinada por la Dra. Etelvina Sandoval y la Mtra. Alicia Carvajal. En dicho proyecto colaboramos alumnos de maestría y doctorado de la citada universidad. El autor de este reporte coordina el trabajo de campo en la entidad chiapaneca. 238 Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> detenimiento la conﬁguración del estilo docente en los primeros años de ejercicio, en tanto es un momento crucial por la complejidad de elementos que lo permean, y entre los cuales se encuentra la historia vivida por el sujeto en la educación Normal, el origen social de los profesores y el contexto donde se ubica la experiencia del ejercicio profesional. Se retoma el planteamiento de considerar los inicios de la profesión docente como una fase de intenso aprendizaje en el oﬁcio del magisterio, donde se va conformando imágenes de la profesión y generando prácticas especíﬁcas, la cual forma parte de un continuo aprendizaje a lo largo de la vida (SANDOVAL, 2011). También se asume que La inserción profesional en la enseñanza (…) es el periodo de tiempo que abarca los primeros años, en los cuales los profesores han de realizar la transición desde estudiantes a docentes. Es un periodo de tensiones y aprendizajes intensivas en contextos generalmente desconocidos y durante el cual los profesores principiantes deben adquirir conocimiento profesional además de conseguir mantener un cierto equilibrio personal. (MARCELO GARCÍA, 2008, p. 14). El trabajo se orienta desde una perspectiva metodológica cualitativa con énfasis en enfoques narrativos y análisis de trayectorias profesionales (BOLIVAR, 2001; TARRÉS, 2004). Iván Alexis Pinto Diaz Para el estudio se consideró a los profesores que se inician en escuelas de Chiapas,2 ya que esa entidad del sureste de México ofrece elementos de gran valía para conocer y reﬂexionar la conﬁguración del proceso de hacerse maestro en contextos rurales y marginados. Esa entidad tiene marcado rezago social y económico. Una alta dispersión de comunidades y los problemas educativos son cuestiones recurrentes. La educación Normal, las condiciones de la formación para el ejercicio profesional En Chiapas, existen diecinueve instituciones formadoras de profesores (las cuales llevan la denominación de escuelas normales) para la educación básica (preescolar, primaria y secundaria). De éstas, solamente una ofrece alguna formación con carácter rural.3 Pero en su conjunto, dichas instituciones se encargan de formar en elementos de orden académico, es decir, en preparación para estar presente en aula y atender los contenidos del nivel educativo que se trate (se deja de lado la formación para entender e incursionar en el ámbito comunitario, es decir, no se prepara para adaptarse al contexto laboral). 2 Se entrevistaron a cuatro profesores de educación primaria del municipio de La Trinitaria, siete profesores de educación secundaria, de los cuales cinco pertenecen al Centro de Educación Básica del Estado de Chiapas (CEBECH) de la ciudad rural sustentable “Juan del Grijalva” en el municipio de Ostuacán, uno de la localidad Golonchan viejo del municipio de Chilón y uno más del municipio de Oxchuc. 3 En México existen dos tipos de normales, las urbanas y rurales. De las escuelas normales rurales solo quedan 16 en el país, de éstas una en Chiapas. La formación de profesores con conocimiento e identidad del contexto rural es cada vez menor, en tanto las instituciones que forman para este campo son pocas y con una reducida matrícula. En el sistema educativo mexicano, los normalistas atienden las escuelas de la educación básica (en años recientes, la presencia de universitarios es más frecuente; su intervención es distante de los requerimientos necesarios para atender las necesidades de los estudiantes y las escuelas). Las normales de licenciatura en educación preescolar preparan a profesoras para el jardín de niños (de tres grados); las normales que ofrecen la licenciatura en educación primaria preparan para atender las escuelas primarias (de seis grados) y las normales superiores forman para atender el nivel de secundaria (se estudia en tres grados). Aquí cabe un comentario como antecedente en el análisis de la situación en las normales mexicanas y especíﬁcamente para entender los procesos formativos que las caracterizan. Desde 1984 la educación Normal fue “elevada” al modelo y nivel de institución de educación superior (IES), lo cual implicó que pasaran del tradicional modelo de organización que los caracterizaba desde su origen, es decir, con procesos orientados en formar a los futuros maestros como técnicos de la enseñanza priorizando el saber hacer del ejercicio docente, para pasar a un modelo de trabajo y organización escolar en donde se impuso dos actividades sustantivas (a parte de la docencia): la investigación y la difusión. Antes de 1984 los formadores que colaboraban en las normales, se dedicaban (como tarea central del contrato laboral) a impartir clase y orientar a los alumnos en cómo deﬁnir un estilo docente en la educación básica; posterior a ese año, los formadores los acicatearon para que entraran en una “competencia” para concursar por las plazas docentes del nuevo modelo de escuela Normal (que las vinculaban a la educación superior mexicana), plazas que imponían nuevas tareas y compromisos. Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> 239 Profesorado principiante: entre montañas y veredas, aprendiendo a ser maestro en el sureste mexicano Los formadores de docentes de las normales dejaron (porque así se los impuso la nueva política y modelo de escuela determinado oﬁcialmente) de tener como eje de su atención el trabajo de preparación para formar técnicos para la enseñanza, empezaron a preocuparse por las demás actividades, que no conocían y que tenían que realizar para cumplir con las condiciones organizativas y laborales de la institución normalista como IES. Dentro de estas actividades, se encontraban las descargas académicas, el formador ya no se ocupaba solo de dar clases, ahora se le imponía cierto tiempo para la investigación o la difusión. La descarga académica fue un tiempo perdido, en tanto el formador no se encontraba preparado para desarrollarla, pero signiﬁcó al cabo del tiempo la posibilidad de realizar cualquier otra cosa, menos actividades que fortalecieran la vida académica de las normales y los procesos de formación de los estudiantes. Empezó a perderse la esencia formativa que preparaba para la docencia. Para los años recientes, la tendencia de las políticas neoliberales del gobierno mexicano ha sido la de constreñir el ﬁnanciamiento al normalismo mexicano, hay una orientación a reducir su presencia como institución formadora, algunas voces oﬁcialistas plantean su desaparición o dejarlas que vayan muriéndose por inanición. Aunado a lo anterior, y en la lógica de que la escuela Normal es una IES, los formadores que prestan servicios en dichas instituciones tienden a ser más universitarios y menos normalistas. Las prácticas y orientaciones se inscriben en la lógica universitaria, en detrimento de la tradicional concepción del normalismo mexicano, es decir, preparar para la enseñanza en las comunidades rurales mexicanas. Los anteriores elementos son nodales para entender la situación de los egresados 240 Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> de las normales, cuando se enfrentan al complejo mundo del trabajo y las primeras experiencias como profesores de la educación básica. Y es que la situación que se observa en la formación de los profesores, está caracterizada por la falta de preparación para entender el ámbito social y cultural de las comunidades en donde le tocará incursionar al egresar. Y bajo esta consideración, el primer elemento a comentar y reﬂexionar en este trabajo es la llegada al mundo laboral de los egresados de las normales. De la educación Normal a la inserción laboral La obtención de la plaza se inscribe en la lógica de presentar examen de oposición que, en los años recientes, se ha convertido en una condición para tener trabajo en el magisterio mexicano. Al examen asisten tanto egresados de las normales como de universidades, y los rezagados en la obtención de una plaza de docente. En la convocatoria y la presentación del examen, el número de aspirantes rebasa por mucho la oferta laboral. La expectativa y el primer momento de conﬂicto del recién egresado, profesor graduado y aspirante a trabajador contratado, es el de competir con una cantidad exorbitante de compañeros por el trabajo (una plaza de profesor que otorga el gobierno). La incertidumbre, después de cuatro años de ser alumno de una Normal, concluye en el momento de publicación de los resultados de quienes se irán a trabajar en cualquiera de las escuelas de educación básica de Chiapas. Los cuatro años de formación de la Normal trascurren en la tranquilidad de las aulas y las prácticas para aprender del hacer del profesor, prácticas que se desarrollan en lugares circunvecinos de la institución Iván Alexis Pinto Diaz que los forma (el plan de estudios establece que el último año de la licenciatura se desarrolle combinando trabajo docente por periodos prolongados en escuelas primarias y periodos cortos de estudio en la Normal); la experiencia estudiantil se circunscribe a estudiar y acreditar las materias. No hay duda que de una u otra manera existe exigencia en la formación, pero no se ofrece elementos para que el futuro profesor comprenda y se arraigue en la comunidad. Poco pasa por el pensamiento del estudiantado el lugar en donde les tocará iniciarse como profesores, en tanto que la institución que los forma soslaya una formación que los prepare en lo elemental de cómo asumir la tarea docente en un contexto que no sea el urbano, por lo que la inserción a la escuela y las comunidades de las apartadas zonas rurales se convierte en un asunto personal y que se deﬁnirá hasta que llegue el momento en que el profesor se enfrente a dicha realidad. El viaje a la comunidad: los primeros problemas Figura 1 - Jolja, Tila, México. Una comunidad para profesor principiante. Pasada la incertidumbre y teniendo la plaza asignada, todo egresado de normales es enviado a una comunidad rural, dependiendo el nivel y la modalidad, el lugar de asignación es deﬁnido por costumbres y prácticas construidas por los profesores que ya tienen experiencia y años de servicio, entre éstas se encuentran dar prioridad a las mujeres o por el número que le corresponde en la lista de quien llega primero a la supervisión escolar, hasta por el estado de ánimo de quien en ese momento le corresponde asignar los lugares;4 estas son de las primeras condiciones con las que tienen que lidiar los nuevos profesores, pero también se constituyen en elementos que preparan para que en el futuro sepan que tienen derechos y que hay un sindicato que los deﬁende, en virtud de que las condiciones de asignación de escuelas los dejan inconformes e inician una búsqueda de razones y argumentos para que no les vuelva a pasar y se les respete como trabajadores. Pero también marca el camino de la competencia y la de cultivar relaciones para mejorar el lugar de asignación. El anhelo, pero sobre todo la necesidad5 de tener trabajo seguro, hace que los noveles profesores (y por la velocidad en que corre el tiempo entre que pasa el examen, saben el resultado y tienen que empezar el complejo proceso administrativo para la aﬁliación) no comprendan la magnitud de lo que les 4 Una de las profesoras entrevistadas reﬁere que cuando se le otorgó la orden de comisión y se presentó ante el supervisor, éste le indicó que por esa ocasión la forma de asignar escuelas cambiaría. En ese momento se permitiría que fueran primero las mujeres las que escogieran a donde querían ir a trabajar. Otro profesor manifestó que cuando se le asignó la escuela, el supervisor lo mandó “a capricho” a la escuela más lejana, tal vez “por que le caí mal”. 5 Regularmente los egresados de normales son gente de clase media baja, que con penurias logran terminar la carrera profesional. Dichos egresados tienen una edad promedio que ﬂuctúa entre los 22 y 24 años. Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> 241 Profesorado principiante: entre montañas y veredas, aprendiendo a ser maestro en el sureste mexicano espera, y que no tiene que ver necesariamente para lo que fueron preparados, es decir, estar frente a grupo y con sus estudiantes (bajo esta aparente tranquilidad y neutralidad que expresa esta última idea) impartiendo clases.6 El lugar de asignación de la escuela representa el inicio de un aprendizaje central en la vida del novel profesor. Regularmente son escuelas enclavadas en lo inhóspito de la geografía chiapaneca y son planteles unitarios. Asignada la escuela y la comunidad, el maestro carga su mochila con ilusiones, esperanzas y sobre todo con ánimos por iniciarse; regularmente son muchas horas de viaje en camionetas o en caballo hasta llegar a su destino: la escuela. La experiencia se empieza a convertir en reveladora. Se inicia por la deﬁnición de dónde quedarse a vivir y qué comer, pasando por qué no tendrá las condiciones de su época de estudiante, hasta el conocer la escuela y los estudiantes. La comunidad se ofrece como un cúmulo de experiencias y aprendizajes que el profesor tiene que ir asumiendo. Regularmente no hay quien le diga qué es lo que tiene que hacer, cómo tiene que comportarse. Pero la cautela y los ánimos por granjearse la conﬁanza de la gente hace que el profesor se acomode a las disposiciones y relaciones que se establecen en las comunidades, se sabe que ante la situación compleja no queda más que aguantar, pues está de por medio la seguridad de la plaza laboral.7 ¿Qué y cómo enseñar?: adaptaciones y aprendizajes docentes Ser maestro novel en el contexto rural de Chiapas signiﬁca la conﬁguración de un hacer mediado por las circunstancias contextuales, escolares y las de orden personal. Al llegar a la comunidad el maestro tiene en mente que pronto estará en su escuela y frente a sus alumnos. Tiene ánimo de iniciar la tarea para la cual se formó y fue contratado. Se sabe de antemano que el salario llegará tarde (tres o cuatro meses después), al cabo de meses de trámite burocrático, por lo que los primeros meses de ir y venir de la comunidad con la mochila que lo acompaña se complementará con las lágrimas y la soledad. Después de llegado el primer pago, se tendrá un hueco en la mochila para colocar una pequeña despensa de víveres que haga más llevadera la estancia en la comunidad. Figura 2 - El aula del profesor principiante. Espacio de formación. 6 La cuestión a la que se alude es que el aula con estudiantes de comunidades rurales está permeada por una gran cantidad de cuestiones de orden cultural, económicos y sociales, que el profesor principiante tendrá que enfrentar. 7 Uno de los profesores entrevistados señaló lo siguiente: “los primeros días como profesor son de incertidumbre, angustia y tristeza. La soledad es terrible. No está uno preparado para lo que le toca a uno vivir en la primera comunidad. Pero no queda de otra más que aguantar, la necesidad es grande y sabe uno que no se puede perder la plaza”. 242 Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> Si es maestro unitario, tendrá que organizar todas las materias y grados. Si es otra modalidad (bidocente o tridocente), Iván Alexis Pinto Diaz al menos tendrá compañía, pero no así la posibilidad de descargar la tarea que tiene frente a él. Los maestros noveles se les castigan y “cargan la mano”, se les imponen tareas extras por parte de los profesores de más años. El profesor principiante se le da el grado escolar más difícil (según la lógica de los profesores con experiencia), no podrá salir con frecuencia de la comunidad y tendrá que organizar los eventos y festividades cívicas y culturales. De cualquier forma, el profesor principiante, solo o con otros compañeros en su primera escuela, enfrentará un cúmulo de adversidades, tareas que desarrollar que le son ajenas, sin orientación y apoyo para realizarlas. Ha llegado el momento de enseñar. Pero el ímpetu empieza a chocar con la realidad. Los alumnos de las comunidades presentan problemas serios en cuanto a su desarrollo escolar, pero sobre todo con graves carencias sociales y económicas. Falta de recursos y materiales en las escuelas, y en algunos casos, no se cuenta con una infraestructura que pueda llamar al espacio en donde conﬂuyen alumnos y maestros, como escuela. En el aula, el maestro se encuentra con que sus alumnos no le entienden y él no entiende a sus alumnos, el ímpetu y la organización (que prepara con esmero y dedicación, en tanto es su carta de presentación) chocan con el hecho de que sus alumnos hablan una lengua indígena. Llega la etapa de incertidumbre y marcado desconcierto. El qué hacer ante la realidad que se le presenta ronda por la cabeza del maestro, deja pasar las horas y los días hasta considerar opciones que le permitan iniciar su labor, este momento es de intensa soledad, se rememora aquellos tiempos de estudiante en donde tenía un maestro de la escuela Normal que le orientara o le indicara qué hacer, ahora se enfrenta a la realidad de falta de acompañamiento, incluso cuando existen compañeros de trabajo y éstos no sugieren ni apoyan, lo único que hacen es asumir una actitud de ﬁscalización. El maestro novel recorre un tramo de su primera incursión profesional entre la improvisación y el diseño de estrategias poco ortodoxas, pero que resultan efectivas. Una profesora entrevistada señalaba que lo común es “tomar al alumno que mejor “medio hable el español” como traductor”. Otro profesor indicaba que no se puede dejar de considerar que el contenido educativo es poco pertinente, en tanto los antecedentes de referencia de los alumnos no les permite entender lo que se les va a enseñar, por lo que los ajustes son permanentes. Y en lo que coinciden todos los profesores es que el grado de marginación y pobreza no les hace “rendir” eﬁcientemente, en tanto la alimentación es precaria, lo que hace que la situación y trabajo docente se complique aun más. En los primeros días y los primeros meses, el profesor principiante vive con carencias y problemas, pero al conocer la realidad de sus alumnos deja de preocuparse por su situación personal, para inmediatamente pasar a la reﬂexión de con qué tipo de población está mediando, y qué esa población requiere de su servicio y él no tiene los elementos, referentes y recursos para apoyarlos. Pero de alguna forma se enamorará (MARCELO GARCÍA, 2008, p. 12) y se comprometerá en la relación con sus alumnos, aunque el trabajo de contenidos no sea precisamente el deseado y el que marca el propósito normativo. Momento de catarsis. Las carencias y problemas que tienen que afrontar los maestros noveles se convierten en acicateadores de reﬂexión y acción ante la Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> 243 Profesorado principiante: entre montañas y veredas, aprendiendo a ser maestro en el sureste mexicano complejidad y profundidad que observan de sus alumnos y de la comunidad en su conjunto. Al momento de llegar a las comunidades y entrar en contacto con los estudiantes y la realidad que los permea, se inicia un proceso de formación y concientización de lo que debe hacer el maestro y también lo que no debe hacer: dejar al garete a los alumnos, ausentarse de la escuela sin justiﬁcación, no respetar las costumbres de las comunidades. Proceso de formación que se genera en la vida cotidiana de la presencia en la comunidad, se da a través de la necesidad de resolver las necesidades inmediatas, por salir al paso de las situaciones problemáticas y por llevar a cabo las acciones escolares que sean posibles. Figura 3 - Alumno de comunidad rural. Motivo para comprometerse en la labor educativa. cambios y se ubica en una mejor comunidad, el maestro novel pasa de aprender a vivir en la comunidad rural, a entender que su labor va más allá de lo que acontece en las cuatro paredes del aula y que es necesario emprender algunas acciones para intentar que por un momento olviden la complejidad y profundidad de los problemas que enfrentan los niños y jóvenes de dichas comunidades.8 Documentar y analizar la conﬁguración del quehacer de los maestros noveles: aprendizaje necesario para plantear rutas de ayuda y mejora. Los primeros años en la docencia son especiales en la enseñanza, pero también los son de aprendizaje para el profesor. El primer año, fundamentalmente, comporta elementos que deﬁnirán la actuación del profesor en su devenir profesional. Para el caso estudiado, los profesores logran una plaza laboral, incentivada por el anhelo de mejorar la situación personal y familiar de la cual provienen. Mientras llega la plaza, no se repara en las condiciones contextuales que permearán la labor docente. Es hasta el momento de obtener la orden de comisión, que el profesor ve una trasformación de su situación personal y de creciente incertidumbre por enfrentarse a realidades desconocidas y complejas. 8 Por lo tanto, es una formación que transcurre en el diario acontecer de los maestros noveles. Formación que da sentido de acción y pensamiento, ubica en su amplia magnitud la labor que deben emprender los maestros de comunidades rurales. En el transcurso del primer año de servicio y hasta el momento en que llega la cadena de 244 Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> Para una de las profesoras noveles de educación primaria, “la experiencia del primer año de trabajo fue formativa y reveladora. Pero además adquirió un sentido afectivo de gran envergadura; los niños y adolescentes de estas comunidades no se les puede abandonar a su suerte, requieren tanto apoyo, comprensión y cariño. Como que el estar con ellos, ver su sufrimiento, la pobreza y el olvido, mueven y sacuden la conciencia. Yo me quedé más de un año con ellos, me fue difícil cambiarme, yo sabía que lo podía hacer, pero me esperé un poco de tiempo”. Iván Alexis Pinto Diaz Entre la necesidad laboral y el obtener una plaza, como sea y en las condiciones que sean, se acepta iniciar un recorrido por veredas y montañas, entre la selva y las comunidades más apartadas de la vida moderna. El profesor principiante conoce del trabajo en la escuela, sabe de contenidos y programas, pero pronto se dará cuenta que en las comunidades rurales e indígenas eso no basta, es necesario tener conocimientos, habilidades y aptitudes para poder lidiar con el entramado de problemáticas y necesidades que las permea. Por lo anterior, no queda más que participar en lo posible (lo que le da su intuición), improvisando y aplicando una carga importante de dedicación a lo que se realice, aunque no corresponda necesariamente a propósitos educativos. Pasa largas horas viajando para llegar y salir de la comunidad, pues la comunidad representa un mundo lejano a sus interpretaciones y actuaciones y por lo tanto, le es necesario salir para poder tomar una bocanada de civilización. En los primeros días y meses, el profesor tiene que adaptarse a las condiciones del contexto, aunque la preocupación está latente porque sus alumnos lleguen a la escuela y aprendan, lo cual queda relegado ante el complejo sistema de procesos culturales y sociales que enfrenta. En la soledad del cuarto donde habita (que le asignan las autoridades de la comunidad), los profesores meditan la realidad que les toca vivir, pero cada día su pensamiento transita hacia la reﬂexión de las condiciones y los sujetos mismos, a los cuales debe atender en el aula. La pobreza y marginación de los alumnos, acompañadas de la precariedad en la alimentación y el vestido, la falta de experiencia de ser niño, en tanto están sometidos desde muy pequeños a situaciones que los hacen madurar muy rápidamente, pero también y fundamentalmente el respeto, aprecio y cariño que profesan padres de familia y alumnos hacia los profesores principiantes, hacen que aquilaten y se comprometan en la presencia temporal de su centro de trabajo. Las condiciones de vida de alumnos y sus familias penetran en el pensamiento del profesorado. Se genera una entrega comprometida por el trabajo áulico, la relación afectiva con el alumnado se profundiza. Pero el profesor carece de “herramientas” para ir más allá del trabajo de los contenidos y establecer un proyecto de intervención educativa que oriente y ayude en la mejora de las condiciones socioculturales de la gente de la comunidad donde trabaja. Aunque se asume comprometido, la tarea del profesor se inscribe en el cumplimiento del horario establecido oﬁcialmente, es decir, de lunes a viernes y por un periodo corto de tiempo, ya que el profesor sabe que toda la angustia terminará cuando concluya el ciclo escolar y se genere la cadena de cambios. Las actividades del profesor principiante que van más allá del aula, se inscriben en jugar un deporte con los niños de la escuela o los adultos de la comunidad o caminar con los alumnos por las veredas para apreciar un rio u observar la exuberante ﬂora y fauna (casi aun intocada) que circunda la comunidad. Al cabo de un año, el profesor está a punto de vivir nuevas experiencias, que ajustarán la práctica profesional que desarrolla. Ya con otra orden de comisión, la de su nuevo centro de trabajo y que es resultado de su solicitud para acercarse a comunidades urbanas con mayores y mejores servicios, y claro para poder alejarse Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> 245 Profesorado principiante: entre montañas y veredas, aprendiendo a ser maestro en el sureste mexicano de la comunidad en donde “ensayó” y conoció lo que signiﬁca ser maestro, y que es la de un maestro que padece inclemencias y carencias, pero sobre todo en donde no hay sanitario, televisión y relaciones con amigos y compañeros de profesión. Ahora tendrá mejores condiciones de vida, pues su antigüedad (un año de servicio) le permite ir escalando en los privilegios del gremio, entre ellos estar ubicado en “una buena escuela”, una comunidad al alcance de la comunicación y los servicios y en mejores condiciones de hospedaje y alimentación. La experiencia vivida en el primer año de servicio se aquilata, se menciona como inolvidable. Las carencias y vicisitudes calan hondo, deﬁnen el pensamiento y la acción, pero contradictoriamente o razonablemente por la forma en que fueron formados, la pretensión es dejar atrás la presencia y actuación como profesor en la primera comunidad y escuela de trabajo. En la nueva realidad y contexto de trabajo, el profesor aplica la experiencia obtenida, se siente comprometido con la labor, sabe de lo complejo de la realidad educativa de las comunidades apartadas, se comenta entre los colegas lo que se vivió y padeció, pero ha llegado el momento, a partir del cambio de adscripción, de iniciar un nuevo proceso de aprendizaje y adaptación. En la memoria queda solo el recuerdo del primer año de experiencia, “recuerdos que los emocionan y enorgullecen, pero que no siempre reconocen como pasos constitutivos de su identidad profesional” (ALEN; VALERIA, 2009: 12). La idea de entrega y preocupación por la tarea escolar no se borra del pensamiento, así como la situación social y económica de los estudiantes. Un maestro de educación primaria entrevistado señalaba de que “yo hubiera seguido en esa escuela, por mis estudiantes, porque me necesitaban”, y otro reﬁere “que las condiciones son 246 Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> difíciles, y no estaba preparado para esto”. En el fondo, la cuestión es ir escalando en las mejoras que el gremio otorga e ir participando de las reglas y prácticas escritas y no escritas que permitan mejorar la condición laboral. Quiero dejar en claro que el problema no radica en dejar la primera escuela, sino que el aprendizaje que se obtiene de la experiencia solo se ubica en el recuerdo y en que “jamás se vuelva a vivir una situación similar”. Otro de los elementos que padecen y carecen los profesores principiantes reﬁere a los conocimientos y habilidades que les permitan sistematizar y reﬂexionar lo vivido, por lo que dicha situación lleva a soslayar la vivencia compleja y articuladora de los procesos culturales, sociales y económicos que deﬁnen el trabajo en una comunidad rural apartada. No se constituye en un referente para delinear un trabajo docente que recupere la experiencia contextual e histórica. Es indudable que de esa experiencia se reproducirá un saber docente, que le permitirá desarrollar la labor en el aula con mayores referentes, tomando en cuenta el manejo de contenidos y la realidad de los estudiantes; es un saber docente basado en elementos vivenciales que le permitirán ubicar en su real dimensión lo que es la escuela, el profesor principiante regularmente señala “que aquí se adquiere lo que la Normal no enseña”. Ahora bien, el alejarse de las comunidades apartadas y ubicarse en pueblos comunicados y con servicios, conlleva asumir una posición de profesor entregado a la labor en la nueva escuela de adscripción, participe de comisiones y tareas. Llegó el momento de atender al grupo asignado y el horario establecido, no hay compromiso por la tarde, ni mucho menos contacto con la comunidad más allá del que se establezca por la atención de los alumnos. El profesor asume las reglas no escritas del comportamiento del Iván Alexis Pinto Diaz gremio, entre las que destacan la del respeto al horario (no quedarse en la escuela más allá de lo establecido oﬁcialmente), aceptar el “liderazgo” de los profesores con más años de servicio, asumir el acuerdo de ausentarse de la escuela para realizar el cobro de la quincena u otros trámites. Estas prácticas (entre otras más) se traducirán al poco tiempo en una forma de intervenir y participar en la tarea escolar. No hacerlo implicará trasgredir el proceso de buen funcionamiento de la escuela y su grupo de profesores. El profesor se concentrará en el trabajo delimitado por las cuatro paredes de su aula, más allá de esos límites son terrenos no propicios para mantener su permanencia y aceptación en el grupo. Es en este contexto que se hace necesario precisar la importancia de investigar y reﬂexionar la realidad de los profesores principiantes. La cuestión implica considerar que es una etapa que puede deﬁnir la ruta de acción del profesional que se inicia en la docencia. El problema es complejo y demanda una revisión de los procesos formativos que se gestan en las normales, sobre todo el hecho de dotar al estudiante de los elementos que le permitan desenvolverse en cualquier contexto de trabajo. También demanda el establecer la importancia y el valor de los primeros años de servicio. Deﬁnir mecanismos de acompañamiento y apoyo. La idea de formación continua y permanente sería base sustantiva en la construcción de la mejora de los procesos de intervención educativa que llevan a cabo los profesores de educación básica en México. Es necesario deﬁnir con claridad roles y perspectivas metodológicas y de interacción educativa, no se puede soslayar la importancia de considerar que para enseñar, es necesario que el profesional posea un cúmulo de conocimientos, habilidades y cualidades humanas que permitan atender y entender a los sujetos con los cuales realizará su labor, pero además para poderse desenvolver en situaciones complejas dentro y fuera del espacio institucional educativo. Marcelo García (1999) señala la importancia de que el profesor principiante tenga información de la cultura, costumbres, idioma y otros factores acerca de la comunidad, para comprender, sobrevivir y ayudar al desarrollo de la comunidad Siguiendo a dicho autor, los profesores principiantes debe ubicárseles y ubicarse en la perspectiva de un aprendizaje permanente, continuo, interactivo, acumulativo, mediante un proceso que requiere de elementos multidimensionales y dinámicos, para lograr una dimensión continua, amplia y reﬂexiva; proceso que lleva al mejoramiento de la práctica docente para lograr la profesionalización de ésta y de la permanencia con gusto y agrado. La cuestión implica pasar de una formación del profesor de ensayo y error a una en que esté mediando un programa de inducción auspiciado por el Estado y por los actores involucrados en el proceso educativo de los diversos niveles, donde el profesor se le brinde la facilidad de conocer el contexto al que se enfrentará en su práctica profesional y que le permita la interiorización de normas, valores, conductas, actitudes, habilidades para la puesta en marcha de estrategias de enseñanza que sean funcionales en el aprendizaje de los estudiantes a los que atiende. Comentario ﬁnal La experiencia del profesor principiante en zonas rurales del estado de Chiapas, se encuentra permeada por Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> 247 Profesorado principiante: entre montañas y veredas, aprendiendo a ser maestro en el sureste mexicano la complejidad de la situación social, económica y cultural en que se encuentra sumida la región. Dichos profesores tienen poca capacidad de maniobra en virtud de los nulos referentes que tienen, como resultado de la formación normalista por la que transitan y que genera una permanente improvisación para sacar adelante el trabajo y que, al ﬁnal de cuentas, van deﬁniendo acciones y procesos de intervención que les permiten incidir en el aula. La comunidad, el trabajo en ella y la interrelación que se pueda establecer se dejan en un segundo término, en virtud de que no se tiene referentes para intervenir y mucho menos para orientar el desarrollo comunitario. Es indudable que la experiencia del primer año de ejercicio profesional es formativa, deﬁne conocimientos y habilidades que habrán de marcar lo subsecuente de la labor del profesor. Pero las posibilidades de que ese aprendizaje se constituya en eje rector de una formación permanente se ve reducida, en tanto el profesor principiante no tiene los elementos y conocimientos para sistematizarlo, es un aprendizaje por la relación directa, que se asienta en el recuerdo y la memoria, pero que no se convierte en material y pre-texto para la discusión y la reﬂexión del quehacer docente. La experiencia del primer año, lo que provoca, por la complejidad y diﬁcultad que representa, es el deseo de jamás volverla a vivir. Y es que el profesor principiante al no saber y conocer lo que representa una comunidad rural, el proceso de adaptación e integración se hace difícil, se siente ajeno a dicha realidad, y al ﬁnal de cuentas la experiencia se piensa como elemento que inaugura la práctica de ser profesor, pero dejando de ser “principiante”, al cabo de “sobrevivir” el primer ciclo y teniendo antigüedad en el servicio (al menos un año) 248 Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> ya se tiene derechos y es lo que permite ir escalando en los privilegios, entre ellos ir a una mejor comunidad y escuela. La cuestión demanda reﬂexionar qué rutas se pueden tomar para aprovechar la experiencia del profesor principiante, es claro que la educación Normal tendría que hacer algo, abrir espacios de formación que permitan conocer el ambiente y realidad de las comunidades rurales. Como también generar un espacio y participación de acompañamiento de profesores noveles, no dejarlos a lo que puedan hacer, desamparados a su suerte. El maestro recién iniciado en las tareas docentes, regularmente busca cursos y programas de actualización y de formación continua. Como en la región no hay instituciones que le ofrezcan algo relacionado con su actividad, “tiene que ir a caer” a programas académicos que le sean posible ﬁnanciar y participar (por que la mayoría son en instituciones particulares), regularmente poco tiene que ver con sus necesidades profesionales y solo cubren cuestiones formales. Por lo tanto, necesario es abrir programas de posgrado que recuperen los requerimientos de formación y actualización que ofrezcan opciones de especialización y profundización en torno a la práctica docente, en sus diversas vertientes. Bajo esta lógica, no solo se estaría atendiendo la mejora de la práctica educativa, sino que también la posibilidad del arraigo de los profesores en sus comunidades de trabajo. El panorama es complejo, pero las posibilidades de mejora son reales. Necesario es revisar la problemática para ir buscando rutas que permitan generar la discusión y abrir los espacios para plantear alternativas. Iván Alexis Pinto Diaz Referencias ALEN, B.; VALERIA, S. (Coords.). Iniciarse como docente en escuelas rurales. Buenos Aires: Ministerio de Educación, 2009. BOLIVAR, A. (Coord.). La investigación biográﬁca-narrativa en educación: enfoque y metodología. Madrid: La muralla, 2001. MARCELO GARCÍA, C. Estudio sobre estrategias de inserción profesional en Europa. Revista Iberoamericana en Educación, n. 19, p. 1-44. 1999. MARCELO GARCÍA, C. M. (Coord.). El profesorado principiante. Barcelona: Octaedro, 2008. SANDOVAL, E. F. (Coord.). Proyecto de investigación: los primeros años de ejercicio docente en educación básica: reconstrucción de prácticas y experiencias en contextos desfavorecidos. México: SEP/ SEB. CONACyT. 146031, 2011. TARRÉS, M. L. (Coord.). Observar, escuchar y comprender sobre la tradición cualitativa en la investigación social. México: El Colegio de México, FLACSO, Miguel Angel Porrúa, 2004. Enviado em: 15. Jul. 2012 Aceito em: 22. Nov. 2012 Olhar de professor, Ponta Grossa, 15(2): 237-249, 2012. Disponível em <http://www.uepg.br/olhardeprofessor> 249
https://openalex.org/W2590675931	https://europepmc.org/articles/pmc5361680?pdf=render	Latin	null	Genetic polymorphisms (rs10636 and rs28366003) in metallothionein 2A increase breast cancer risk in Chinese Han population	Aging	2,017	cc-by	4,898	AGING 2017, Vol. 9, No. 2 www.aging‐us.com AGING 2017, Vol. 9, No. 2 Research Paper Research Paper Genetic polymorphisms (rs10636 and rs28366003) in metallothionein 2A increase breast cancer risk in Chinese Han population Di Liu1,, Meng Wang1,, Tian Tian1,, Xi‐Jing Wang1, Hua‐Feng Kang1, Tian‐Bo Jin2, Shu‐Qun Zhang1, Hai‐Tao Guan1, Peng‐Tao Yang1, Kang Liu1, Xing‐Han Liu1, Peng Xu1, Yi Zheng1, Zhi‐Jun Dai1 1Department of Oncology, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, China 2National Engineering Research Center for Miniaturized Detection Systems, School of Life Sciences, Northwest University, Xi’an 710069, China Equal contribution Correspondence to: Zhi‐Jun Dai; email: dzj0911@126.com Keywords: metallothionein 2A; polymorphism; breast cancer Received: November 14, 2016 Accepted: February 9, 2017 Published: February 22, 2017 ABSTRACT Genetic polymorphisms of MT2A are frequently observed in many different cancers. We performed this case‐ control study, including 459 breast cancer (BC) patients and 549 healthy controls from Northwest China, to evaluate the associations between two common MT2A polymorphisms (rs10636 and rs28366003) and BC risk. The MT2A polymorphisms were genotyped via Sequenom MassARRAY. The individuals with the rs28366003 A/G, A/G‐G/G genotypes underwent a higher risk of BC (P＜0.0001). And, the minor allele G of rs28366003 was related to an increased BC risk (P＜0.0001). We also found a significantly increased BC risk with rs10636 polymorphism among homozygote and recessive models (P＜0.05). Further subgroup analysis by clinical characteristics of BC patients showed that Scarff, Bloom and Richardson tumor grade (SBR) 1‐2 have a higher expression of the minor allele of these two MT2A loci than SBR 3. Our results indicated that the rs10636 and rs28366003 polymorphisms in MT2A increased BC risk in Northwest Chinese Han population. RESULTS rs1610216, and rs10636) and the risk of prostate cancer. And only the rs28366003 SNP in MT2A was observed to be correlated with the prostate cancer risk in Polish population [19]. Similarly, Krzeslak’s data suggested that the rs28366003 polymorphism in MT2A was related to the BC risk in a Polish population [13]. Although several poly-morphisms have been identified in MT2A, the function of its polymorphisms in BC not being fully understood [20]. In this study, we investigated and comprehensive-ly assessed the association between two SNPs of MT2A (rs10636 and rs28366003) and BC risk in Chinese Han population. INTRODUCTION several crucial mechanisms including modulating p53 zinc-dependent activity, inhibiting NF-κB signaling, and regulating the PIK3/AKT and Rb/E2F pathways [15-17]. The functional isoforms of MT2A mRNA transcript have been reported to express at the highest levels in breast tissues and is positively related to the cell proliferation and histological grade of BC [18]. several crucial mechanisms including modulating p53 zinc-dependent activity, inhibiting NF-κB signaling, and regulating the PIK3/AKT and Rb/E2F pathways [15-17]. The functional isoforms of MT2A mRNA transcript have been reported to express at the highest levels in breast tissues and is positively related to the cell proliferation and histological grade of BC [18]. Breast cancer (BC) is the most frequent cancer among women, expecting to be 252,710 new BC cases and 40,610 death cases among American women in 2017 [1]. And, the incidence of BC increased dramatically in Asian population in recent years [2]. It is widely known that genetic factors contribute to BC susceptibility [3-7]. Recent studies showed the expression of the Metallothionein 2A (MT2A) gene increased in some human neoplasms [8-10]. And, it has been reported that the high expression of MT2A associated with the stage and prognosis of tumors [9, 11, 12]. MT2 belongs to metallothioneins (MT), which have four groups (MT1, MT2, MT3 and MT4 proteins) and are encoded by a family of genes located on chromosome 16q13 [13]. The expression of MT1 and MT2 are the most active isoforms in human cells [14]. And, the researchers found that MT1/MT2 may play an important role in tumors via Rs28366003 and rs10636 polymorphisms are the most common loci of MT2A, having been found to be associated with many different cancers. Rs28366003 locates in the core promoter region, while rs10636 polymorphism is in the 3’UTR region of the MT2A gene [17]. A recent study found that the single nucleotide polymorphism (SNP) rs28366003 stood apart from rs1610216 and rs10636 is significantly related to the laryngeal cancer risk in a Polish population [12]. Forma studied the association between MT2A (rs28366003, AGING (Albany NY) 547 www.aging‐us.com ER: Estrogen receptor; PR: Progesterone receptor; Her‐2: human epidermal growth factor receptor 2; SBR: Scarff, Bloom and Richardson tumor grade 48 AGING (Albany NY) Characteristics of the study population No significant difference was observed in the distribution of age, menopausal status, body mass index, and procreative times between the cases and controls (P ＞0.05), which suggested that the cases and controls of this study were matched adequately on general characteristics (Table 1). The genotypic frequencies of the MT2A rs10636 and rs28366003 polymorphisms among the controls were in accord with HWE (P = 0.343 and P = 0.363, respectively). Table 1. The characteristics of breast cancer cases and cancer‐free controls. Characteristics Cases Control P value Number 459 549 Age (mean ± SD) 49.09±11.02 48.80±8.28 0.61 Age of menarche (mean ± SD) 14.37±1.57 Menopausal status Premenopausal 237 267 0.376 Postmenopausal 222 282 Body mass index (kg/m2) (mean ± SD) 23.06±2.92 22.45±2.53 0.274 Procreative times <2 242 298 0.657 ≥2 217 251 Tumor size <2 cm 152 ≥2 cm 307 Lymph node involvement Negative 184 Positive 275 Histological grade SBR 1-2 244 SBR 3 215 Venous invasion None–little 292 Moderate–severe 167 Immunohistochemistry results ER – 202 + 257 PR – 208 + 251 Her-2 – 330 + 129 Ki67 ≥ 14% 294 < 14% 165 ER: Estrogen receptor; PR: Progesterone receptor; Her‐2: human epidermal growth factor receptor 2; SBR: Scarff, Bloom and Richardson tumor grade Table 1. The characteristics of breast cancer cases and cancer‐free controls. AGING (Albany NY) 548 www.aging‐us.com www.aging‐us.com Table 2. Association between MT2A polymorphisms (rs10636 and rs28366003) and breast cancer risk. Characteristics of the study population Model Genotype Case(459) Control(549) OR (95% CI) P-value rs10636 HWE=0.343 Codominant G/G 241 (52.5%) 290 (52.8%) 1.00 G/C 164 (35.7%) 224 (40.8%) 0.88 (0.68-1.15) 0.35 C/C 54 (11.8%) 35 (6.4%) 1.86 (1.17-2.94) 0.008 Dominant G/G 241 (52.5%) 290 (52.8%) 1.00 G/C-C/C 218 (47.5%) 259 (47.2%) 1.01 (0.79-1.30) 0.92 Recessive G/G-G/C 405 (88.2%) 514 (93.6%) 1.00 C/C 54 (11.8%) 35 (6.4%) 1.96 (1.26-3.05) 0.003 Overdominant G/G-C/C 295 (64.3%) 325 (59.2%) 1.00 G/C 164 (35.7%) 224 (40.8%) 0.81 (0.62-1.04) 0.10 Allele G 646 (%) 804 (%) 1.00 C 272 (%) 294 (%) 1.15 (0.95-1.40) 0.16 rs28366003 HWE=0.363 Codominant A/A 378 (%) 508 (%) 1.00 A/G 70 (%) 41 (%) 2.29 (1.53-3.45) <0.0001 G/G 11 (%) 0 (%) - - Dominant A/A 378 (%) 508 (%) 1.00 A/G-G/G 81 (%) 41 (%) 2.66 (1.78-3.96) <0.0001 Recessive A/A-A/G 447 (%) 547 (%) 1.00 G/G 11 (%) 0 (%) - - Overdominant A/A-G/G 390 (%) 510 (%) 1.00 A/G 70 (%) 41 (%) 2.23(1.48-3.35) 0.0001 Allele A 826 (%) 1057 (%) 1.00 G 92 (%) 41 (%) 2.87 (1.97-4.20) <0.0001 Table 2. Association between MT2A polymorphisms (rs10636 and rs28366003) and breast cancer risk. ble 2. Association between MT2A polymorphisms (rs10636 and rs28366003) and breast i k MT2A polymorphisms and the breast cancer risk In terms of genotype and allele distributions of MT2A polymorphisms, two polymorphisms in MT2A (rs10636 and rs28366003) showed positive associations with BC risk (Table 2). Rs10636 was identified to increase the BC risk in codominant and recessive models (C/C vs. G/G: OR = 1.86, 95% CI: 1.17- 2.94, P = 0.008; C/C vs. G/G+G/C: OR = 1.96, 95% CI: 1.26- 3.05, P = 0.003). In addition, the rs28366003 polymorphism showed an association with BC risk in codominant, dominant, recessive and overdominant models ( A/G vs. A/A, OR = 2.29, 95%CI = 1.53-3.45, P ＜ 0.0001; A/G+G/G vs. A/A, OR = 2.66, 95% CI = 1.78-3.96, P ＜ 0.0001; A/G vs. A/A+G/G, OR = 2.23, 95% CI = 1.48-3.35, P ＜ 0.0001). And, the minor allele G of rs28366993 was also related to increasing BC risk (G vs. A, OR = 2.87, 95% CI = 1.97-4.2, P ＜ 0.0001). We also analyzed the relationships between MT2A SNPs and clinical features of BC, including tumor size, lymph node metastasis, ER/PR/HER-2 status, his- tological grade, and venous invasion. As shown in Table 3, when the G/G genotype was used as a reference, there was no significant relation between the rs10636 polymorphism and clinical parameters except the histological grade (C/C vs. G/G: OR = 0.46, 95% CI = 0.29- 0.74, P = 0.001; G/C+C/C vs. G/G: OR = 0.63, 95% CI = 0.42- 0.93, P = 0.02). The same result was found in the analysis of the rs28366003 polymorphism and the histological grade of BC (G/G vs. A/A, OR = 0.52, 95% CI = 0.33- 0.82, P = 0.005, shown in Table 4). AGING (Albany NY) 549 www.aging‐us.com Table 3. The associations between the MT‐2A rs10636 polymorphism and clinical characteristics of BC patients. MT2A polymorphisms and the breast cancer risk Variables GG(%) GC(%) p OR(95%CI) CC(%) p OR(95%CI) GC+CC (%) p OR(95%CI) Tumor size <2CM 57(37.5) 54(35.5) 41(27) 95(62.5) ≥2CM 98(31.9) 104(33.9) 0.63 1.12(0.71- 1.78) 105(34.2) 0.11 1.49(0.92- 2.42) 209(68.1) 0.23 1.2(0.85-1.92) LN metastasis Negative 64(34.8) 65(35.3) 55(30) 120(65.2) Positive 84(30.5) 103(37.5) 0.41 1.2(0.77- 1.89) 88(32) 0.41 1.22(0.76- 1.95) 191(69.5) 0.34 1.2(0.82-1.8 ER Negative 77(38.1) 63(31.2) 62(30.7) 125(61.9) Positive 79(30.4) 91(35.4) 0.13 5 1.41(0.9- 2.21) 87(33.9) 0.174 1.3(0.87- 2.15) 178(69.3) 0.1 1.3(0.94-2.05) PR Negative 90(43.3) 96(46.2) 22(10.5) 118(56.7) Positive 111(44.2) 106(42.2) 0.58 0.90(0.61- 1.33) 34(13.5) 0.46 1.25(0.69- 2.29) 140(55.8) 0.84 0.96(0.66-1.39) Her-2 Negative 100(30.3) 112(33.9) 118(35.8) 230(69.7) Positive 47(36.4) 44(34.1) 0.48 0.84(0.51- 1.37) 38(29.5) 0.14 0.69(0.41- 1.13) 82(63.6) 0.21 0.76(0.49-1.16) Ki-67 ﹤50% 80(27.2) 85(28.9) 129(43.9) 214(72.8) ≥50% 42(25.5) 47(28.5) 0.84 1.05（0.63- 1.77） 76(46.1) 0.63 1.12（0.7- 1.79） 123(74.5) 0.68 1.1（0.71-1.69） Histological grade SBR 1-2 65(26.6) 85(34.8) 94(38.5) 179(73.4) SBR 3 79(36.7) 83(38.6) 0.34 0.8（0.51- 1.26） 53(24.6) 0.001 0.46（0.29- 0.74） 136(63.3) 0.02 0.63（0.42-0.93） Venous Invasion Non-little 34(11.6) 64(21.9) 194(66.4) 258(88.4) Moderate-severe 24(14.4) 65(38.9) 0.25 1.44（0.77- 2.69） 78(46.7) 0.06 0.57（0.32- 1.02） 143(85.6) 0.4 0.79（0.45-1.38） LN: Axillary lymph node; ER: Estrogen receptor; PR: Progesterone receptor; Her‐2: human epidermal growth factor receptor 2; SBR: Scarff, Bloom and Richardson tumor grade Table 3. The associations between the MT‐2A rs10636 polymorphism and clinical characteristics of BC patients. ns between the MT‐2A rs10636 polymorphism and clinical characteristics of BC patients Table 3. The associations between the MT‐2A rs10636 polymorphism and clinical cha Similarly, no significant correlation was detected between the rs28366003 polymorphism and other clinical features. signal [27, 28], in which MT could suppress free- radial- induced oxidative damage of tissues and cells. The dysfunction of MT2A might be linked with the zinc release blockage and the reduction of intracellular zinc concentrations, which resulted in an increased risk of oxidative damage, as well as abnormal breast cells genesis [25]. As few studies concern to the relationship between the polymorphisms of MT2A and BC, we performed this case-control study in a Chinese population. Krzeslak, et al. have reported an association between rs28366003 polymorphism and the risk of ductal BC, prostate cancer, and laryngeal cancer in a Polish population [12, 13, 19]. We observed significant differences in genotypes distribution and allele DISCUSSION It was reported that MT acts as a regulator in cell proliferation, apoptosis, and differentiation, which imply that MT may involve in carcinogenesis of BC [9, 11, 21-24]. The biological effects of MT are connected with its physiochemical properties [25, 26]. Under the stress situation, MT regulates cell apoptosis, inhibits cell death and improves cell survival. Recent studies suggested the regulation of MT were completed by preventing oxidative progress and binding with apoptotic AGING (Albany NY) 550 www.aging‐us.com frequencies of two MT2A gene polymorphisms locus (rs10636 and rs28366003) between BC patients and control groups (P < 0.05). The MT2A SNP rs28366003 is an A/G substitution that is situated in the core promoter region of the MT2A gene sequence between the TATA box and transcription initiation site [13]. The transition may reduce the binding to the core promoter region, which is a nuclear molecule regulates MT2A gene transcription [29]. In this way, MT2A SNPs might be associated with functional changes, which imply that they may be involved in the interactions with the behavior of BC cell. Finally, the biological features of BC cells are gradually influenced. In addition, MT2A rs10636 polymorphism is located in the 3’ untranslated region, which implies that it may be involved in interactions with other nucleotide polymorphisms [29]. Our results suggest that both of the two MT2A SNPs rs10636 and rs28366003 significantly influence the susceptibility of BC. frequencies of two MT2A gene polymorphisms locus (rs10636 and rs28366003) between BC patients and control groups (P < 0.05). The MT2A SNP rs28366003 is an A/G substitution that is situated in the core promoter region of the MT2A gene sequence between the TATA box and transcription initiation site [13]. The transition may reduce the binding to the core promoter region, which is a nuclear molecule regulates MT2A gene transcription [29]. In this way, MT2A SNPs might be associated with functional changes, which imply that they may be involved in the interactions with the behavior of BC cell. Finally, the biological features of BC cells are gradually influenced. In addition, MT2A rs10636 polymorphism is located in the 3’ untranslated region, which implies that it may be involved in interactions with other nucleotide polymorphisms [29]. Our results suggest that both of the two MT2A SNPs rs10636 and rs28366003 significantly influence the susceptibility of BC. As indicated by Ki-67 immunohistochemistry, MT2A is related to the proliferation of BC[25]. DISCUSSION And, the down- regulation of MT2A arrests growth in MCF-7 cell lines also suggested the involvement of MT2A in the proliferation of BC [29, 30]. Moreover, it has been demonstrated that MT2A regulates endothelial cell migration through transcriptional regulation of the expression of vascular endothelial growth factor- c(VEGF-c) [31]. And it is believed that MT2A affects the histological differentiation grade in BC. The expression of MT2A mRNA in histological grade 3 tumors were higher than grade 1 and 2 tumors [20]. But in our study, BC patients with SBR 1-2 have a higher expression of the minor allele of MT2A SNPs loci than patients with SBR 3. So, we assume MT2A poly-morphisms may mitigate the aggressive behavior of BC cell. However, according to our results, there were no associations between ER/PR/HER-2 status, lymph node metastasis and MT2A polymorphisms (rs10636 and rs28366003). Table 4. The associations between the MT‐2A rs28366003 polymorphism and clinical characteristics of BC patients. Variable GG(%) GC(%) p OR(95% CI) CC(%) p OR(95%CI) GC+CC (%) p OR(95%CI) Tumor size <2CM 57(37.5) 54(35.5) 41(27) 95(62.5) ≥2CM 98(31.9) 104(33.9) 0.63 1.12(0.7 1-1.78) 105(34.2) 0.11 1.49(0.92- 2.42) 209(68.1) 0.23 1.14(0.74-1.75) LN metastasis Negative 45(24.5) 56(30.4) 83(45.1) 139(75.5) Positive 67(24.4) 78(28.4) 0.8 0.94(0.5 6-1.56) 130(47.3) 0.83 1.05（0.66- 1.68） 208(75.6) 0.98 1.01(0.65-1.55) ER Negative 70(34.7) 65(32.2) 67(33.2) 132(65.3) Positive 69(26.8) 88(34.2) 0.18 1.37(0.8 7-2.18) 100(39) 0.07 1.51（0.96- 2.39） 188(73.2) 0.07 1.44(0.97-2.16) PR Negative 55(26.4) 67(32.2) 86(41.3) 153(73.6) Positive 77(30.7) 81(32.2) 0.54 0.86(0.5 4-1.39） 93(37.1) 0.26 0.77(0.49- 1.22) 174(69.3) 0.32 0.81 (0.54-1.22) Her-2 Negative 71(21.5) 82(24.8) 177(53.6) 259(78.5) Positive 35(27.1) 33(25.6) 0.49 0.82(0.4 6-1.45) 61(47.3) 0.16 0.7(0.43- 1.15) 94(72.9) 0.2 0.74(0.46-1.18) Ki-67 <14% 44(145) 105(35.7) 145(49.3) 250(85) ≥14% 27(16.3) 64(38.8) 0.98 0.99(0.5 6-1.76) 74(44.8) 0.52 0.83(0.48- 1.45) 138(83.6) 0.69 0.9(0.53-1.52) Table 4. The associations between the MT‐2A rs28366003 polymorphism and clinical characteristics of BC patients. etween the MT‐2A rs28366003 polymorphism and clinical characteristics of BC patients le 4. The associations between the MT‐2A rs28366003 polymorphism and clinical charac AGING (Albany NY) 551 www.aging‐us.com Subjects The consent of all the participants was obtained after they were informed that their blood samples would be used for research purpose. The blood samples of 459 Chinese women with sporadic BC (mean age: 49.09 ± 11.02 years) were selected from the Second Affiliated Hospital of Xi’an Jiaotong University, Shaanxi Province, China. Table 5. Primers used for this study. SNP_ID 1st-PCRP 2nd-PCRP UEP_SEQ rs10636 ACGTTGGATGAGAACG CGACTTCCACAAAC ACGTTGGATGCATAGAAAAAGG AATATAGC GACGGAATATAGCAAA CGGTCA rs28366003 GCCGCCTTACATCGCG GTTCAGGGAACTG GAAGCAACCGCCCTTGGAGGAG GCGTGGT AACTCAGGTCAACTGG ATGCA Table 5. Primers used for this study. SNP_ID 1st-PCRP 2nd-PCRP UEP_SEQ rs10636 ACGTTGGATGAGAACG CGACTTCCACAAAC ACGTTGGATGCATAGAAAAAGG AATATAGC GACGGAATATAGCAAA CGGTCA rs28366003 GCCGCCTTACATCGCG GTTCAGGGAACTG GAAGCAACCGCCCTTGGAGGAG GCGTGGT AACTCAGGTCAACTGG ATGCA Table 5. Primers used for this study. Subjects In terms of the current study limitations, the sample size was inadequate for a stratified analysis and analysis of mix-type BC. Besides, we did not investigate other predisposing factors, including high-dose radiation exposure, alcohol consumption, and postmenopausal obesity. The further study should assess these factors as well for a more precise evaluation. The consent of all the participants was obtained after they were informed that their blood samples would be used for research purpose. The blood samples of 459 Chinese women with sporadic BC (mean age: 49.09 ± 11.02 years) were selected from the Second Affiliated Hospital of Xi’an Jiaotong University, Shaanxi Province, China. In addition, 549 age- and sex- matched healthy individuals (mean age: 48.80 ± 8.28 years) without any history of autoimmune or malignant diseases formed the control group. All of the BC cases and controls were Han population and from Northwest China. All the patients were diagnosed by histology or pathology, as described in our previous studies [32]. The data of clinicopatho- logical characteristics of patients, including tumor size, clinical stages, lymph node metastasis, menopausal status, procreative times, estrogen receptor (ER) status, progesterone receptor (PR) status, and human epidermal growth factor receptor type2 (HER-2) status, were obtained from the patients' medical records (Table 1). In conclusion, this study showed that MT2A polymorphisms rs10636 and rs28366003 are associated with BC risk in Chinese Han population. And, the relationship of MT2A polymorphisms and the histological grade may guide us to judge prognosis of BC. Further functional studies and large population- based prospective studies are needed to provide accurate evidence about the influence of MT2A variants on BC. DISCUSSION In addition, 549 age- and sex- matched healthy individuals (mean age: 48.80 ± 8.28 years) without any history of autoimmune or malignant diseases formed the control group. All of the BC cases and controls were Han population and from Northwest China. All the patients were diagnosed by histology or Histological grade SBR 1-2 49(20) 34(13.9) 161(66) 195(79.9) SBR 3 57(26.5) 61(28.4) 0.13 1.54(0.8 8-2.72) 97(45.1) 0.005 0.52(0.33- 0.82) 158(73.5) 0.1 0.79(0.45- 1.08) Venous Invasion None-little 94(32.2) 102(34.9) 96(32.9) 198(67.8) Moderate- severe 51(30.5) 57(34.1) 0.9 1.03(0.6 4-1.65) 59(35.3) 0.6 1.1(0.71- 1.81) 116(69.5) 0.71 1.08(0.72-1.63) LN: Axillary lymph node; ER: Estrogen receptor; PR: Progesterone receptor; Her‐2: human epidermal growth factor receptor 2; SBR: Scarff, Bloom and Richardson tumor grade DNA extraction and genotyping This study was approved by the Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong university (Xi’an, China). The research protocol was completed according to the approved guidelines. The samples were contained in tubes coating with ethylene-diaminetetraacetic acid and were stored at AGING (Albany NY) 552 www.aging‐us.com −80Ԩ after centrifugated until analysis. Genomic DNA was extracted from whole blood using the Universal Genomic DNA Extraction Kit Ver. 3.0 (TaKaRa Bio Inc., Shiga, Japan). DNA concentration was measured by spectrophotometry (DU530 UV/VIS spectro-photometer, Beckman Instruments, Fullerton, CA, USA). Two tag- SNPs (rs10636 and rs28366003) were selected in this study. A multiplexed SNP MassEXTEND assay was designed by Sequenom MassARRAY Assay Design 3.0 Software (Agena Bioscience, Inc., San Diego, CA, USA). SNP genotyping was performed by the Sequenom MassARRAY RS1000, and the primers were listed in Table 5. The data analyses were accomplished by Sequenom Type 4.0 [33]. and Tian-Bo Jin analyzed and interpreted the data. Di Liu, Meng Wang wrote the paper. Xi-Jing Wang, Hua- Feng Kang, Shu-Qun Zhang, Hai-Tao Guan, contributed materials/analysis tools. Zhi-Jun Dai supervised the entire study. Statistical analysis All the statistical analyses were completed using the SPSS software package (version 20.0; SPSS Inc., Chicago, IL, USA). Hardy-Weinberg equilibrium (HWE) was examined by comparing expected and observed frequencies using Alrlquin 3.1 program (L. Excoffier, CMPG, University of Bern, Switzerland). The genotype frequencies of observed values were compared with expected values obtained from HWE theory (p2+2pq+q2=1; p is the frequency of the wild- type allele and q is the frequency of the variant allele). The calculation was performed by χ2 test and the degree of freedom was 1 in the cases and controls. The significant difference in allele and genotype frequencies between cases and controls was determined by Pearson's χ2 test [34, 35]. And the cancer risk linked with alleles and genotypes was calculated with an odds ratio (OR) and 95% confidence interval (CI). We evaluated the risk in the codominant model (Aa vs. AA and aa vs. AA), dominant model (AA+ Aa vs. aa), recessive model (aa vs. Aa+AA), overdominant model (AA+ aa vs. Aa) and the allele model (a vs. A) respectively (A: the major allele, a: the minor allele). A two-sided P-value < 0.05 was considered statistically significant in all the tests. REFERENCES 1. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin. 2017; 67:7–30. doi: 10.3322/caac.21387 1. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin. 2017; 67:7–30. doi: 10.3322/caac.21387 2. Ghoncheh M, Momenimovahed Z, Salehiniya H. Epidemiology, Incidence and Mortality of Breast Cancer in Asia. Asian Pac J Cancer Prev. 2016; 17:47– 52. 3. Easton DF, Pooley KA, Dunning AM, Pharoah PD, Thompson D, Ballinger DG, Struewing JP, Morrison J, Field H, Luben R, Wareham N, Ahmed S, Healey CS, et al, and SEARCH collaborators, and kConFab, and AOCS Management Group. 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https://openalex.org/W4220749071	https://www.researchsquare.com/article/rs-745223/latest.pdf	English	null	Early detection of plant virus infection using multispectral imaging and spatial–spectral machine learning	Scientific reports	2,022	cc-by	10,358	Version of Record: A version of this preprint was published at Scienti¦c Reports on February 24th, 2022. See the published version at https://doi.org/10.1038/s41598-022-06372-8. Early Detection of Plant Virus Infection Using Multispectral Imaging and Spatial-Spectral Machine Learning Yao Peng University of Manchester Mary Dallas North Carolina State University José T. Ascencio-Ibáñez North Carolina State University Steen Hoyer Rutgers University James Legg International Institute of Tropical Agriculture Linda Hanley-Bowdoin North Carolina State University Bruce Grieve University of Manchester Hujun Yin (  hujun.yin@manchester.ac.uk ) University of Manchester ABSTRACT Cassava brown streak disease (CBSD) is an emerging viral disease that can greatly reduce cassava productivity, while causing only mild aerial symptoms that develop late in infection. Early detection of CBSD enables better crop management and intervention. Current techniques require laboratory equipment and are labour intensive and often inaccurate. We have developed a handheld active multispectral imaging (A-MSI) device combined with machine learning for early detection of CBSD in real-time. The principal beneﬁts of A-MSI over passive MSI and conventional camera systems are improved spectral signal-to-noise ratio and temporal repeatability. Information fusion techniques further combine spectral and spatial information to reliably identify features that distinguish healthy cassava from plants with CBSD as early as 28 days post inoculation. Application of the device has the potential to increase farmers’ access to healthy planting materials and reduce losses due to CBSD in Africa. It can also be adapted for sensing other biotic and abiotic stresses in real-world situations where plants are exposed to multiple pest, pathogen and environmental stresses. Yao Peng1, Mary M. Dallas2, Jos´e T. Ascencio-Ib´a˜nez3, J. Steen Hoyer4, James Legg5, Linda Hanley-Bowdoin2, Bruce Grieve1, and Hujun Yin1,* 1Dept. of Electrical and Electronic Engineering, University of Manchester, Manchester, UK 2Dept. of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA 3Dept. of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, USA 4Dept. of Ecology, Evolution, and Natural Resources, Rutgers University, New Brunswick, NJ, USA 5International Institute of Tropical Agriculture (IITA), Tanzania hujun.yin@manchester.ac.uk Research Article Posted Date: August 2nd, 2021 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Scienti¦c Reports on February 24th, 2022. See the published version at https://doi.org/10.1038/s41598-022-06372-8. Early detection of plant virus infection using multispectral imaging and spatial-spectral machine learning Yao Peng1, Mary M. Dallas2, Jos´e T. Ascencio-Ib´a˜nez3, J. Steen Hoyer4, James Legg5, Linda Hanley-Bowdoin2, Bruce Grieve1, and Hujun Yin1, Introduction The advent of digital technology has been making an impact on growing number of areas including agriculture. There is a pressing need for better management of limited resources and optimisation of cultivation practice, including early detection of plant diseases. While end-point PCR is often the preferred diagnostic method for detection of viral nucleic acid in ﬁeld-collected samples, it is dependent on expensive instrumentation, time consuming and often cannot reliably detect virus early in infection, as seen for cassava brown streak disease (CBSD)1. Imaging technology has been applied to the analysis of plant conditions and nutrition levels, either based on visual traits or certain spectral properties reﬂected by the conditions or diseases2. Hyperspectral imaging (HSI) and multispectral imaging (MSI) have become increasingly available and affordable techniques that offer many advantages over conventional RGB imaging. RGB imaging has been used to recognise visual symptoms of the disease3, while plant nutritional conditions and metabolic or biotic changes due to disease may be reﬂected in certain spectral wavelengths beyond the RGB channels3–5. These subtle signs in vast amounts of spectral and spatial imaging data can be successfully detected using advanced machine learning techniques. With the rapid advancement of imaging sensors, MSI systems have become smaller and are able to be applied in real-time and in-ﬁeld. This paper describes the application of a custom built active MSI (A-MSI) device and a machine learning method that leverages both spectral and spatial information of the imagery data for early detection of CBSD. Cassava, Manihot esculenta Crantz, produces starchy tuberous roots and is one of the important staple food crops in the developing world6,7. It is cultivated primarily by smallholder farmers. Cassava production in Africa is limited by two viral diseases, cassava mosaic disease (CMD) and CBSD. Together these diseases cause severe economic losses and threaten food security8. CMD has been extensively studied and sources of endogenous resistance have been identiﬁed and deployed9–15. Unfortunately, many farmer-preferred cassava cultivars, like the CMD2-resistant cultivar TME204, are highly susceptible to CBSD. CBSD, which was ﬁrst reported in the coastal areas of Tanzania16, has emerged recently as serious threat to food production13,17. The rapid spread of CBSD throughout east and central Africa resulted in research targeting development of new cassava cultivars that are resistant to both CBSD and CMD7,18–20. Introduction CBSD is caused by two closely related RNA viruses, cassava brown steak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), in the Ipomovirus genus of the Potyviridae family. These viruses are transmitted from plant to plant by the whiteﬂy Bemisia tabaci21. The viruses are also propagated from one season’s crop to the next through the use of stem cuttings obtained from infected plants. CBSD typically induces only mild foliar symptoms that can be difﬁcult to discern. The subtle symptoms can make it difﬁcult even for experts to identify infected plants in the ﬁeld, and the call is often complex and based on many visual cues. However, the tuberous roots of infected cassava plants have prominent necrotic lesions that can spread throughout the entire root structure, rendering it inedible. Because the necrotic lesions or root rot are only discovered when cassava is harvested, farmers often do not know that their crop is infected with CBSD until harvest at 9-12 months after planting. p g Although considerable effort has been devoted to the search for strong sources of resistance to CBSD, the progress has been slow because of the lack of a rapid, reliable method for diagnosing CBSD during early stages of infection. Historically, diagnosis is based on subtle symptoms22 and scoring requires visible symptoms characteristic of established infections and does not distinguish resistant from tolerant plants. The use of molecular techniques to screen for the presence or absence of viral RNA has only recently been implemented in a few African national research programmes because of the technical requirements, cost, and time constraints involved in screening large numbers of plants7,20,23. To address these constraints, we have developed an active multispectral imaging (A-MSI) sensor system enhanced with machine learning as a screening platform for virus infection. Multispectral imaging of plant viral infections generates massive amount of data. Machine learning techniques have become the most efﬁcient means of extracting useful information from the wealth of the data available and for detecting the underlying relationships between certain mechanisms or functions under study and a large number of contributing parameters. Our previous studies24,25 have used feature selections and a novelty detection classiﬁer to distinguish healthy sugar beet plants from rust-diseased plants or stressed from control Arabidopsis plants using laboratory-based hyperspectral imaging systems. Performance was signiﬁcantly higher than conventional vegetation indices such as NDVI (the normalized difference vegetation index). Introduction In the study reported here, an A-MSI device and machine learning were combined to probe the early detection of CBSD. Such an approach alleviates the burden of using an expensive and precise MSI system. Machine learning techniques can effectively make sense of plant conditions even with a low-cost, compact and less precise MSI device. Combining spectral and spatial features of the scans, machine learning identiﬁed signiﬁcant differences at a high conﬁdence between healthy cassava plants and plants inoculated with UCBSV in three experimental trials. The approach reliably detects CBSD much earlier and in a faster and much less invasive manner than end-point PCR. The integrated handheld device with advanced machine learning should make it possible to detect disease in cassava ﬁelds early in the growing season, at a time when farmers can replant with virus-free cassava cuttings and should improve the efﬁciency of the work of cassava breeders in selecting for resistance to CBSD. Table 1. Experimental design of the Cassava-TME204-UCBSV A-MSI trials. Trial Cultivar Susceptibility Groups Treatment # Plant Leaf position CMD UCBSV 1 TME204 Resistant Susceptible Control Not inoculated 24 Leaf 2 for 7 dpi, leaf 3 for 28 and 53 dpi, and leaves 2 and 6 for 88 dpi. Mock Empty injection 12 Infected Inoculated by UCBSV 12 2 Control Not inoculated 18 Leaf 2 for 14, 28 and 54 dpi. Mock Empty injection 18 Infected Inoculated by UCBSV 18 3 Control Not inoculated 18 Leaf 2 for 7, 14, 21, 28, 52 and 59 dpi. Mock Empty injection 18 Infected Inoculated by UCBSV 18 Table 1. Experimental design of the Cassava-TME204-UCBSV A-MSI trials. Experimental Settings The white arrow points to faint yellow blotches characteristic of a symptom score of 2. b: Average symptom scores from 7-87 days. c: End-point RT-PCR of total RNA extracts isolated from untreated (U), mock-inoculated (M), and UCBSV-inoculated plants at 88 dpi. The upper panel shows a 445-bp band corresponding to UCBSV. The lower panel shows a 619-bp band corresponding to a cassava RbcS transcript, which served as a positive control for the isolation of ampliﬁable RNA. Figure 1. Infection results for Trial 2. a: Images of leaves from TME204 plants at 7, 28, 52 and 87 dpi. The symptom score of each leaf is in parentheses. The white arrow points to faint yellow blotches characteristic of a symptom score of 2. b: Average symptom scores from 7-87 days. c: End-point RT-PCR of total RNA extracts isolated from untreated (U), mock-inoculated (M), and UCBSV-inoculated plants at 88 dpi. The upper panel shows a 445-bp band corresponding to UCBSV. The lower panel shows a 619-bp band corresponding to a cassava RbcS transcript, which served as a positive control for the isolation of Figure 1. Infection results for Trial 2. a: Images of leaves from TME204 plants at 7, 28, 52 and 87 dpi. The symptom score of each leaf is in parentheses. The white arrow points to faint yellow blotches characteristic of a symptom score of 2. b: Average symptom scores from 7-87 days. c: End-point RT-PCR of total RNA extracts isolated from untreated (U), mock-inoculated (M), and UCBSV-inoculated plants at 88 dpi. The upper panel shows a 445-bp band corresponding to UCBSV. The lower panel shows a 619-bp band corresponding to a cassava RbcS transcript, which served as a positive control for the isolation of ampliﬁable RNA. dpi, a few plants (15%) inoculated with UCBSV displayed very mild symptoms on a single leaf. For plants showing symptoms, their severity slowly increased over the next 8 weeks. By 87 dpi, all of the plants in the UCBSV-inoculated treatment group displayed symptoms with an average symptom score of 2.6 (out of 4), highlighting the mild nature of the symptom phenotype (Fig. 1, panel A). UCBSV infection was conﬁrmed in 10 plants at 88 dpi by end-point RT-PCR of viral RNA (Fig. 1, panel C). We did not analyze viral RNA in the other 8 UCBSV-inoculated plants because the RcbS positive control could not be ampliﬁed from the samples. Experimental Settings Viral RNA could also be detected at 52 dpi in some plants but with variable results. During the timeframe of the experiment, no symptoms were seen on plants in the other treatment groups and the RT-PCR results were negative for the control and mock groups. These results indicate that inoculation was highly efﬁcient, consistent with the original report of this infectious clone28, but do not guarantee that 100% of plants in the virus-inoculated group were infected. Some of the classiﬁcation results presented below were therefore done using only the 10 plants with PCR-conﬁrmed infection (“Trial 2 with PCR”). Scanning of Cassava Leaves A single leaf (usually the second visible leaf from the apex) from each plant was screened using the A-MSI device, producing leaf images across 14 wavelengths. Twelve patches were automatically cropped from random spatial locations in the leaf area of each image at each wavelength, avoiding the leaf clapping grids and main leaf veins. This represents a simple approach to considering spatial variation in contrast to using the whole leaf. Cropped patches were of various sizes, varying from 16×16 to 48×48 pixels, which covered sufﬁcient spatial variations. Examples are shown in Fig. 2. Experimental Settings Experimental Settings In three independent trials, we used the cassava cultivar TME204, which is susceptible to CBSD, to generate three treatment groups - control, infected and mock-inoculated (18 plants in each group, except for Trial 1 – see Table 1). The infected group was inoculated with plasmid DNA corresponding to an infectious clone for the UCBSV Kenyan isolate 12526–28. The mock group was inoculated with an ‘empty’ control E. coli plasmid using the same protocol and the untreated group was not subjected to inoculation. All of the plants were grown together in an insect-free plant growth chamber for the duration of each experiment. The three treatment groups were visually indistinguishable at 7, 14 and 21 days post inoculation (dpi) (Fig. 1, panel B). At 28 p g In three independent trials, we used the cassava cultivar TME204, which is susceptible to CBSD, to generate three treatment groups - control, infected and mock-inoculated (18 plants in each group, except for Trial 1 – see Table 1). The infected group was inoculated with plasmid DNA corresponding to an infectious clone for the UCBSV Kenyan isolate 12526–28. The mock group was inoculated with an ‘empty’ control E. coli plasmid using the same protocol and the untreated group was not subjected to inoculation. All of the plants were grown together in an insect-free plant growth chamber for the duration of each experiment. The three treatment groups were visually indistinguishable at 7, 14 and 21 days post inoculation (dpi) (Fig. 1, panel B). At 28 2/15 Figure 1. Infection results for Trial 2. a: Images of leaves from TME204 plants at 7, 28, 52 and 87 dpi. The symptom score of each leaf is in parentheses. The white arrow points to faint yellow blotches characteristic of a symptom score of 2. b: Average symptom scores from 7-87 days. c: End-point RT-PCR of total RNA extracts isolated from untreated (U), mock-inoculated (M), and UCBSV-inoculated plants at 88 dpi. The upper panel shows a 445-bp band corresponding to UCBSV. The lower panel shows a 619-bp band corresponding to a cassava RbcS transcript, which served as a positive control for the isolation of ampliﬁable RNA. Figure 1. Infection results for Trial 2. a: Images of leaves from TME204 plants at 7, 28, 52 and 87 dpi. The symptom score of each leaf is in parentheses. Performance for Cassava Disease Detection From each cropped patch region of a scanned leaf, a variety of spectral and spatial features were explored, including six vegetation indices (VIs), patch-based spectra, as well as patch-based texture extracted by Markov random ﬁeld (MRF) probabilistic model. Support vector machine (SVM) was used as the basic classiﬁer, along with various information fusion 3/15 (a) (b) (c) (d) (e) Figure 2. Randomly selected patches of various sizes sampled from leaf areas, avoiding leaf clapping grid and main veins. Patch sizes (pixels): (a) 16×16, (b) 24×24, (c) 32×32, (d) 40×40 and (e) 48×48. (a) (e) (d) (b) (c) (d) (a) (e) (b) (c) Figure 2. Randomly selected patches of various sizes sampled from leaf areas, avoiding leaf clapping grid and main veins. Patch sizes (pixels): (a) 16×16, (b) 24×24, (c) 32×32, (d) 40×40 and (e) 48×48. Performance for detecting CBSD has been explored with various methods for utilising these spectral and spatial features and classiﬁer fusions, divided into to the following three categories, with corresponding results shown in Table 2. Performance for detecting CBSD has been explored with various methods for utilising these spectral and spatial features and classiﬁer fusions, divided into to the following three categories, with corresponding results shown in Table 2. 1. Conventional spectral methods (1st sub row of three under “Methods” in the table). In the vegetation indices method, each of six VIs was calculated and averaged from cropped patch regions and six VIs were then concatenated for classiﬁcation. The spatial reﬂectance (whole leaf) method refers to using the averaged spectral reﬂectance (spectrum) from each leaf. 2. Spatial-spectral methods (2nd sub row under “Methods” in the table). Spectral reﬂectance (patch-based) refers to using patch-based spectra for classiﬁcation. Instead of using averaged spectra from entire leaves, patch-based spectra represents the simplest spatial-spectral approach. The spatial-spectral fusion (patch-based) method refers to the use of concatenated patch spectrum and MRF texture parameters for classiﬁcation. 3. Spatial-spectral & classiﬁer fusion methods (3rd sub row under “Methods” in the table). Decision fusion (average) refers to fusion of three classiﬁers trained on VIs, spectral reﬂectance and MRF texture parameters extracted from patches. The patch-based voting scheme regards a leaf as infected when at least half of the patches extracted from the leaf are classiﬁed as infected. The scheme was used with either spectral information (spectral reﬂectance (patch-based voting)) or spatial-spectral information, i.e. concatenated patch spectrum and MRF parameters (spatial-spectral fusion (patch-based voting)). Performance for Cassava Disease Detection The proposed probabilistic decision fusion method (ProbDecFus) combines VIs, MRF texture features and spectral reﬂectances to generate reliable classiﬁcation. When the method is used on patches, we refer to it as ProbDecFus (patch-based). When it is used with patch-based voting, the method becomes ProbDecFus (patch-based voting). For a fair and comprehensive comparison, we separately trained the classiﬁers on three pairs of sample sets: control vs. infected, mock vs. infected, and control vs. mock. A leave-one-leaf-out scheme was adopted in Trial 2 and Trial 3 as the numbers of leaf samples in the three conditions (control, infected and mock) were 18:18:18. Trial 1 however had unbalanced sample numbers (24:12:12). We therefore randomly selected half of the control leaves each time and then performed the leave-one-leaf-out training. The random selection process of control samples was repeated at least 5,000 times and the averaged classiﬁcation results were produced. For ﬁnding the best hyperparameters of the SVM in each training, we randomly chose half of the training samples as the validation set and optimised the SVM classiﬁer using the grid search algorithm. As shown in Table 2, at 28 dpi the proposed ProbDecFus with patch-based voting achieved a classiﬁcation accuracy of 87.3% for Trial 1, 98.5% for Trial 2 and 93.6% for Trial 3 on control vs. infected. The methods using patch-based spatial information (fusion or not) greatly boosted classiﬁcation compared to using the whole leaf. Using additional MRF texture features further improves and stablises the performance. For the “Trial 2 with PCR” results, the classiﬁers were trained only from those leaves with detected UCBSV by end-point RT-PCR (Fig. 1 panel C). The similarities between this column and “Trial 2” column conﬁrm the effectiveness of the A-MSI device and classiﬁcation method in detecting CBSV at 28 dpi. The progressive detection performances on Trial 1 are shown as an example as it has the longest time course. Fig. 3 depicts the classiﬁcation results of various methods over leaf samples at 7, 28, 53 and 88 dpi. These graphs illustrate again that combining spatial and spectral information gives an edge over other ways of utilising the available information and signiﬁcantly outperforms the use of vegetation indices. It is worth noting that although MRF is a powerful model for describing spatial dependence, it does not deliver convincing results when used alone. Classifying Other HSI/MSI Data To demonstrate the generalizability of the developed spatial-spectral machine learning method, a public benchmark HSI dataset was used. The Indian Pine dataset is a HSI image dataset on land coverage29. Each image is of 145 by 145 pixels with a spatial 4/15 7 28 53 88 DPI 30 40 50 60 70 80 90 Classification rate (%) (a) 7 28 53 88 DPI 45 50 55 60 65 70 75 Classification rate (%) (b) 7 28 53 88 DPI 40 50 60 70 80 90 100 Classification rate (%) (c) Figure 3. Classiﬁcation accuracy (%) from leaf scans on Cassava-TME204-UCBSV dataset Trial-1, at 7, 28, 53 and 88 dpi, respectively, (a) control vs. infected, (b) mock vs. infected, and (c) control vs. mock. 7 28 53 88 DPI 30 40 50 60 70 80 90 Classification rate (%) 7 28 53 88 DPI 45 50 55 60 65 70 75 Classification rate (%) 7 28 53 88 DPI 40 50 60 70 80 90 100 Classification rate (%) (c) Figure 3. Classiﬁcation accuracy (%) from leaf scans on Cassava-TME204-UCBSV dataset Trial-1, at 7, 28, 53 and 88 dpi, respectively, (a) control vs. infected, (b) mock vs. infected, and (c) control vs. mock. 5/15 Table 2. Classiﬁcation accuracy (%) at 28 dpi on leaf scans of three trials of Cassava-TME204-UCBSV. ‘Trial 2 with PCR’ was the classiﬁcation results of the models re-trained only on those leaves that were later conﬁrmed of infection with PCR at 88 dpi. Groups Methods Trial 1 2 2 with PCR 3 Vegetation indices 56.0±13.1 87.7±10.0 89.4± 9.3 73.9±12.0 Spectral reﬂectance (whole leaf) 67.6±25.0 97.6± 8.3 96.9± 9.8 88.8±16.4 Spectral reﬂectance (patch-based) 78.9±13.7 95.8± 5.8 96.1± 5.0 90.8±11.3 Control Spatial-spectral fusion (patch-based) 67.7±13.6 94.1± 7.3 95.9± 6.3 89.1±11.9 vs. Decision fusion (average) 76.9±13.8 95.1± 6.6 95.6± 6.1 90.3±11.5 Infected Spectral reﬂectance (patch-based voting) 87.2±17.5 98.7± 5.6 98.2± 6.4 93.5±14.5 Spatial-spectral fusion (patch-based voting) 78.6±20.9 98.3± 6.3 98.0± 5.8 91.7±15.7 ProbDecFus (patch-based) 79.0±13.6 95.7± 6.3 96.5± 5.8 90.8±11.4 ProbDecFus (patch-based voting) 87.3±17.4 98.5± 6.9 98.6± 5.1 93.6±14.8 Vegetation indices 53.6±16.2 57.0±14.9 56.3±14.8 56.6±17.5 Spectral reﬂectance (whole leaf) 52.1±21.8 75.3±26.3 67.3±22.3 76.1±27.0 Spectral reﬂectance (patch-based) 65.7±13.3 72.6±19.2 67.7±17.6 76.9±18.4 Mock Spatial-spectral fusion (patch-based) 52.8±13.3 71.9±17.3 66.9±17.7 74.7±16.5 vs. Classifying Other HSI/MSI Data Decision fusion (average) 63.8±15.0 70.9±17.9 66.0±16.9 74.2±19.9 Infected Spectral reﬂectance (patch-based voting) 75.5±20.8 75.4±24.7 70.9±24.1 81.0±22.8 Spatial-spectral fusion (patch-based voting) 57.1±25.8 78.4±25.6 71.1±24.6 81.3±21.8 ProbDecFus (patch-based) 66.0±13.3 72.3±18.7 67.3±17.4 77.6±18.2 ProbDecFus (patch-based voting) 75.9±20.8 75.1±24.5 70.2±23.7 81.8±22.6 Vegetation indices 55.3±16.9 74.6±15.5 N/A 67.8±14.7 Spectral reﬂectance (whole leaf) 60.8±27.7 88.6±18.9 79.4±25.6 Control Spectral reﬂectance (patch-based) 63.6±17.7 91.4± 9.9 76.3±15.5 vs. Spatial-spectral fusion (patch-based) 59.8±15.5 90.4±11.0 77.1±15.2 Mock Decision fusion (average) 62.0±17.9 88.5±12.3 75.9±15.3 Spectral reﬂectance (patch-based voting) 70.7±25.5 93.4±11.5 84.3±20.2 Spatial-spectral fusion (patch-based voting) 66.9±26.2 93.3± 14.0 84.7±19.7 ProbDecFus (patch-based) 63.2±17.7 91.4±10.4 76.5±15.4 ProbDecFus (patch-based voting) 79.0±25.6 93.1±11.9 84.5±19.9 resolution of 20 m covering 16 different crops, provided in the ground truth reference as detailed in29. The dataset was captured by the AVIRIS sensor in June 1992 at the Indian Pines test site. The data contains a subset of a full scene that covers portions of Northwestern Tippecanoe County, IN, USA. The dataset is widely used in HSI analysis for validating classiﬁcation efﬁciency. The classiﬁcation was performed using a 5-fold cross-validation strategy, and repeated 10 times to achieve satisfactory resolution of 20 m covering 16 different crops, provided in the ground truth reference as detailed in29. The dataset was captured by the AVIRIS sensor in June 1992 at the Indian Pines test site. The data contains a subset of a full scene that covers portions of Northwestern Tippecanoe County, IN, USA. The dataset is widely used in HSI analysis for validating classiﬁcation efﬁciency. resolution of 20 m covering 16 different crops, provided in the ground truth reference as detailed in29. The dataset was captured by the AVIRIS sensor in June 1992 at the Indian Pines test site. The data contains a subset of a full scene that covers portions of Northwestern Tippecanoe County, IN, USA. The dataset is widely used in HSI analysis for validating classiﬁcation efﬁciency. The classiﬁcation was performed using a 5-fold cross-validation strategy, and repeated 10 times to achieve satisfactory i i The classiﬁcation was performed using a 5-fold cross-validation strategy, and repeated 10 times to achieve satisfactory precision. Convolutional neural networks (CNNs) were used on the extracted spectral-spatial features. The baseline model, named 2D-CNN, consisted of seven main blocks (architecture: 1×20-(8C3-8C3)-16R3-32R3-64R3-128R3-256R3-(512FC-16FC)). The ﬁrst block used two convolutional layers, each containing 8 ﬁlters of 1×3 with zero padding. Classifying Other HSI/MSI Data After subsequent ﬁve residual blocks, two fully connected layers were used with a softmax layer to produce probabilistic output over each class. Batch normalisation was inserted in each convolutional layer between the convolutional and ReLU activation. A max-pooling layer was also inserted in each residual block after the addition function. The developed Spatial-Spectral Net (SSFNet2D) had the same architecture as the baseline except that the input was further combined with MRF texture parameters extracted from the 25×25 surrounding neighbourhood of the centre pixels. The networks were trained using the Adam optimiser for 200 epochs with a batch size of 4. The learning rate started at 0.001 and was decreased using the polynomial scheduler. Again, the inclusion of spatial features was beneﬁcial, resulting in more accurate and more stable classiﬁcation results. Comparisons with the state-of-the-art methods are presented in Table 3 and classiﬁcation maps are shown in Fig. 4. Discussion Several technologies have been used for diagnosis of plant viral diseases, including, enzyme-linked immunosorbent assay (ELISA), loop mediated isothermal ampliﬁcation (LAMP)35, and PCR. All three have been used to detect CBSV and UCBSV, with PCR being the most widely used. RNA isolation from non-model plants like cassava is often technically challenging, whereas the scanning approach described here does not require RNA isolation. To date, the high cost and need for laboratory resources have been prohibitive for mass deployment of these technologies for real-time, in-ﬁeld detection of CBSD. Imaging offers an alternative to indirectly sense subtle biological changes reﬂected in plant leaves as a result of viral infection. Such an approach is almost instant once conﬁgured and can be portable, hence offering the potential for widespread, low-cost deployment. The results in this study also show that such changes can be detected at a very early stage, well before symptoms 6/15 Table 3. Class-speciﬁc accuracies (%) on Indian Pines dataset. Table 3. Class-speciﬁc accuracies (%) on Indian Pines dataset. Class 3D-CNN-LR RNN-GRU Feature-ensemble CNN-MRF HSINet UHfeSRVAE11 2D-CNN SSFNet2D 30 -PReTanh31 ND-SVM24 32 33 34 1 100 70.6 99.9±0.1 86.5 100 89.6 90.7±7.5 95.3±4.3 2 96.3±1.1 70.3 66.4±1.4 91.5 66.9 89.4 97.6±1.3 98.4±1.0 3 99.5±0.7 81.5 82.8±1.0 96.4 62.4 85.1 97.8±1.6 98.7±1.2 4 100 90.2 89.9±1.2 96.2 100 82.0 97.8±2.4 99.0±1.8 5 99.9±0.2 92.0 94.6±0.6 99.5 83.2 92.6 96.4±2.5 97.8±2.4 6 99.8±0.3 96.1 99.3±0.1 99.8 98.0 96.7 98.6±1.3 99.3±0.8 7 100 84.8 99.9±0.1 78.0 100 34.8 84.6±17.6 93.0±9.6 8 100 59.6 99.6±0.1 98.8 99.7 98.6 99.9±0.3 99.9±0.1 9 100 86.2 99.9±0.1 100 100 93.8 84.9±17.5 93.9±9.8 10 98.7±1.0 99.4 92.2±0.7 94.3 77.5 89.9 97.1±1.9 98.4±1.4 11 95.5±1.2 85.0 77.7±1.0 96.5 78.4 93.2 99.0±0.6 99.4±0.5 12 99.5±0.4 77.6 83.2±1.2 91.9 75.0 85.5 96.8±2.0 97.6±2.7 13 100 95.6 99.8±0.1 98.9 99.5 99.0 98.7±2.2 99.1±1.6 14 99.6±0.6 84.6 95.7±0.2 98.4 96.5 96.7 99.7±0.5 99.9±0.2 15 99.5±1.3 90.9 86.2±1.1 91.5 69.1 80.1 98.9±1.4 99.2±1.3 16 99.3±1.08 100 99.9±1.0 97.9 100 92.9 87.8±7.5 95.3±5.9 OA 97.6±0.4 88.6 - 96.1 83.0±0.2 91.4 98.1±0.4 98.9±0.4 AA 99.2±0.2 85.3 91.7±0.1 94.8 87.9±0.2 87.5 95.4±3.1 97.8±2.8 κ ×100 97.0±0.5 73.7 - 95.8 81.9±0.2 90.2 (a) (b) (c) Figure 4. Classiﬁcation maps of Indian Pines dataset obtained by compared methods. (a) Ground truth, (b) 2D-CNN and (c) SSFNet2D. Discussion Class 3D-CNN-LR RNN-GRU Feature-ensemble CNN-MRF HSINet UHfeSRVAE11 2D-CNN SSFNet2D 30 -PReTanh31 ND-SVM24 32 33 34 1 100 70.6 99.9±0.1 86.5 100 89.6 90.7±7.5 95.3±4.3 2 96.3±1.1 70.3 66.4±1.4 91.5 66.9 89.4 97.6±1.3 98.4±1.0 3 99.5±0.7 81.5 82.8±1.0 96.4 62.4 85.1 97.8±1.6 98.7±1.2 4 100 90.2 89.9±1.2 96.2 100 82.0 97.8±2.4 99.0±1.8 5 99.9±0.2 92.0 94.6±0.6 99.5 83.2 92.6 96.4±2.5 97.8±2.4 6 99.8±0.3 96.1 99.3±0.1 99.8 98.0 96.7 98.6±1.3 99.3±0.8 7 100 84.8 99.9±0.1 78.0 100 34.8 84.6±17.6 93.0±9.6 8 100 59.6 99.6±0.1 98.8 99.7 98.6 99.9±0.3 99.9±0.1 9 100 86.2 99.9±0.1 100 100 93.8 84.9±17.5 93.9±9.8 10 98.7±1.0 99.4 92.2±0.7 94.3 77.5 89.9 97.1±1.9 98.4±1.4 11 95.5±1.2 85.0 77.7±1.0 96.5 78.4 93.2 99.0±0.6 99.4±0.5 12 99.5±0.4 77.6 83.2±1.2 91.9 75.0 85.5 96.8±2.0 97.6±2.7 13 100 95.6 99.8±0.1 98.9 99.5 99.0 98.7±2.2 99.1±1.6 14 99.6±0.6 84.6 95.7±0.2 98.4 96.5 96.7 99.7±0.5 99.9±0.2 15 99.5±1.3 90.9 86.2±1.1 91.5 69.1 80.1 98.9±1.4 99.2±1.3 16 99.3±1.08 100 99.9±1.0 97.9 100 92.9 87.8±7.5 95.3±5.9 OA 97.6±0.4 88.6 - 96.1 83.0±0.2 91.4 98.1±0.4 98.9±0.4 AA 99.2±0.2 85.3 91.7±0.1 94.8 87.9±0.2 87.5 95.4±3.1 97.8±2.8 κ ×100 97.0±0.5 73.7 - 95.8 81.9±0.2 90.2 (a) (b) (c) gure 4. Classiﬁcation maps of Indian Pines dataset obtained by compared methods. (a) Ground truth, (b) 2D-CNN and FNet2D. (c) (b) (a) (c) (b) (a) Figure 4. Classiﬁcation maps of Indian Pines dataset obtained by compared methods. (a) Ground truth, (b) 2D-CNN and (c) SSFNet2D. emerge, whereas end-point PCR depends on viral RNA accumulation to detectable levels, which occurs later in infection. Across the three trials, the available common sampling points were at 7, 28 and 53 dpi, as shown in Table 1. Trials 1 and 3 were also scanned at 14 dpi and detection of differences was also highly probable (>80%). Further optimization of the spectral proﬁle of the A-MSI device may enable still earlier detection of CBSD. In principle, early detection of infection in primary whiteﬂy-infected leaves could even enable removal of infected tissue before the virus can move systemically thus eliminating infection to preserve the yield of individual plants. emerge, whereas end-point PCR depends on viral RNA accumulation to detectable levels, which occurs later in infection. Across the three trials, the available common sampling points were at 7, 28 and 53 dpi, as shown in Table 1. Trials 1 and 3 were also scanned at 14 dpi and detection of differences was also highly probable (>80%). Discussion Further optimization of the spectral proﬁle of the A-MSI device may enable still earlier detection of CBSD. In principle, early detection of infection in primary whiteﬂy-infected leaves could even enable removal of infected tissue before the virus can move systemically thus eliminating infection to preserve the yield of individual plants. Machine learning approaches for analysis of MSI data can effectively even out the inaccuracies of the imaging to some extent. Even with intuitively chosen wavelengths, as compared to those strictly optimized through many rounds of cross- validation process, machine learning has proven useful and effective. This in principle is in line with its broad deviation from the traditional orthogonal approach in information processing. With an active MSI system, more crop or growth conditions could be investigated using the modulation properties of various wavelengths of electromagnetic spectrum in response to metabolic changes in the organism, as well as translucent properties of the plant leaves, extending the imaging approach from spectral and spatial to temporal and transitive. We are optimistic that reﬁnements of this approach may be useful for early detection of infection in a wide range of crop pathosystems. 7/15 Active Multispectral Imaging (A-MSI) System p g g ( ) y A handheld active multispectral imaging (A-MSI) prototype, developed at the e-Agri Sensors Centre, the University of Manchester, was used to obtain the data presented in this study. The sensor system exploits a modiﬁed proprietary digital imaging detectors appropriately engineered within the active optical assembly, also to enable the vastly overlapping ‘Nth- order’ molecular vibrational harmonics, from the near-infrared and visible 2D time-series data, to be deconvoluted. Isotropic illumination is achieved, with minimised specular reﬂectance, via a combination of an integrating hemisphere, optical diffuser and appropriately arranged narrow-band semiconductor sources (LEDs). The latter cover 15 wavebands using 10 LEDs per waveband, at the wavelengths detailed in the Table 4. Custom drive electronics are then used to enable the multispectral frames to be compiled within a parallel processing unit (NVDIA Jetson Nano). The variant of A-MSI adopted in the study is distinct from more traditional passive multispectral imaging (MSI) systems36 as closed-loop control of the illumination power at each detection band enables highly repeatable measurements to be undertaken with signiﬁcantly greater signal-to-noise ratio (SNR) than that by a ﬁltered or dispersive-element based passive MSI sensor-system. This is due, in part, to the variability in illumination angle, spectral composition and polarisation of ambient illumination. The prototype instrument used in the study is shown in Fig. 5, which depicts the as-built unit and the inset measurement chamber, relative position of the LED array, and camera assembly within the integrating hemisphere. The system automatically calibrates the illumination level for each band at the start of each scanning process. (a) (b) Figure 5. (a) Photo of the developed A-MSI system and (b) its LED ring and sensing chamber. (a) (b) (a) (b) Figure 5. (a) Photo of the developed A-MSI system and (b) its LED ring and sensing chamber. Data Preprocessing In the three experiments, multispectral scans of cassava leaves were sampled by automatically cropping out patches. Twelve patches were cropped out at random spatial locations of the leaf region from each leaf scan. For spectral analysis, the number of pixels from the cropped leaf area were averaged over each wavelength range to reduce the variability in pixel intensities and produce the spectral signature. Examples are shown in Fig. 2. Such patch-based spectral information represents the simplest approach to consider spatial variation. Averaged reﬂectance across the entire leaf was used for comparisons, in which leaf segmentation was performed and average reﬂectance calculated. Cassava Leaf MSI Acquisition Leaves were detached from the plants at 7, 28, 53 and 88 dpi, and the adaxial and abaxial surfaces were scanned using a handheld multispectral imaging instrument. The leaves were sampled from the same position on each plant (leaf 2 or leaf 6 counting from the plant apex). The plants were also scored for symptom development at the same dpi using a scale from 1 (no visible symptoms) to 4 (severe symptoms). Each leaf was ﬂash frozen and stored at -80◦C for future analysis of viral DNA titres. Details of the experimental design are shown in Table 1. Table 4. Detailed wavelength information used in the A-MSI system. LED No. Band No. Wavelength (centre band) 1 8 395 nm 2 9 415 nm 3 10 470 nm 4 11 528 nm 5 12 532 nm 6 13 550 nm 7 14 570 nm 8 0 585 nm 9 1 590 nm 10 2 610 nm 11 3 625 nm 12 4 640 nm 13 5 660 nm 14 6 700 nm 15 7 880 nm null 15 No LED M-MuLV reverse transcriptase (200U/µL) using the following conditions: 5 min at 25◦C , 60 min at 42◦C, 20 min at 65◦C. cDNA (5 µL) was ampliﬁed in PCR reactions comprising 10 pMol primers, 10 mM dNTPs, and Hot Start Taq polymerase (NEBioLabs). UCSBV RNA was ampliﬁed across the 3 ′ NIa-Pro and 5 ′ NIb coding sequences using the primer pair – UCBSV-F (GGGTTCCATAGTGGTGTGATTAG) and UCBSV-F (CTCGAACTGGCTCATTGTACTT). The cassava RbcS transcript (Manes.05G137400.1), which served as a positive control for mRNA and cDNA quality, was ampliﬁed using the primer pair – RBC-1 (CTACTATGGTGGCTCCGTTC) and RBC-2 (CCGTTCAGTCGGAGAAACTC). Both sequences were ampliﬁed for 30 cycles using the following conditions: 95◦C denaturation for 60 sec, 51◦C annealing for 60 sec, 68◦C extension for 60 sec. The PCR products were resolved on 1% agarose gels. The UCBSV products were puriﬁed using the Qiagen PCR puriﬁcation kit and veriﬁed by Sanger sequencing. Cassava Cultivation and Virus Inoculation Cassava Cultivation and Virus Inoculation Cassava Cultivation and Virus Inoculation Cassava cuttings (Manihot esculenta cultivar TME204) were originally provided by J. Ndunguru (Tanzania Agricultural Research Institute). Import and containment of plant cuttings and the UCBSV infections clone (pLX-UCBSVi) followed international, U.S. (Department of Agriculture Animal and Plant Health Inspection Service), and institutional guidelines. Plants were propagated at 28◦C in a 12-h light/12-h dark cycle. For each experiment, 18 plants at the 6-leaf stage were inoculated under the apical meristem area using a microsprayer and 40 psi helium to deliver gold particles coated with plasmid DNA37 (Venganza, Inc.). Each plant was inoculated with 1.67 µg of plasmid DNA corresponding to pLX-UCBSVi (infected)27,28 or pUC119 (mock). pLX-UCBSVi contained an E35S expression cassette driving transcription of UCBSV Ke_125 (GenBank accession KY82516626). Untreated plants (18) were not subjected to the inoculation treatment. Plants were monitored for symptoms from 7 – 88 dpi, and were scored on a scale of 1 (no symptoms), 2 (small yellow blotches on 1 leaf), 3 (yellow blotches on two leaves), and 4 (yellow blotches and yellowing along veins on multiple leaves). Samples (1 mg) were collected at 88 dpi near the petiole of leaf 2 (relative to the plant apex), ﬂash-frozen in liquid nitrogen, and stored at 80◦C. Leaf samples were ground in a Retsch Mixer Mill, followed by RNA extraction using a Qiagen RNeasy Plant Mini kit. RNA concentration was measured using the Qubit RNA BR Assay Kit (Invitrogen). cDNA was synthesized in reactions containing 0.5 µg total RNA, oligo d(T)18 mRNA primer (60 µM), RNasin (40U/µL), dNTPs (10mM), and Samples (1 mg) were collected at 88 dpi near the petiole of leaf 2 (relative to the plant apex), ﬂash-frozen in liquid nitrogen, and stored at 80◦C. Leaf samples were ground in a Retsch Mixer Mill, followed by RNA extraction using a Qiagen RNeasy Plant Mini kit. RNA concentration was measured using the Qubit RNA BR Assay Kit (Invitrogen). cDNA was synthesized in reactions containing 0.5 µg total RNA, oligo d(T)18 mRNA primer (60 µM), RNasin (40U/µL), dNTPs (10mM), and 8/15 Vegetation Indices (VIs) Calculation The spectral signatures from each cropped patch were extracted and averaged to calculate empirical VIs. Based on the 14 wavelength bands provided by the A-MSI, six empirical indices were extracted and analysed to study plant properties and conditions. The primary formulation is the Carter index (CI), a strong indicator for plant stress, which measures the ratio between reﬂectance at 695 nm and 420 nm38, CI = R695 R420 . (1) CI = R695 R420 . (1) The modiﬁed chlorophyll absorption in reﬂectance index (MCARI) measures the depth of chlorophyll absorption at 670 nm relative to the green reﬂectance peak at 550 nm and the reﬂectance 700 nm39, MCARI = [(R700 −R670 −0.2(R700 −R550)]R700 R670 . (2) 0 −0.2(R700 −R550)]R700 R670 . (2) MCARI = [(R700 −R670 −0.2(R700 −R550)]R700 R670 . (2) 9/15 The optimised index transformed chlorophyll absorption in reﬂectance index (TCARI) was studied as being more sensitive to chlorophyll content, thus avoiding the inﬂuence by canopy and soil reﬂectance values40, TCARI = 3[(R700 −R670 −0.2(R700 −R550)(R700 R670 )]. (3) TCARI = 3[(R700 −R670 −0.2(R700 −R550)(R700 R670 )]. (3) The photochemical reﬂectance index (PRI) measures the normalised difference VI of reﬂectivity at 531 nm and 570 nm and has been developed for disease detection41, The photochemical reﬂectance index (PRI) measures the normalised difference VI of reﬂectivity at 531 nm and 570 nm and has been developed for disease detection41, PRI = (R531 −R570) (R531 +R570). (4) PRI = (R531 −R570) (R531 +R570). (4) The disease water stress index (DSWI) is the ratio between reﬂectances at 550 nm and 680 nm42, The disease water stress index (DSWI) is the ratio between reﬂectances at 550 nm and 680 nm42, The disease water stress index (DSWI) is the ratio between reﬂectances at 550 nm and 680 nm42, DSWI = R550 R680 . (5) And the healthy index (HI)43 can be expressed by, HI = (R534 −R698) (R534 +R698) −0.5R704. (6) Conventional Decision Fusion Techniques q The general idea of classiﬁer/decision fusion can be summarised as merging multiple learners or classiﬁers to produce the best possible decision so as to enhance the prediction performance over a single classiﬁer. By taking into account the outputs of all classiﬁers, combinations of multiple classiﬁers minimise the risk of choosing a weak classiﬁer, stabilise results of random classiﬁers and increase the robustness of the decisions44. Classiﬁer/decision fusion has been an active research topic in machine learning since the late twentieth century and much of the effort has been devoted to combining classiﬁers for decision making in several pattern recognition applications45–48. Typically, in a multiple classiﬁer system, there are two general approaches to obtaining the ﬁnal decision44: 1. Selection: Assuming complementary classiﬁers, only a single selected classiﬁer contributes to the ﬁnal decision. 2. Fusion: Assuming competitive classiﬁers, the integration of all classiﬁers determines the ﬁnal decision. Based on the output information of classiﬁers, fusion can be divided into three levels49: Based on the output information of classiﬁers, fusion can be divided into three levels49: 1. Abstract level: Each classiﬁer only outputs the predicted class label for each input. An abstract level combiner includes weighted or unweighted versions of the majority vote. 2. Rank level: For each input, classiﬁers rank all labels or classes and produce a list of possible predictions. 3. Measurement level: Instead of class labels, each classiﬁer outputs the probability or conﬁdence score for each class. The measurement level contains the most information among these three levels, making it possible to incorporate with various combiners (e.g. average, maximum, minimum and product), by using either selection or fusion methods. Various methods in the literature are also concerned with how the ﬁnal outputs can be combined. Majority vote is the simplest and most used combiner, in which the ensemble of classiﬁers choose the class that receives the highest number of votes. The fusion scheme for the unweighted majority voting can be described as, Various methods in the literature are also concerned with how the ﬁnal outputs can be combined. Majority vote is the simplest and most used combiner, in which the ensemble of classiﬁers choose the class that receives the highest number of votes. Markov Random Field Texture Analysis y As a fundamental image property descriptor, image texture models brightness variations in a local neighbourhood. Furthermore, image texture features are associated with various image properties such as orientation, coarseness and smoothness and quantify the spatial arrangements of pixel intensities in an image or an image region. Texture-based image analysis has been shown to be helpful in various applications such as remote sensing, medical imaging and industrial inspection. MRFs are generative, ﬂexible and stochastic image texture models, in which contextual dependencies and spatial interrela- tionships are established among image pixels or other correlated features51. Due to the random nature of imaging and noise, pixels are naturally considered as random variables that are conditionally related to neighbouring variables. As undirected probabilistic graph models, MRFs not only specify the conditional dependencies between these random variables, but also interpolate the joint probability distributions with useful potential functions52. MRF based texture analysis plays an important role in modern texture modelling and synthesis53–55 as well as helps visual interpolation and image understanding52,56,57. A typical Gaussian MRF model is a stationary noncausal two-dimensional autoregressive process that can be expressed by a set of difference equations51,58 as fs = ∑ s+r∈Ns βr fs+r +es, (13) where r is the relative position with respect to central pixel s, and {es} is a stationary Gaussian noise sequence with zero mean and standard deviation σ2 characterised by where r is the relative position with respect to central pixel s, and {es} is a stationary Gaussian noise sequence with zero mean and standard deviation σ2 characterised by E(eses+r) =    σ2 if r = (0,0) −σ2βr if r ̸= (0,0) 0 otherwise , (14) E(eses+r) =    σ2 if r = (0,0) −σ2βr if r ̸= (0,0) 0 otherwise , if r = (0,0) if r ̸= (0,0) otherwise , (14) (14) where βr is the model parameter describing the relationship between pixels fs and fs+r. All the parameters βr in the neighbourhood system Ns form the parameter vector βββ. where βr is the model parameter describing the relationship between pixels fs and fs+r. All the parameters βr in the neighbourhood system Ns form the parameter vector βββ. In model-based texture methods, model parameters can be used as features for distinguishing textures. Model parameter estimation plays an signiﬁcant role in analysing image properties and the least squares estimation is commonly used to estimate Gaussian MRF models58. Conventional Decision Fusion Techniques The fusion scheme for the unweighted majority voting can be described as, ˆy = arg max θ j∈{θ1,θ j,···,θC} L ∑ i=1 ˆyi, j, ˆy = arg max θ j∈{θ1,θ j,···,θC} L ∑ i=1 ˆyi, j, (7) (7) where {θ1,θj,··· ,θC} are the C possible classes that an input is to be assigned to, L denotes the total number of classiﬁers, ˆyi, j is the predicted output of the i-th classiﬁer for the j-th class, and ˆy represents the ﬁnal decision. In cases where each classiﬁer contributes unequally to the fusion output, a weighted majority vote scheme can be employed by associating a weighting wi for i-th classiﬁer, and the decision becomes, where {θ1,θj,··· ,θC} are the C possible classes that an input is to be assigned to, L denotes the total number of classiﬁers, ˆyi, j is the predicted output of the i-th classiﬁer for the j-th class, and ˆy represents the ﬁnal decision. In cases where each classiﬁer contributes unequally to the fusion output, a weighted majority vote scheme can be employed by associating a weighting wi for i-th classiﬁer, and the decision becomes, ˆy = arg max j∈{θ1,θ j,···,θC} L ∑ i=1 wi ˆyi, j. (8) 10/15 Apart from the majority voting, multiple rules can be applied at the measurement level47,50. The maximum, minimum or average rule ﬁnds the maximum, minimum or average probability of each class among the classiﬁers and assigns the input to the class with the maximum score among the maximum, minimum or average scores, respectively. These rules can be expressed as, ˆy = arg max θ j∈{θ1,θ j,···,θC}max L P(θ j\|xi), (9) ˆy = arg max θ j∈{θ1,θ j,···,θC}(1−min L P(θ j\|xi)), (10) ˆy = arg max θ j∈{θ1,θ j,···,θC} 1 L L ∑ i=1 P(θj\|xi), (11) where P(θ j\|xi) represents the estimated probability for input x that the i-th classiﬁer output xi belongs to the j-th class θj. Similarly, the product rule multiplies the probabilities or conﬁdence scores generated by each classiﬁer and assigns the class label with the maximum score to given input pattern, ˆy = arg max θ j∈{θ1,θ j,···,θC} L ∏ i=1 P(θ j\|xi). (12) Markov Random Field Texture Analysis The quadratic difference Θ between the centre pixel and its neighbours can be deﬁned as Θ = ∑ s fs −∑ s+r∈Ns βr fs+r 2 . (15) Θ = ∑ s fs −∑ s+r∈Ns βr fs+r 2 . (15) The least squares problem can be resolved by a close-form solution, The least squares problem can be resolved by a close-form solution, ˆβββ = (FT s+rFs+r)−1FT s+rΘ, (16) where fs+r represents the neighbouring pixels of fs. 11/15 ProbDecFus: Probabilistic Decision Fusion Support vector machines (SVMs) are commonly used machine learning algorithms for classiﬁcation and regression. Given training vectors XXX = {xxx(1),xxx(2),··· ,xxx(N)} = {xxx(k)}N k=1 ∈RM×N and its corresponding class labels YYY = {y(1),y(2),··· ,y(N)} = {y(k)}N k=1, the ν-SVM59 solves the quadratic optimisation problem min ωωω,b,ξξξ,ρ 1 2ωωωTωωω −νρ + 1 N N ∑ k=1 ξk s.t. y(k)(ωωωTφ(xxx(k))+b) ⩾ρ −ξk ν ∈(0,1], ξk ⩾0, ρ > 0 , (17) min ωωω,b,ξξξ,ρ 1 2ωωωTωωω −νρ + 1 N N ∑ k=1 ξk s.t. y(k)(ωωωTφ(xxx(k))+b) ⩾ρ −ξk ν ∈(0,1], ξk ⩾0, ρ > 0 , (17) where ωωω denotes the weight vector, b is the learning bias, ξ is a non-zero slack variable, and ν is the regularisation parameter that controls the trade-off between smaller training errors and larger margins. ν ∈(0,1] represents an upper bound on the fraction of training margin errors as well as a lower bound of the fraction of support vectors59,60. Training vectors xxxi are mapped into a high-dimensional space by function φ though the kernel trick K(xxx(i),xxx(j)) = φ(xxx(i))Tφ(xxx(j)). A radial basis function (RBF) is a typical kernel function where ωωω denotes the weight vector, b is the learning bias, ξ is a non-zero slack variable, and ν is the regularisation parameter that controls the trade-off between smaller training errors and larger margins. ν ∈(0,1] represents an upper bound on the fraction of training margin errors as well as a lower bound of the fraction of support vectors59,60. Training vectors xxxi are mapped into a high-dimensional space by function φ though the kernel trick K(xxx(i),xxx(j)) = φ(xxx(i))Tφ(xxx(j)). A radial basis function (RBF) is a typical kernel function K xxx(i),xxx(j) = exp −γ∥xxx(i) −xxx( j)∥2 , γ > 0, (18) K xxx(i),xxx(j) = exp −γ∥xxx(i) −xxx( j)∥2 , γ > 0, (18) where γ is the kernel parameter. Markov Random Field Texture Analysis Hence the predicted class labels ˆYYY = {ˆy(1), ˆy(2),··· , ˆy(N)} = {ˆy(k)}N k=1 can be obtained through the decision function, where γ is the kernel parameter. Hence the predicted class labels ˆYYY = {ˆy(1), ˆy(2),··· , ˆy(N)} = {ˆy(k)}N k=1 can be obtained through the decision function, ˆy = sigmoid N ∑ k=1 αky(k)K(xxx(k),xxx)+b , k)K(xxx(k),xxx)+b , (19) ˆy = sigmoid N ∑ k=1 αky(k)K(xxx(k),xxx)+b , (19) (19) where αk is the Lagrange multiplier. In addition to predicted class labels, it is also possible to obtain an estimated probability for each class, P(θj\|xxx(k)), by minimising the negative log likelihood and optimising the quadratic problem60–62. In this study, three independent SVM classiﬁers were constructed based on the spectral reﬂectances, VIs and MRF spatial features, respectively. The spectral reﬂectance proﬁles, xxx(k) 0 , extracted from selected areas of leaves, were averaged within the regions over the entire wavelengths. The empirical VI information, xxx(k) VI , refers to the concatenation of six VIs (CI, MCARI, TCARI, PRI, DWSI and HI) calculated on the spectral reﬂectance. The MRF spatial features, xxx(k) MRF, were produced by estimating the texture parameters in each of the selected area. The classiﬁer built on the spectral reﬂectances was considered as the baseline model, and we proposed a probabilistic decision fusion scheme, ProbDecFus, for integrating spectral and spatial information for further classiﬁcation. Firstly, we calculated a threshold value µ based on the classiﬁcation accuracy of the validation set (accval), (20) µ = µ1 ∗accval + µ2 ∗(1−accval) where, max ∈{θ1 θj ··· θC}Pval(θj\|xxx(k) 0 ) ( µ1 = min k∈{y(k)=ˆy(k)} max θj∈{θ1,θj,···,θC}Pval(θj\|xxx(k) 0 ) (21) max 1,θj,···,θC}Pval(θj\|xxx(k) 0 ) (22) µ2 = max k∈{y(k)̸=ˆy(k)} max θ j∈{θ1,θj,···,θC}Pval(θj\|xxx(k) 0 ) (22) max {y(k)̸=ˆy(k)} max θ j∈{θ1,θj,···,θC}Pval(θj\|xxx(k) 0 ) (22) µ2 = max k∈{y(k)̸=ˆy(k)} max θ j∈{θ1,θj,···,θC}Pval(θj\|xxx(k) 0 ) Then according to the probability estimations, the ﬁnal classiﬁcation results were generated by weighting and fusing the three classiﬁers using Then according to the probability estimations, the ﬁnal classiﬁcation results were generated by weighting and fusing the three classiﬁers using w(k) 0 = 1− minθ j∈{θ1,θ j,···,θC} P(θj\|xxx(k) 0 ) maxθ j∈{θ1,θ j,···,θC} P(θj\|xxx(k) 0 ) \|xxx(k) 0 ) j\|xxx(k) 0 ) (23) (23) w(k) i =      1− minθ j∈{θ1,θ j,···,θC} P(θj\|xxx(k) i ) maxθ j∈{θ1,θ j,···,θC} P(θj\|xxx(k) i ) w(k) 0 ⩽wµ 0 w(k) 0 > wµ (24) 12/15 References 1. 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All authors contributed to analysis of the results and drafting and approving the submitted manuscript. M.M.D., J.T.A-I., J.S.H. and L.H.B. designed the laboratory experiments. M.M.D. collected the data. Y.P., B.G. and H.Y. analysed the data and designed ML methods, and B.G. designed the MSI device. All authors contributed to analysis of the results and drafting and approving the submitted manuscript. M.M.D., J.T.A-I., J.S.H. and L.H.B. designed the laboratory experiments. M.M.D. collected the data. Y.P., B.G. and H.Y. analysed the data and designed ML methods, and B.G. designed the MSI device. All authors contributed to analysis of the results and drafting and approving the submitted manuscript. Competing Interests The authors declare no competing interests. The authors declare no competing interests. 15/15 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. Fig1C.docx Supplementaryinformation.pdf

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